WO2019084008A2 - Plzf+ regulatory cd8 t cells for control of inflammation - Google Patents

Plzf+ regulatory cd8 t cells for control of inflammation

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Publication number
WO2019084008A2
WO2019084008A2 PCT/US2018/057112 US2018057112W WO2019084008A2 WO 2019084008 A2 WO2019084008 A2 WO 2019084008A2 US 2018057112 W US2018057112 W US 2018057112W WO 2019084008 A2 WO2019084008 A2 WO 2019084008A2
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treg
cells
cds
mice
specific
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WO2019084008A3 (en
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Vipin Kumar Chaturvedi
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • G—PHYSICS
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
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    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
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    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3061—Blood cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0637—Immunosuppressive T lymphocytes, e.g. regulatory T cells or Treg
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/2302—Interleukin-2 (IL-2)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)

Definitions

  • T cells are controlled by both T cell-intrinsic and extrinsic cell-based mechanisms that prevent them from causing excessive tissue damage: immune ignorance, anergy, exhaustion, phenotype skewing and antigen-induced apoptosis are part of the intrinsic mechanisms whereas regulatory T cells (Treg), including Foxp3 " CD4 " and Trl, natural killer T (NKT) cells and CD8 " T cells comprise cell-based mechanisms.
  • CD8 + T cells play an important role in immune regulation of autoimmune diseases, transplant tolerance and in homeostasis of cellular and humoral immune responses.
  • mice genetically deficient in or depleted of CD8 ⁇ T cells by treatment with anti-CD8 mAb an important role for CD8 T cells in regulation of EAE and arthritis has been shown.
  • a critical regulatory role of CD122 ⁇ CD8 ⁇ T cells involving a cytolytic mechanism has been shown in IL-2-/- and IL-2RP-/- mice, interestingly, IL-2/IL-15Rp-deficiency in humans also leads to a severe combined immunodeficiency syndrome as observed for IL-2Ra.
  • IL-2-/- and IL-2RP-/- mice interestingly, IL-2/IL-15Rp-deficiency in humans also leads to a severe combined immunodeficiency syndrome as observed for IL-2Ra.
  • CD122 " CD8 " T ceils can provide barriers to stem cell engraftment, indicating the clinical relevance of CD8 Treg in humans.
  • a critical role for CD8 + T cells in IL-2-/- mice lacking CD8 + T cells was shown as these animals develop colitis with an accelerated kinetics.
  • CD8 + T ceils also have been implicated in various conditions in humans, e.g. transplant survival, prevention of inflammatory bowel disease, and the treatment of multiple sclerosis with either glatiramer acetate (GA) or vaccination with irradiated, myelin basic protein-activated CD4 + T cells.
  • GA-induced CD8 + Treg have been shown to eliminate CD4 + T cells in a HLA-E-restricted manner.
  • anti-CD3 mAb anti-CD3 mAb
  • CD8 + T regulatory ceils have been a subject of study for many years, but this cell population has been difficult to define and identify.
  • immunosuppressive cells can be very important in the initiation and/or the maintenance of diseases when their numbers are skewed (high or low), such as in cancer, 1BD, lupus, rheumatoid arthritis and various autoimmune diseases. The hardest part so far has been to identify and target these cells efficiently to
  • CD8 + T regulator ' cell An immunosuppressor type of unconventional CD8 + T regulator ' cell, which is more prevalent in the liver and in the gut, is identified.
  • Adoptive transfer of these CD8 + T regulatory ceils protect mice from developing antigen- induced EAE (a model for multiple sclerosis) and also protect mice from developing T cell- induced-colitis (a model for IBD).
  • administration of anti- 4-iBB antibody increases the number of these CDS" T regulatory in the liver.
  • the anti-4-lBB antibody was administered in mice with antigen -induce EAE, these mice show lesser symptoms than the negative controls. Similar results were obtained when anti- CD3 was administered in these EAE mice.
  • innate-like PLZF + CD8aa Treg enriched in liver and in intestine play an important role in the homeostasis of immunity.
  • the disclosure provides for the use of antibodies to identify a CD8 + T regulatory cell population (e.g., by flow cytometry) in a subject with the following minimal phenotype : TC a(3 + CD 8 axr NK 1.1 + PLZF * CD 161 ⁇ in humans, CD1 lc + , though other markers are usually present too, for example one or more of CD137 + ( ' 1)244 ' . or one or more of the NK-inhibitory receptors.
  • the disclosure provides for a method of treating cancer patients by administering monoclonal antibodies that bind to this CD8+ T reg cell population with the goal of lowering the number of this T cell population or eliminating these altogether in patients, allowing them to mount a proper immune response to the tumor.
  • the disclosure provides for a method of treating IBD, lupus, RA patients and patients suffering from other autoimmune diseases by administering Qa- l/HLA-E binding peptides that will stimulate CD8T regs.
  • the disclosure provides for a method of treating IBD, lupus, RA patients and patients suffering from other autoimmune diseases by administering anti- CD3 in patients.
  • the disclosure provides for a method of treating IBD, lupus, RA patients and patients suffering from other autoimmune diseases by administering anti- CD137 (also known as anti-4-lBB).
  • anti- CD137 also known as anti-4-lBB
  • the disclosure provides for sorting and expanding patient ceils with cytokines IL-2/IL-15 and a combination of antibodies mentioned above.
  • the cells are identified using monoclonal antibodies specific for CDSota, NKl . l, PLZF, or CD161 , and optionally antibodies specific for one or more of CD I lc, CD 137, CD244, TCRctfl or one or more of NK- inhibitory receptors or a combination thereof.
  • the cells are human cells.
  • the method includes isolating the identified cells and optionally expanding the isolated cells.
  • the cells are from a patient with an autoimmune disease.
  • the cells are cultured with IL-2, IL-15, Qa-l/HLA-E binding peptides, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof.
  • the composition is systemicaiiy administered. In one embodiment, the composition is locally administered.
  • the method includes administering to the mammal an effective amount of a composition comprising one or more antibodies specific for CD8aa, specific for NK 1.1, specific for PLZF, or specific for CD 161, or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD 11c, specific for CD 137, specific for CD244, specific for TCRaP, or specific for one or more of NK -inhibitory receptors, or a combination thereof.
  • the cancer is neck cancer.
  • the cancer is melanoma
  • the mammal is a human.
  • the composition is systemicailv administered. In one embodiment, the composition is locally administered.
  • a method to pre vent, inhibit or treat autoimmune disease in a mammal includes administering to the mammal a composition comprising one or more Qa-l/HLA-E binding peptides, anti-CD3 antibodies or anti-CD137 antibodies in an amount effective to stimulate CDSa+T regs.
  • the disease is IBD, colitis, lupus or RA.
  • the mammal is a human.
  • the composition is systemicailv administered.
  • the composition is locally administered.
  • FIGS 1A-H Liver enriched CD 8 Treg are PLZF positive and utilize an mnate-like PLZF-driven transcription program.
  • FIGS 1A-H Representative flow cytometry plots showing gate strategy for murine CDS Treg in liver
  • MNCs mononuclear cells from naive B6 mice.
  • CDS Treg were identified as B220 ⁇ CD4 ⁇ TCRp + CD8a + P ⁇ ceils.
  • B Histograms show CD69, NK 1.1, CD44 and CD62L expression by CDS Treg (top) and CDScmw (bottom) compared with isotypes (filled gray) as determined by flow cytometry. Numbers in histograms indicate the percentage of positive cells.
  • C Representative Dot plots showing CD8 Treg and CD8 C onv (top) as well as iNKT cells and CD4 T cells (bottom). Histograms show expression of PLZF or GFP (reporting PLZF) by CD 8 Treg and CD8 C onv (top) as well by iNKT cells (CD4 ⁇ aGaiCer/CDld tetramer + ) and CD4 + T cells
  • CD4 ⁇ aGalCer/CDl d tetramef " (bottom) from naive B6 mice or PLZF-eGFP reporter (PEG) mice.
  • CDS Treg and iNKT cells (open); CD8 C onv and CD4 j" T cells (filled gray).
  • D Quantitative RT-PCR analysis of PLZF expression on sorted CDS Treg and CDSconv following normalization using ⁇ actin gene.
  • E Expression of TdTomato in CDS Treg and CDSconv from livers of B6 (top) and Pcre x R2.6T (26T) (bottom) mice.
  • CDS Treg and CDSconv were stimulated for 3 days with anti-CD3/anti-CD28 mAbs and TdTomato expression was measured by FACS on sorted cells. Numbers indicate percentage.
  • F Quantitative RT- PCR analysis of selected transcription factors expression on sorted CDS Treg. The expression of each transcription factor is represented by mRNA fold change to that on CDSconv.
  • G Numbers of CDS Treg in liver and spleen of homozygous PLZF-/- mice and littermates PLZF+/+ and PLZF+/- mice as determined by FACS. Data represent mean ⁇ 2SD. *p ⁇ 0.05, **p ⁇ 0.01, impaired t test.
  • FIGS 2A-I Hepatic CDS Treg are innate-like and a substantial portion of CDS Treg co-express CD244 and CD 11c.
  • A Representative dot plot showing CD244 and CD 11c expression by hepatic CDS Treg (left) and CDSconv (right) from naive B6 mice following gate strategy. Numbers indicate % of CDS Treg or CDSconv that were CD244 + or CD244 ⁇ CDl lc + .
  • ⁇ SEM Mean disease scores ⁇ SEM are shown on the y-axis versus days post- immunization on the x-axis.
  • D Representative dot plot showing NK1.1, NKG2D, CD137 (4- IBB) and CD200 expression by hepatic CD244 " CDl le + CD 8 Treg from naive B6 mice.
  • E CDS Treg are not Foxp3 " and represent a distinct population of Treg. Histograms show Foxp3 and GITR expression by CDS Treg (black line) and CDSconv (gray line) in liver MNCs from naive B6 mice.
  • (F) Histograms show CD25, CD 122, PD-1, CD28, CD27, OX40, CD200 and 4-lBB expression by CDS Treg (top) and CDSconv (bottom) compared with isotypes (filled gray). Numbers indicate percentage of positive cells.
  • (G) Histograms show ICOS, CXCR5, CD 127, Eomes and CD 103 expression by CD8 Treg (top) and CDSconv (bottom) compared with isotypes (filled gray). Numbers indicate percentage of positive cells.
  • FIGS 4A-G Hepatic CDS Treg have immune regulatory properties and are physiologically expanded during EAE.
  • A Adoptive transfer of sorted CDS Treg protects mice from MOG -induced EAE (a model for multiple sclerosis). Sorted hepatic CDS Treg or CD8 ⁇ uv (lxl0 5 /mouse) from naive B6 mice were adoptively transferred i.v. into groups of naive B6 mice one day prior to EAE induction with MOG35--55/CFA/PT. As a positive control, EAE was also induced in non-transferred naive B6 mice. EAE clinical severity (disease scores) was monitored daily in all groups.
  • Mean disease scores ⁇ SEM are shown on the y- axis versus days post-immunization on the x-axis. *p ⁇ 0.05, Student t test.Data representative of 3-5 mice analyzed in each group.
  • B Sorted hepatic CDS Treg or CDSconv (2xl0-Vmouse) from naive CDld-/- mice were adoptively transferred i.v. into groups of naive B6 mice one day prior to EAE induction as described in A. Mean disease scores ⁇ SEM are shown. *p ⁇ 0.05, Student t test. Data representative of 3-5 mice analyzed in each group.
  • C CDS Treg are increased during the recovery phase of EAE.
  • EAE was induced in groups of naive B6 mice with either MOG35--55 or hen egg iysozyme (HEL) peptide along with CFA/PT and the clinical disease scores were evaluated daily.
  • C Percentage of CDS Treg in liver MNCs from CD8a-/-, CD8p-/-, ⁇ -/-, CD Id-/-, Jal8-/-, Qa- ⁇ -l- and TAP1-/- mice compared with B6 mice as determined by FACS. Each dot represents an individual mouse. Data representative of 3-5 mice per group. Data are represented as mean ⁇ SEM. *p ⁇ 0.05, ***p ⁇ 0.001, ****p ⁇ 0.0001, unpaired t test.
  • E H&E staining of liver sections of B6 and Qa-1-/- mice following ConA injection as before. Magnification X 100. Data representative of three independent experiments.
  • FIGS 6A-F Hepatic CDS Treg are polyclonal.
  • Va chain expression on hepatic CD8 Treg from naive B6 mice Liver MNCs were stained with anti-TCR , anti-CD8a, anti-CD8 .2 and available anti- TCR Va mAbs.
  • F RT-PCR analysis of TCR Va gene segments expression in CD8 Treg and CD8 C onv. Total R A was extracted from sorted CDS Treg and CDSconv from liver MNCs of naive B6 mice. The TCR Va chain was amplified using specific primers for each Va gene and a common Ca primer. The PCR products were electrophoresed in 1.2% agarose gel and visualized by ethidium bromide staining.
  • FIGS 7A-B CD8aa Treg are enriched in colonic IEL and colonic IEL CDS Treg express NK-inhibitory receptors.
  • CD45 TCRp ' CD8aa/aP live cells from colonic lELs were gated for lymphocyte activation marker, CD44 and IL-2 receptor ⁇ chain, CD 122.
  • the expression of NK-inhibitory receptors was analyzed on CD44 + CD122 + colonic IEL CDS Treg and IEL CD8o$ T cells.
  • Numbers indicate the frequency of events.
  • FIGS 8A-C Spontaneous inflammation in Qa-I b -/- mice genetically deticient in CDS Treg.
  • A Inflammation in liver of Qa-l b -/- mice. Representative H&E and Sirius-red staining of liver sections from naive Qa-l b -/- mice.
  • FIGS 9A-H Activation/expansion of CDS Treg by TCR-derived peptides protect mice from MOG-induced EAE as well as from DSS-induced colitis in a Qa-1 -dependent fashion.
  • A Proliferative response of splenocytes from B6 (Qa-l b +/+) (white bar) and Qa-l b -/- (black bar) mice against in vitro stimulation with individual TCR-derived peptides. Proliferation was measured by incorporation of pH] -thymidine in triplicate cultures. Bar graph depicts mean ⁇ 2SD of stimulation index. *p ⁇ 0.05, paired t test.
  • EAE was induced with MOG35 -55/CFA/PTX, (Right). EAE incidence in SJL mice after vaccination with peptide 8.2L. SJL mice were vaccinated i.p. with 50 ⁇ g of peptide 8.2L in IFA or PBS/IF A (control). One week later, EAE was induced with PLPi39-isi/CFA/PTx. All mice were monitored for disease symptoms until day 25. Results represent the mean ⁇ SD. These data are representative of three independent experiments. H. Treatment with a CD 8 Treg -inducing peptide confers protection from DSS- induced colitis in WT but not in Qa-l b -/- mice.
  • mice Groups of B6 (WT) (upper panels) or Qa-l b -/- (lower panels) mice were supplied with 2.5% DSS containing water for 7 days following prophylactic (3 days prior) i.p. injection of the Qa-l - binding peptide p8.2L (50 ⁇ / ⁇ ) as indicated (+P) or control (-P).
  • the relative reduction in colon length (gross morphology) as well as patches of cellular infiltrates in epithelial damage (white arrows) using H&E staining of colonic section are shown. Bar graphs (middle panels) indicate cumulative clinical scores in one representative experiment (each dot represents indiv idual mouse).
  • Cumulative clinical scores were calculated on the day of sacrifice (peak disease by body weight) by the following: i) grading loss in body weight (0 to 4% as 0: 4 to 10% as 1; 10 to 15% as 2; 15 to 20% as 3 and a loss of >20% as 4); is) grading colon length decrease (0.5 to 1 cm as 1 ; 1.1 to 1.5 cm. as 2; 1.6 to 2 cm as 3 and > 2 cm as 4); iii) average colon thickness of proximal and distal colon in mm; iv) stool softness on a scale of 0 to 3 with 0 for regular hard stool and 3 for liquid stool.
  • FIG. 10 Requirement of migratory DC in CD 8 Treg-mediated protection from DSS-induced colitis.
  • Treatment with peptide p8.2L (50 ⁇ ;/ ⁇ 8 ⁇ ) fails to prevent colitis in BatB-/- mice deficient in migrator ⁇ - CD 103 " DC but not in other DC populations.
  • Age matched WT and Batf3-/ ⁇ male mice were treated with peptide prophyiactically and challenged with 2.5% DSS in drinking water. Gross photography (left) and cumulative clinical disease scores (right) are shown. Each dot in the bar graphs represents one mouse. ****p ⁇ 0.0001, Student's t test.
  • Figures 11A-F Figures 11A-F.
  • Agonistic anti-4-l BB Ab administration leads to expansion/activation of CDS Treg and protection from EAE in a Qa-1 dependent manner.
  • C. Groups of B6 mice (n 5) were injected i.p.
  • FIGS. 12A-E Induction of CDS Treg following administration of anti-
  • CD3 mAb and subsequent protection from EAE are representative dot plots and cumulative data of CDS Treg in spleen and liver of B6 mice after anti-CD3 mAb treatment.
  • Female B6 mice were immunized with anti-CD 3 mAb i.p. on day I and day 3. On day 7, mice were sacrificed and spleen and liver mononuclear cells were isolated and stained for FACS.
  • B The frequency of CDS Treg in liver mononuclear cells was monitored daily by FACS after immunization with 200 ⁇ g of anti-CD3 mAb.
  • C Groups of female B6 mice were injected i.p. with 200 ⁇ -g of anti-CD3 mAb and five days later challenged with MOG35-55/CFA/PTX for the induction of EAE. Clinical scores were recorded daily as shown .
  • mice Female B6 mice were immunized with anti-CD3 mAb i.p. on day 1 and day 3. On day 7, mice were sacrificed and spleen cells were isolated and stained with various fluorochrome labeled antibodies. CDS Treg and CDap T cell proliferation was measured by BrdU incorporation.
  • Anti- CD3 mAb is the best anti-T cell surface antibody for induction of CDS Treg.
  • Anti-CD3 (clone 2C11) mAb activates/expands CDS Treg in liver. Naive female B6 mice were administered i.p. with various antibodies (200 ⁇ g) or just with PBS (control).
  • liver mononuclear cells were isolated and stained with CDSa, CDSp and TCRp fluorochrome labeled antibodies.
  • H57 anti- TCRP
  • GK1.5 anti-CD4
  • 2.43 anti-CD8
  • YTS177 and YTS 05 are also different anti-CD4 mAbs.
  • Figure 13 Low frequency of CDS Tregs in liver mononuclear cells from Yaa lupus mice. Representative dot plot showing hepatic CDS Treg in two 10 weeks old BXSB-Yaa mice (right) and one age-matched B6 mouse (right). Numbers indicate % of CDS Treg.
  • FIG. 14 Upregulation of several proinflammatory genes in liver of Qa- l -/- mice deficient in CDS Treg. Volcano plot displaying each gene's -log 10 (p- value) and log2 fold change with the selected covariate. Highly statistically significant genes fall at the top of die plot above the horizontal lines, and highly differentially expressed genes fall to either side. Horizontal lines indicate various False Disco very Rate (FDR) thresholds or p-value thresholds if there is no adjustment to the p-values. Genes are colored if the resulting p-value is below the given FDR. or p-value threshold. ' The 40 most statistically significant genes are labeled m the plot.
  • FDR False Disco very Rate
  • Figure 15 We have successfully generated several founders (mice) in which the allele of Zbtb l6 (PLZF) containing loxp sites flank the exon that encodes the BTB domain and the zinc fingers. The place where the loxp oligos were inserted and the sequencing scans showing the successful insertion of the loxp sites are shown. These founders are currently being bred with CD4-Cre to generate mice lacking PLZF+ T cells, including CDS Treg.
  • PLZF Zbtb l6
  • FIGS 16A-E CDS Treg are also present in human peripheral blood and express similar cell surface markers.
  • A Representative flow cytometiy plots showing gating strategy for the identification of circulating CDS Treg in human PBMC.
  • the total CDS 1" gate was identified using anti-CD8a and anti-CDSp mAbs within the TCRaP + gate: after intracellular staining for PLZF,
  • CD8TPLZF ⁇ T cells were gated within the CD8 ⁇ population; using anti-CD8ce and anti-CDSp mAbs, CD8a + p ⁇ T cells were identified within the CD8 + PLZF + gate; finally, based on expression of CD 161 and the invariant TCR Va7.2, CDS Treg were defined as PLZF + TCR.aP + CD8aa + T cells negative for TCR
  • Each TRBV gene segment is represented by a slice proportional to its average frequency.
  • the CDR3 was defined as starting at the last cysteine encoded by the 3' portion of the ⁇ gene segment and ending at the phenylalanine in the conserved TRB J segment motif FGXG.
  • FIGS 17A-B A. Cumulative data of Geo MFI of IFNy, IL- i 7A, TNFa and IL-4 (top) as well as IL-18R i, RORyt, CXCR.6 and CCR6 (bottom) expression by circulating CD8 Treg compared with both CDSconv and MAIT cells. Each dot represents individual donors. Data were obtained from 6-12 healthy donors. Results shown as mean ⁇ SEM. *p ⁇ 0.5, * *p ⁇ 0.01, ***p ⁇ 0.001, one-way ANO VA with Bonferroni's multiple comparisons test. B.
  • FIGS 18A-E Circulating CDS Treg are increased in PBMCs from chronic Rheumatoid Arthritis (RA) patients. Cumulative data of (A) percentage of CDS T cells in total TCRap ⁇ T cells, (B) percentage of CDS Treg cells in the TCRaP + PLZF + CD8aa + Va7.2 " CD 16 gate and (C) percentage of
  • CD244O31 lc + CDS Treg in PBMC of healthy individuals and RA patients CD8 Treg from RA patients significantly secrete more Granzyme B than healthy controls in i O- l 1) (**p ⁇ 0.01, Mann Whitney test). Scatter plots show Geo MFI values for Granzyme B, IL-17A, Perforin, CD244 and CDl lc expression.
  • E High frequency of circulating Granzyme B + and CD244 " CDS Treg in RA patients. Scatter plots show percentage of CDS Treg that were positive for Granzyme B, 1L-17A, Perforin and CD 11c.
  • PBMC were analyzed by multi- parameter flow cytometry. Each dot represents an individual donor. Results shown as mean ⁇ SEM.
  • CDS Treg from SLE patients have significantly high expression of RORyt. No differences were found between groups regarding to IL-lSRa, CCR6 and CXCR6 expression. Scatter plots show Geo MFI values in CDS Treg for IL-18Ra, CCR6, CXCR6 and RORyt. PBMC were analyzed by multi-parameter flow cytometry Each dot represents an individual donor. Results shown as mean ⁇ SEM.
  • E High frequency of circulating TNFa " CDS Treg in SLE patients. Scatter plots depict percentage of CDS Treg are positive for the corresponding cytokine. PBMC were analyzed by multi-parameter flow cytometry. All data are represented as mean ⁇ SEM. *p ⁇ 0.05, Mann -Whitney test.
  • Figures 20A-C No significant alterations in the frequency of CDS Treg in PBMCs from head and neck squamous cell carcinoma patients.
  • FIGS 21A-D A significant increase in the frequency of circulating CDS Treg in PBMCs from chronic melanoma patients.
  • Cumulative data of (A) percentage of CDS T cells in total TCRaP ⁇ + T cells, (B) percentage of CDS Treg cells in TC aP ⁇ PLZF ⁇ CDSaaVaJ ⁇ CDiei ⁇ gate and (C) percentage of (1)244 ( D i lc + CD8 Treg from PBMC of healthy individuals and melanoma patients.
  • the frequency of CD8 Treg in PBMC from melanoma patients was significantly increased compared to healthy controls.
  • PBMC were analyzed by multi -parameter flow cytometry. Each dot represents an individual donor. Results shown as mean ⁇ SEM.
  • D Identification of infiltrating CDS Treg into human melanoma tumor by flow cytometry.
  • FIG. 22 A working model of the negative feedback regulatory mechanism mediated by a novel innate-like PLZF ⁇ CD8aa + TCRc$ + Treg population (CDS Treg) enriched in liver and colon of naive mice, (a) During chronic inflammation, CDS Treg recognize Qa-1 -expressing activated CD4 + T cells; (b) the CDS Treg TCR bind to the non-classical Qa-1 -bound peptides on the surface of activated CD4 + T cells; (c) following recognition, CDS Treg mediate release of granzyme B and perforin and induce apoptosis in the target activated CD4 + T cells. Since naive CD4 + T cells do not express Qa-1 molecules on the surface, they are not susceptible to CDS Treg-mediated apoptosis allowing the immune response to proceed.
  • CDS Treg novel innate-like PLZF ⁇ CD8aa + TCRc$ + Treg population
  • an antibody consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N-terminal variable (Vi-i) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C -terminal constant (CL) region.
  • the variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
  • An antibody, or antigen-binding fragment thereof can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinam y) and in vivo sources (e.g., rodents).
  • in vitro sources e.g., a hybridoma or a cell line producing an antibody recombinam y
  • in vivo sources e.g., rodents
  • a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
  • antigen-binding fragments include but are not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody.
  • compositions can be via any of suitable route of administration, particularly parente rally, for example, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularJy, intraureth rally, intrasternally, intracranially, intramuscularly, or subcutaneously.
  • parente rally for example, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularJy, intraureth rally, intrasternally, intracranially, intramuscularly, or subcutaneously.
  • Such administration may be as a single bolus injection, multiple injections, or as a short- or long-duration infusion.
  • Implantable devices e.g., implantable infusion pumps
  • the compounds may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents.
  • the solution may contain other substances such as salts, sugars (particularly glucose or mannitoi), to make the solution isotonic with blood, buffering agents such as acetic, critric, and/or phosphoric acids and their sodium salts, and preservatives.
  • buffering agents such as acetic, critric, and/or phosphoric acids and their sodium salts, and preservatives.
  • compositions alone or in combination with other active agents can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
  • compositions alone or in combination with another active agent may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, maybe compressed into tablets, or may be incorporated directly with the food of the patient's diet.
  • a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
  • the composition optionally in combination with an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
  • amount of conjugate and optionally other active compound in such useful compositions is such that an effective dosage level will be obtained.
  • the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin;
  • excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
  • a liquid carrier such as a vegetable oil or a polyethylene glycol.
  • Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form.
  • tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like.
  • a syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor.
  • any material used in preparing any unit dosage form should he pharmaceutically acceptable and substantially non-toxic in the amounts employed.
  • the phospholipid conjugate optionally in combination with another active compound may be incorporated into sustained-release preparations and devices.
  • compositions optionally in combination with another active compound may also be administered intravenously or intraperitoneally by infusion or injection.
  • Solutions of active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
  • the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
  • the liquid carrier or vehicle can be a solvent or li uid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • the prevention of the action of microorganisms during storage can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, buffers or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating compound(s) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • one method of preparation includes vacuum dr ing and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • the antigen(s) and adjuvant(s) optionally in combination with another active compound may be applied in pure form, e.g., when they are liquids.
  • a dermatologically acceptable carrier which may be a solid or a liquid.
  • Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
  • Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
  • Adjuvants such as fragrances and antimicrobial agents can be added to optimize the properties for a given use.
  • the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
  • Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
  • the invention provides various dosage formulations optionally in combination with another active compound for inhalation delivery.
  • formulations may be designed for aerosol use in devices such as metered-dose inhalers, dry powder inhalers and nebulizers.
  • Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
  • the concentration of the active compound in a liquid composition will be from about 0.1-25 wt-%, e.g., from about 0.5-10 wt-%.
  • the concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, e.g., about 0.5-2.5 wt-%.
  • the active ingredient may be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 ⁇ , e.g., about 1 to 50 ⁇ , such as about 2 to about 30 ⁇ .
  • This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.0 -5.0 mg/kg/lir or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).
  • a suitable dose will be in the range of from, about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, for instance in the range of 6 to 90 mg/kg/day, e.g., in the range of 15 to 60 mg/kg/day.
  • the active compound may be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
  • the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
  • the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
  • the dose, and perhaps the dose frequency will also vary according to the age, body weight, condition, and response of the individual patient.
  • the total daily dose range for an active agent for the conditions described herein may be from about 50 mg to about 5000 mg, in single or divided doses.
  • a daily dose range should be about 100 mg to about 4000 mg, e.g., about 1000-3000 mg, in single or divided doses, e.g., 750 mg every 6 hr of orally administered compound. This can achieve plasma levels of about 500-750 uM, which can be effective to kill cancer cells.
  • the therapy should be initiated at a lower dose and increased depending on the patient's global response.
  • a method to identify or detect immune cells having
  • CD8CKX+ Nkl. l t, PLZF+, CD161+, and optionally having one or more of CD1 lc+, CD 137+ CD244+, or one or more of NK-inhibitory receptors is provided.
  • the method includes contacting a sample having mammalian immune cells with a ligand that binds CD8aa, a ligand that binds NK 1.1, a ligand that binds PLZF, and a ligand that binds CD 161, and optionally a ligand that binds CD 11c, a ligand that binds CD137, a ligand that binds CD244, a ligand that binds ⁇ , or a ligand that binds NK-inhibitory receptors; and identifying or detecting an amount of a population of cells CD8aa+, Nkl .
  • the cells are identified using antibodies specific for
  • the cells are human cells.
  • the method further comprises isolating the identified cells.
  • the method further compr ses expanding the isolated cells.
  • the cells are from a patient with an autoimmune disease.
  • the isolated cells ae cultured with IL-2, IL-15, Qa-l HLA-E binding peptides, anti-CD 3 antibodies or anti-CD 137 antibodies, or any combination thereof.
  • a method to decrease the number of immune cells having ⁇ , CDSotot, Nkl . l , PLZF, and CD 161, and optionally having one or more of CD1 lc, CD 137, CD244, or one or more of NK-inhibitory receptors in a mammal is provided.
  • the method includes adm inistering to the mammal an effective amount of one or more antibodies specific for CD8aa, specific for Nk 1.1 , specific for PLZF, or specific for CD 161 , or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD1 lc, specific for CD137, specific for CD244, specific for TCRa , or one or more of NK-inhibitory receptors, or a combination thereof.
  • a.method to prevent inhibit or treat cancer in a mammal comprising: administering to the mammal an effective amount of one or more antibodies specific for CD8aa, specific for Nk 1.1, specific for PLZF, or specific for CD161, or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD1 lc, specific for CD 137, specific for CD244, specific for ⁇ ⁇ , or specific for one or more of NK- inhibitory receptors, or a combination thereof.
  • the cancer is neck or head cancer.
  • the cancer is melanoma.
  • a method to prevent, inhibit or treat autoimmune disease in a mammal comprising: administering to the mammal one or more Qa- 1/HLA-E binding peptides, anti-CD 1 lc antibodies, anti-CD3 antibodies or anti- CD137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a+T regs.
  • the one or more antibodithat are specific for CD1 lc and CD137, CD 1 lc and CD3, or CD3 and CD137.
  • the disease is IBD, colitis, lupus or RA.
  • the disease is an autoimmune liver disease.
  • the disease is autoimmune hepatitis or primary biliary cirrhosis.
  • the T or B cells in the mammal with the disease are increased relative to a mammal without the disease.
  • a method to prevent, inhibit or treat orgn or graft rejection in a mammal comprising:
  • administering to the method includes administering to a mm ami in need theref one or more Qa-l/HLA-E binding peptides, anti-CD 1 lc antibodies, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a.+T regs.
  • the mammal is a human.
  • composition is systemically administered.
  • the liver has a central role not only in the metabolism and clearance of the diet and intestinal microbe-derived toxins but also mount immunity against infectious agents and cancer. Chronic infections in the liver indicate that several tolerance mechanisms are evolved to protect the hepatic tissue from excessive immune stimulation (1-3). Liver has several residential T cells, B cells and macrophages or Kupffer cells and in steady state immune cells are
  • liver uniquely harbors both conventional T cells and several unconventional T cells (4).
  • the unconventional T cells are comprised of NKT cells, MATT cells, and gd T cells in the liver.
  • adaptive T cells are class la MHC-restricted
  • innate-like unconventional T cells are reactive to non-classical MHC-Ib molecules and recognize a class of antigens.
  • NKT cells and MAIT cells recognize both self and microbial antigens in the context of CD id and in the context of the MR-1 molecules, respectively and could provide immunity against microbes (5-7).
  • a detailed understanding of cellular and molecular interactions in liver among different innate and conventional T cells involved in the maintenance of immune tolerance is lacking.
  • T cells are controlled by both intrinsic (e.g., PD1, anergy and exhaustion) and extrinsic cell-based (regulatory T cells or Treg) mechanisms that prevent them, from causing excessive tissue damage.
  • An important role for FoxP3+CD4+ Treg has been well-defined, a phenotypic characterization of CD8+ cells with regulatory activity is poorly studied.
  • immune regulatory nature of CD8 ⁇ T cells in homeostasis of cellular and humoral immune responses has been suggested (8-10) (1 1 -13), ability to distinguish them (CD8 Treg) from non- regulatory conventional CD8 ⁇ T cells (CDSconv) has not sufficiently advanced as happened for CD4 Treg following the discovery of Foxp3 (14).
  • CD8+ Treg Similar to CD4+Treg, an important role for IL-2 signaling for CD8+ Treg has been shown in mice deficient in IL-2 and IL-2Rb chain (15-17) (18). Regulatory CD8 ⁇ T cells also have been implicated in various conditions in humans, e.g. transplant survival (19), inflammatory bowel disease (20) and in multiple sclerosis (21-25). In a clinical trial with anti-CD3 mAb (Teplizumab), increased frequency of memory CD8 + T cells with regulatory gene expression was found to be associated with a positive clinical response in type i diabetes patients (26). Interestingly, IL ⁇ 2/IL-15Rp ⁇ deficiency in humans also leads to a severe combined immunodeficiency syndrome as observed for IL-2Rcx (27).
  • CD8 + Treg have a unique ceil surface phenotype in that they are CD I lc- ⁇ - 2B4- ⁇ -NKG2D+ and, despite having innate- like features, they are distinct from other innate-like T cells, including NKT cells or mucosal associated invariant T (MAIT) cells, CD8 + Treg do not express Foxp3 but they express glucocorticoid-induced tumor necrosis factor-related receptor (GITR), a marker of active Tregs cells (28). A large number of them are dependent upon Qa-lb molecules as a significant reduced frequency is foind in Qa-P-/- mice. They are TAP-independent but dependent upon IL15 signaling for their development.
  • GITR glucocorticoid-induced tumor necrosis factor-related receptor
  • CD8 + Treg do not secrete IL-10 or TGF[3 but secrete typical cytokines produced by cytotoxic T cells (TNFa, IL-17A) as well as PLZF -driven secretion of both IL-4 and IFNy similar to that in NKT cells. It is noteworthy that these cells very rapidly secrete large amounts of IL-2 as well .
  • CD8 Treg shares several features with oilier unconventional T cells, including expression of memoiy/activation markers, a common transcription program imprinted by the expression of PLZF and their enrichment in non-lymphoid organs.
  • Liver enriched CD8 Treg are PLZF + and dependent upon PLZF and related transcription factors
  • CD8 Treg represent -3.25% of TCRap + T cells (within the B220-CD4- CD8b- gate) in liver mononuclear cells (MNCs) in naive B6 mice. They are also present in other lymphoid compartments, including bone marrow, spleen, peripheral blood and lung.
  • CDS Treg are not readily detectable in adult thymus, they are present in the neonatal period ( ⁇ 0.5%) until day 5 after birth (data not shown), Athymic nude mice are deficient in CD8 Treg, indicating their thymic origin. In contrast to hepatic CD8 ⁇ nv, the majority of CD8 Treg are CD69 + and N K ⁇ I . suggesting an activated profile similar to other innate-like T cells, including NKT cells. Additionally, CDS Treg are mostly CD44 h,gh and CD62L iow compared to CD8 C onv, suggesting a memory phenotype.
  • PZF promyelocytic leukemia zinc finger
  • PLZF- /-, RORa-/- and Id3-/- mice showed a significant reduction in the number of CD8 Treg. These results indicate that CDS Treg are PLZF + and are dependent upon PLZF and related transcription factors.
  • Hepatic CD8 Treg have immune regulatory properties and are physiologically expanded during an experimental autoimmunity
  • CDS Treg have regulator ⁇ ' potential to control autoimmunity.
  • An equal number of sorted hepatic CDS Treg from naive B6 mice were transferred into B6 recipients that were immunized next day with myelin-oligodendrocyte glycoprotein (MQG)35-JJ peptide in completed Freund's adjuvant (CFA) plus pertussis toxin (PTx) (MOGss-ss/CFA/PTx) for the induction of experimental autoimmune encephalomyelitis (EAE).
  • CFA myelin-oligodendrocyte glycoprotein
  • PTx pertussis toxin
  • EAE experimental autoimmune encephalomyelitis
  • hepatic CDS Treg or CDSconv were sorted from CD Id-/- mice deficient in both populations and used in adoptive transfer experiment as above. Mice that received CDS Treg but not CDSconv from CD Id-/- mice showed significant protection from EAE. These data indicate regulatory property of CDS Treg independent of NKT ceils or innate-like memory CD 8 T cells.
  • CDS Treg are physiologically altered in liver during the course of EAE.
  • B6 mice were immunized with either MOG35-55 for the EAE induction or an irrelevant peptide derived from hen egg iysozyme (HEL) and hepatic CDS Treg were analyzed on day 10 at the onset of disease (EAE dlO) or at day 25 during the recovery phase of the disease (EAE d25).
  • CD8 Treg were significantly ' expanded during the recovery phase (day 25) but not at the onset of EAE.
  • CDS Treg numbers were not altered in non-diseased mice immunized with HEL peptide.
  • CD8 Treg expansion inversely correlates with the severity of EAE.
  • CD8 Treg are physiologically expanded during recovery phase of EAE and their higher numbers correlating with less svere disease suggest their physiological role in spontaneous recovery as suggested earlier for the CD8 T cells .
  • CD4 CD25 ⁇ CD45RB iilg!i T cells mediated colitis into Rag 17- mice and co-transfered CDS Treg from B6 or perforin-/- mice
  • ARa l-/- recipients that received CD 8 Treg from perforin-/- mice developed colitis similar to the control mice whereas mice that received CD8 Treg from perforin+/+ mice were significantly protected as indicated by increase in body weight ratio and histological analysis of colon using H&E staining.
  • CFSE-labeled OT- II CD4 + T cells were transferred to naive B6 mice alone or co-transferred with positive-selected CD8 + T cells or liver MNCs from B6 or perforin-/- mice.
  • recipients were i.p. challenged with OVA323-339 peptide or PBS and, 4 days later, proliferation of CFSE-labeled OT-I1 CD4 + T cells was analyzed by flow cytometry.
  • CDS Treg have regulatory activity and able to control activation/expansion of disease-causing CD4 T cells.
  • the addition to the culture of anti-Qa- l b rnAb but not anti-MHC class I rnAb significantly blocked the suppressive function of CDS Treg (data not shown), suggesting a dependence on Qa-l b molecules.
  • Hepatic CDS Tree secrete typical PLZF-driven cytokines and are dependent on TL-15/IL-2R.P signaling for their development
  • CDS Treg cytokine secretion profile of sorted hepatic CDS Treg and compared to the sorted CDSconv from naive B6 following in vitro stimulation with the plate-bound anti-CD3 rnAb for 96 hr and cytokine secretion measured in culture
  • CDS Treg secreted typical proinflammatory cytokines produced by cytotoxic T cells (TNFa, IL-17A and IL-6) as well as PLZF-driven secretion of both IFNy and 1L-4.
  • CDS Treg secreted significantly higher levels of IL-2 within 24 hr while CDSconv secreted only a minimal amount.
  • CDS Treg do not secrete the suppressive cytokine IL-10 and the expression of ⁇ was significantly down-regulated in these cells. Consistently, CDSconv showed a typical cytotoxic T cell profile, including secretion of IFNy and TNFa.
  • CDS Treg Hepatic CDS Treg were significantly reduced in both CD 122-/- and IL-15-/- mice in comparison to B6 mice. Considering that CD 122, the ⁇ chain receptor for IL-2 and IL-15, is essential for CD8 + T cell response to IL-15, these results indicate that IL-15 signaling is necessary for CD8 Treg development. In contrast, CD 8 Treg are not affected in CD25-/-, IL-7- /-, IL-6-/-, and IFNy-/- mice. Surprisingly, CDS Treg are T-bet independent and are not reduced in T-bet-/- mice.
  • Qa-1 -restricted T cells can be either TAP-dependent or TAP- independent, no differences in the frequency CDS Treg between TA 1-/- and B6 mice indicate that TAP-dependent antigen processing is not required for the development of CD 8 Treg.
  • ConA concanavalin A
  • Hepatic CD8 Tree are polyclonal and use diverse TCR a and ⁇ chains
  • TRBV20 ( ⁇ 8.1/8.2), TRBV20 ( ⁇ 15), TRBV12-1/12-2 ( ⁇ 5.1/5.2), TRBV5 ( ⁇ ) and TRBV4 ( ⁇ ). Additionally, TRBJ usage, another indicator of the
  • CD8 Treg was also diverse and almost similar among CDS Treg and CDSconv, except for an increase in 1 ⁇ 2.5 usage by CD8 Treg.
  • CD8 Treg also showed a skewed bell-shaped distribution of CDR3 lengths with a peak at 39 nucleotides while CDScom- show a more typical Gaussian-like distribution with a peak at 42 nucleotides.
  • TCR Va analysis of CDS Treg by FACS and RT-PCR revealed diverse Va usage similar to that in CD8 C onv. Together, these data indicate that the TCR repertoire of the hepatic CDS Treg in nai e mice is polyclonal.
  • Hepatic CD8 Treg are CD1 lc+ and CD244+ and express several markers typical of innate-like T cells
  • CD8 Treg To further differentiate CD8 Treg from CD8 C on», we determined the expression of different cell surface markers using flowcytometry and quantitative RT-PCR., A large proportion of CDS Treg but not CD8 ⁇ nv CD1 lc. In addition, the majority of CDS Treg express CD244 (2B4). Co-staining of CDS Treg indicates that all CD I lc + CDS Treg are also CD244 + while none of the CD8 CO nv express CD1 lc or CD244.
  • CDS Treg co-expressing CD244 and CD 11c are dependent on PLZF.
  • Both percentage and numbers of CD244 " CDl lc + CDS Treg were significantly reduced in PLZF- /- mice. Consistent with the correlation of regulatory activity with CD1 l c expression in Treg, CD1 lc-bead depletion resulted in loss of CDS Treg ability to control EAE upon adoptive transfer (data not shown).
  • CDS Treg have distinguishing features, they are NK1.1+PLZF + , CD244 1" and CD I lc + .
  • CDSconv, CD8 Treg were negative for FoxP3 but do express glucocorticoid- induced tumor necrosis factor-related receptor (GITR), also present on CD4 Treg (Ronchetti S, J Immunol Res 2015). Consistent with the role of IL-15 signaling, CDS Treg were CD122(lL-2Rp) hi 8 h CD25(IL-2Ra) low , similar to innate-like CD8 + T cells (Walzer T, JI 2002), and most of them were CD27 s h , marker associated with memory CDS '1" T cells. Interestingly, CDS Treg were CD28 + , PD-l ⁇ and also express another regulatory signaling molecule CD200.
  • GITR glucocorticoid- induced tumor necrosis factor-related receptor
  • CDS Treg were negative for OX-40. Notably, CDS Treg also expressed low levels of 4- IBB (CD137), a co-stimulatory molecule belonging to the TNF- receptor superfamiiv 9 and expressed only on activated T cells. In addition, CDS Treg did not express ICOS, CXCR5, Eomes as well as CD103, the a-chain of the ⁇ 7 integrin that characterize tissue-resident memory (TRM) T cells (Ariotti S, Adv Immunol 2012). CDS Treg are not typical memory cells as they do not express CD 127 (IL-7Ra), which is required for memory CDS T cells.
  • IL-7Ra IL-7Ra
  • CDS Treg are PLZF " .
  • CDS Treg express only NK inhibitory receptors (Ly49E/F>Ly49G2>Ly49A>Ly49I>Lv49G), but not activating receptors (Ly49D and Ly49H). Consistent with an innate-like behavior, CDS Treg also express NKG2D.
  • NK inhibitory receptors Ly49E/F>Ly49G2>Ly49A>Ly49I>Lv49G
  • CDS Treg also express NKG2D.
  • CDS Treg showed significant down-regulation of €08 ⁇ and up-regulation of CD8a, CD244 (2B4), CD200, Ly49A, Granzyme A (Gzma), 4-1BB, CD25, CD 122 and CD28.
  • Fibrinogen-like 2 (Fgl2), which is a negative regulator of the immune response was up-regulated in CDS Treg.
  • Fgl2 Fibrinogen-like 2
  • LAGS Lymphocyte-activation gene 3
  • PLZF + CDS Treg are also present in human peripheral blood and have several features similar to the murine counterpart
  • PBMC peripheral blood mononuclear cells
  • M AIT mucosal-associated invariant T
  • CD161 ⁇ CD161 ⁇
  • Pl FTCRaP + CD8a + CD8p- T cells that express low to intermediate levels of CD161 ⁇ (1) ! 61
  • Human circulating CD 8 Treg represent -0.24% ( ⁇ SEM 0.06) of the total TCRc$ +' T cells and -0,87% ( ⁇ 0.21) of the total CDS '" T cells.
  • CD244 (2B4) and CD l ie expression was significantly higher in circulating CD8 Treg than in both CD8 ⁇ nv.
  • circulating CDS Treg also secrete higher levels of Granzyme B than both CD8 C om- and MAIT cells.
  • human CDS Treg Similar to murine CDS Treg, human CDS Treg also secrete IFNy, TNFcc, IL-4 and IL-17A.
  • IL-18 receptor a subunit (IL-18Ral ), RORyt, CXCR6 and CCR6, known to be up-regulated in MAIT cells were significantly reduced in CDS Treg.
  • CD244 and CD1 lc Similar to murine CD 8 Treg, a subset of human CD8 Treg also co- express CD244 and CD1 lc (CD244 "1" CD1 lc + ) that was significantly higher in CDS Treg than in MA IT cells (15.7% ⁇ 3.0 vs 1 .4% ⁇ 0.7). Interestingly, -69 % of CD244 "1" CD 1 lc + CDS Treg secrete both Granzyme B and perforin .
  • TRBV20 (V 2), were expressed at almost similar frequencies by both subsets. Both CDS Treg and CDSconv do not show any preferential usage of specific TRBJ gene segment and showed a Gaussian-like CDR3 length distribution profiles. Collectively, these results indicate that circulating human CDS Treg express a diverse and polyclonal TCRJ3 repertoire. The proportion of circulating CD 8 Treg within total PLZF DSacc Diei ⁇ T cells was -12.7% ( ⁇ 2.2) that represent ⁇ 2xIQ 3 ( ⁇ 0.6x10 3 ) CDS Treg. In contrast, MAIT cells represent
  • CD8alphaalpha TCRalphabeta + T ' reg enri ched in liver and colon of naive mice, hereafter referred to CD 8 Treg, expressing the promyelocyte leukemia zinc finger (PLZF) transcription factor that distinguishes it from conventional CDS " T ceils.
  • PZF promyelocyte leukemia zinc finger
  • CDSacfTCRap* IEL population and are memory-like (CD44 h!3 ⁇ 4h CD62L iow ), activated (CD 122 + CD25 + CD69 + ), innate-like ( ⁇ 1.
  • ⁇ ) cells that express NK- inhibitory (Ly49C/1/F/H) but not NK-activating receptors; 2) are distinct from MAIT or NKT cells; 3) do not secrete IL-10 and TGF-b but express features of cytotoxic CDS T ceils with enhanced perforin/granzyme B expression; 4) protect Ragl-/- mice from CD4 ⁇ T cells-induced colitis; and 5) are Qa-lb restricted and can be induced with a Qa-lb-binding TCR leader peptide and protect DSS- mduced colitis in a Qa- lb-dependent manner.
  • PLZF + CD8 Treg displaying a unique phenotype
  • PZF ⁇ TCRaiphabeta + NKl .1 + CD44 D 122 + expressing NK-mhibitory receptors are PLZF -dependent and have a transcriptional signature similar to CDS Treg in the liver.
  • CDS Treg use cytolytic mechanism to control colitis in a Qa- lb-dependent manner and that CDS Treg can be activated/expanded following immunization with Qa- lb-binding peptides. Also CDS Treg can inactivate or kill key dendritic cell populations involved in colitis.
  • Colonic CDS Treg have a similar phenotype and function: Consistent with earlier data, CD8aTCRap+ 1 cells are present in both the small intestine (SI) and colon. It also has been shown that SI CDSaaTCRotP T cells and not conventional CDS TCR T cells are regulatory and maintain g t homeostasis. We propose that colonic CDS Treg with a phenotype similar to liver (PLZF + , NK1.1 + , expressing NK-inhibitory receptors and memory phenotype,
  • CD44 " CD 122 + ) are present within the CDSaaTCRcxp" intraepithelial lymphocytes (IEL) population.
  • CDS Treg are able to protect from CD4 + T cell- induced colitis in Ragl-/- mice.
  • Adoptive transfer of CDS Treg protect Ragl-/- miee from CD4 + CD45Rb b3 ⁇ 4u T cell-induced colitis.
  • CDS Treg are important in the maintenance of gut homeostasis, we predict that Qa-lb-/- mice, lacking CD8aa Treg, should become more susceptible or sensitive to dextran sulfate sodium (DSS)-induced colitis.
  • DSS dextran sulfate sodium
  • a Qa-lb-binding peptide induces CDS Treg and protect DSS colitis: Since CDS Treg are Qa- lb-restricted, we have synthesized several peptides with the potential Qa-lb-binding motif derived from, the conserved regions of the TCR ⁇ chains, heat shock proteins (HSP), and QDM. Next, we have determined their potential to induce Qa-1 -dependent CD8 + T ceil response in naive B6 mice. We found 6 peptides that are able to bind to the Qa-1 molecule (in in vitro Qa-lb folding assays) and induce a CD8 + T cell proliferative response (CFSE-dilution) in naive B6 mice.
  • CDS Treg are Qa- lb-restricted
  • HSP heat shock proteins
  • TNF- ⁇ LVJIFNy and IL-17A decreased significantly in comparison to the control mice.
  • CDS Tregs are involved in a dominant immune regulatory mechanism able to control both DSS-induced colitis, which is primarily mediated by innate cells, and CD45Rb hlgh CD4 + T cell-induced colitis, which is mediated by adaptive Thl/Thl? cells.
  • CDS Treg population in PBMCs from healthy donors that are PLZF + TCR "t' CD8 "t' CD161 ⁇ iQt) with a memory phenotype expressing NK-inhibrtory receptors similar to the phenotype in mice.
  • PLZFTCRa + NKl .1 + CD44 D122 + expressing NK-inhibitory receptors are PLZF -dependent and have a transcriptional signature similar to CDS Treg in the liver.
  • colon will be cut longitudinally and transversely into small pieces and washed twice in HBSS solution without calcium and magnesium containing 10 mM HEPES, 5 mM EDTA, 5% FBS, lmM DTT using the MACSmix tube rotator at 37°C.
  • the washed fluid is the IEL fraction.
  • colon pieces will be treated with an enzyme mixture following manufacturer's protocol at 37°C for 30 min.
  • the enzyme-treated colonic pieces will be dissociated into single cell suspension using the gentleMacs tissue dissociator. This is the LP fraction.
  • the IEL and the LP fractions will be labeled with different fluorescent labeled mAbs to identify CDS Treg within the TCRp + CD4-B220 " CD45 + gate and their CDS counterpart using flow cytometry.
  • We will include staining for several activation markers, NK cell-specific markers, memory T cell markers as well as CD1 lc expression and compare these profiles to conventional 038 ⁇ T cells as well as to CD8 Treg derived from liver.
  • Sorted CD 8 Treg and CD8o$ T cells will be used for the extraction of total RNA using the RNAeasy Micro Kit (Qiagen) followed by its integrity analysis using Agilent RNA 600 Nano Kit (Agilent). Poly-A mRNA enrichment will be used for library preparation and 50 bp-reads will be generated by sequencing on an Alumina HiSeq4000 analyzer using the TruSeq v3 Cluster kit at the UCSD Institute for Genomic Medicine core facility. RNA sequence data will be analyzed by the Bioinformatics core facility at UCSD and the log2 RPKM (Read per kilobase per million) will be quantile normalized.
  • the R statistical software will be used to calculate differentially expressed genes between CD8aa Treg and CD8ap T cells. Genes satisfying the following criteria will be chosen for analysis: first, the average count is more than 1 0 in at least one sample group, and second, the global False Discovery Rate (FDR) is controlled at p values of 0.05 with a minimum fold-change of 2. This should generate a CD8aa Treg vs. CD8ap T cells "signature gene set" for further analysis. To determine PLZF expression and its role in the development of colonic CD8aa Treg.
  • FDR False Discovery Rate
  • PLZF-Cre mice which express a bacterial artificial chromosome transgene in which the gene encoding Cre recombinase is knocked into gene encoding PLZF
  • Rosa26 fl/fl mice which express the fluorescent marker tdTomato and cany a lox -flanked stop codon at the ubiquitous Rosa26 locus. Therefore, in the resultant PLZF-Cre X Rosa26 fl/fl mice, cells that express PLZF (and therefore Cre) are permanently tdTomato+.
  • CD8aa Treg from gut, liver and spleen are tdTomato+.
  • CD8 T cells as negative and type I NKT cells as positive controls in these
  • mice are available from Dr. Derek Sant'Angelo who will help us with these experiments.
  • a clear pronounced tdTomato staining will confirm that colonic CD8aa Treg also had expressed PLZF during development.
  • chimeric mice using CD45.1 and CD45.2 markers will be generated to study CD8aa Treg.
  • WT B6 mice will be reconstituted with a 50:50 mix of bone marrow cells from PLZF-/- and WT mice and the development of CDScux Treg analyzed 12 to 16 weeks after transfer by cell surface markers, including CD45.1 vs. CD45.2 to differentiate WT from PLZF-/- cells.
  • CD44 + CD 122 ⁇ and CD 1 lc ⁇ a marker thought to be present on suppressor T cells (29).
  • CD1 lc + Treg a marker thought to be present on suppressor T cells (29).
  • CD1 lc + Treg a marker thought to be present on suppressor T cells (29).
  • CD1 lc + Treg a marker thought to be present on suppressor T cells (29).
  • CD1 lc + Treg results in loss of protection from EAE in adoptive transfer experiments (data not shown). Therefore, attempts will be made to sort and compare gene expression between CD1 lc + Treg and CD1 lc " populations. If there is a difference it would indicate that within CD8aa T cells only those that are also CD1 lc + are CDSoux Treg. It is possible that PLZF requirement is not intrinsic to CD8aa Treg. In this case, CD8aa Treg could still develop in chimeric mice generated with bone marro cells from PLZF-/- mice.
  • CD8cece Treg in these chimeric mice may not have fully developed their innate-like properties, including cytokine secretion, NKi . l expression or expression of other cytotoxicity genes, like perforin and granzyme B. Therefore, we will use flow cytometry to analyze all relevant markers after in vitro stimulation with plate-bound anti-CD3/CD28 mAbs as shown.
  • CD8aa Treg use cytolytic mechanism to control colitis in a Qa- lb-dependent manner and that CD8aa Treg can be activated/expanded following immunization with Qa-lb-binding peptides.
  • CD8aa Treg utilize cytolytic mechanism to kill activated target CD4 + T cells based upon the following observations: (a) they do not secrete detectable levels of suppressive cytokines, such as IL-10 and ⁇ ; but produce cytokines typical of cytotoxic CDS T cells and NKT cells; (b) RT-PCR analysis show that CD8aa Treg express enhanced levels of perforin and granzyme B, but not granzyme A; (c) earlier data using bulk CD 122 + CD8 + T cells or cloned TCR-peptide-reactive CDS ' " " T cells show killing of activated target cells. Since there is similarity between hepatic CD8aa Treg and colonic CD8aa Treg in all the features analyzed so far, we propose to compare whether both Treg populations use similar regulatory mechanisms involving perforin/granzyme B
  • colonic CD8aa Treg are able to provide protection in Rag l-/- mice, then in next experiments, colonic CD8aa Treg will be isolated from WT, Perforin-/- and Granzyme B-/- mice and use in adoptive transfer experiments into Rag l-/- mice. Next, we will use sorted colonic CD8aa Treg from PLZF-GFP mice to determine their ability to homing into gut to protect colitis.
  • CDS Treg are expanded in H-2 11 mice following capture of apoptotic T cells by conventional DCs that mediate cross-presentation of TCRp-cham-derived peptides to the CDS Treg, We will determine whether Qa-l is required for the CD8aa Treg -mediated regulation of colitis.
  • mice will be sacrificed to measure colitis using parameters as described before.
  • we will administer the peptide on day 5 after beginning of DSS treatment when the body weight loss start.
  • we will determine the priming or activation of CD8aa Treg following peptide- immunization by examining CD69 increase and cytokine expression by intracytoplasmic staining.
  • An irrelevant peptide from hen-egg lysozyrne will be used as a negative control in these experiments.
  • CD8aa Treg Physiological induction of colonic CDSaa Treg in the regulation of colitis. Rationale: We will examine the hypothesis that the frequency of colonic CD8aa Treg may differ in different phases of colitis and may correlate with the disease severity. Since CD8cece Treg appear to be involved in the maintenance of homeostasis, we will determine whether the number CD8aa Treg in colon is significantly decreased during the peak of disease and whether it is restored to normal values during the relapsing phase indicating their physiological importance.
  • mice will investigate both acute and chronic models of DSS-induced colitis.
  • DSS Affymetrix
  • mice will add 2.5% DSS (Affymetrix) to the feeding water and expose the mice for 7 days. After that, mice will be put on regular water.
  • mice will be put to 3 cycles of 7 days with 1.5% DSS plus 14 days with regular water.
  • 1.5% DSS gives a minimal response in the acute model of the disease.
  • Mice will be sacrificed and colonic (LP and IEL) and MLN tissue will be dissociated into single cell suspensions and analyzed by flow cytometry.
  • days of sacrifice for phenotyping will be 6 th , 9 th , !6 m and 25* and, in the chronic model, will be at the end of each cycle, i.e. days 21 st , 42 nd and 63 rd
  • mice Following input in the chronic phase, we will observe mice and sacrifice after every 15 days for phenotyping of CD8aa T cells in colon until the time when body weights are restored. To measure colitis we will use body weight loss, gross colonic features and histopathologic score of colon and cytokine measurement in colon explant culture. In addition, we will use FITC- Dextran tracer to detect epithelial barrier malfunction and Myeloperoxidase (MPO) assay, whenever required.
  • MPO Myeloperoxidase
  • CD8aa Treg rapidly secrete IL-2, there is a possibility that they may recruit CD4 " CD25 + Foxp3 ⁇ Treg. Attempts will be made to examine this in DSS colitis model following peptide treatment and examining intestinal tissues by FACS on days 2, 4, 8 and 16 for the induction of CD4 + Treg and compare to the control peptide-treated mice. If there is paucity of colonic CD8aa Treg for adoptive trsanfer studies, we may use sorted liver derived CD8aa Treg and then compare their gene signature and other properties. Since DSS colitis is primarily mediated by innate cells, alternatively regulatory mechanism(s) may need to be explored (37)(9)( 11).
  • CD8 Treg are also enriched in colon and play an important role in limiting the intensity or duration of the inflammatory immune response in gut This mechanism is different but complimentary from the one mediated by Foxp3 + CD4 + Treg that prevents a damaging response from occurring.
  • CD8aa Treg-mediated regulation is a negative feedback response in the sense that it allows operation of the natural mechanisms of defense or healing but shuts down these mechanisms after a delay in order to avoid excessive tissue damage (29, 38).
  • CD8aa Treg A similar phenotype, gene signature and regulatory function of CD8aa Treg in human peripheral blood.
  • Patients with active autoimmune disease may have altered frequency of CD8aa Treg in PBMC and this will be examined after their characterization, including their frequency, cell surface phenotype, cytokine and gene expression profile, MHC-restriction, TCR repertoire and their regulator ⁇ ' function. It is important to investigate whether the CD8 a Treg equivalent to the mouse counterpart are also present in humans.
  • Our preliminary data using multiparameter flow cytometry analysis suggest that in peripheral blood of healthy individuals, potential CD8aa Treg are present.
  • CD8aa Treg are defined as PLZF + TCRaP + CD8aa + T cells that do not express the TCRVa7.2/Ja33, which identifies MAIT cells, and have intermediate or very low expression of CD161, different from MAIT cells that are CD161 !li ! ⁇
  • the frequency of CD8aa Treg was 0.20% ⁇ 0,05 (mean ⁇ sem) that correspond to 2 10 3 ⁇ 0.5 x 10 3 in a million of PBMC.
  • the expression of RORyt, the transcription factor of MAIT cells, as well as CXCR6 and CCR6 in CDSaa Treg was significantly lower than both in MAIT cells and in CD8ap T cells (data not shown).
  • CD8aa Treg Similar to murine CD8aa Treg, CD244 or 2B4, and CD1 lc were also significantly high expressed in human CD8aa Treg. Though IF y, IL-17 and IL- 4 showed no differences between CD8aa Treg and either € ⁇ )8 ⁇ T cells or MAIT cells, granzyme B, perforin and T Fa secretion by human CD8aa Treg were significantly increased similar to murine CD8aa Treg (data not shown).
  • CD8aa Treg will be identified within the TCRaP ⁇ CD8 ⁇ PLZF + gate as
  • CD 122 IL- 2Rp
  • CD28 CD 127, Foxp3, GITR
  • CTLA-4 CXCR3, CD45RC, CCR8 and TGF- ⁇
  • activation markers such as CD25 and CD69.
  • Our preliminary data on murine CD8aa Treg show high expression of CD25, CD28, CD 122 and GITR, but not CD127 and FoxP3 in comparison with conventional CDScxp T cells. However, it is unknown whether human CD8aa Treg also express some of these markers.
  • CCR7 + CD45RA " Fluorescent monoclonal antibodies will be purchased from BD Bioscience, BioLegend or eBioscience.
  • CD8aa Treg in humans also constitutively secrete significantly more granzyme B, TNFa and perforin than MAIT cells and conventional € ⁇ 8 ⁇ T cells (data not shown), however IFNy, IL-4 and IL17 secretion was similar between subsets.
  • sorted CD8cece Treg and CD8a(3 T cells will be stimulated with phorbol 12-myristate 13 -acetate (PMA) and ionomycin for 4-6 hours with Monensin (BD GolgiStop).
  • PMA phorbol 12-myristate 13 -acetate
  • BD GolgiStop Monensin
  • cytokine- producmg cells IL-2, IFNy, IL-4, IL-10, IL-13, IL-17, IL-22, ⁇ , TNFa, granzyme B and perforin
  • ELISPOT enzyme-linked immunospot
  • the culture supernatant will be collected and analyzed using BDTM CBA Human Chemokine Kit: IL-8 (CXCL8/IL-8), RANTES (CCL5/RANTES), monokine induced by interferon-y (CXCL9/MIG), monocyte chemoattractant protein- 1 (CCL2/MCP-1), and interferon-y--mduced protein-10 (CXCLlO/IP-10).
  • IL-8 CXCL8/IL-8
  • RANTES CL5/RANTES
  • CXCL9/MIG monokine induced by interferon-y
  • CXCL9/MIG monocyte chemoattractant protein- 1
  • CXCLlO/IP-10 interferon-y--mduced protein-10
  • cytokine gene transcripts will be analyzed by real-time PCR using RNA isolated from sorted CD8aa Treg and CD8ap T cells after stimulation with PMA and ionomycin.
  • RNA sequencing RNA sequencing
  • CD8aa Treg RNA sequencing
  • conventional €'08 ⁇ T cells RNA sequencing
  • RNA-seq data of sorted CD8CKX Treg and CD8ap T cells will be generated by an Alumina HiSeq4000 analyzer using the TruSeq v3 Cluster kit at the UCSD Institute for Genomic Medicine as in the case of murine CD8aa Treg.
  • Irnmimoregidatory properties First, we will examine suppression of T cell proliferation as measured by the dilution of CFSE-labeled (CellTraceTM Thermo Fisher Scientific) sorted autologous CD4 + CD25 " T cells cultured either alone (3 x K /well) or with sorted CD8aa Treg (1 x 10 ' /w ell) in the presence of anti-CD3/CD28 microbeads. After 72h of stimulation, cells will be labeled with fluorochrome -conjugated anti-CD8 mAb and intracellular staining with anti- human IFNy.
  • CD8aa Treg will be able to suppress both T cell proliferation when co-cultured and the Thl cytokine secretion profile.
  • a purified anti -human HLA-E antibody (clone 3D 12) to the cultures to block antigen presentation by HLA-E and determine whether the suppressive activity is blocked.
  • CD8aa Treg we will examine different mechanisms that potentially can be involved in suppression of CD4 T cell proliferation by CD8aa Treg, including neutralizing antibodies against specific soluble factors to reverse inhibition of proliferation and Trans- well experiments to examine whether cell-cell contact is required for apoptosis induction using annexm V staining.
  • CD8aa Treg The ability of CD8aa Treg to suppress allogeneic response in a mixed lymphocyte reaction (MLR) is determined. Briefly, PBMCs (responder cells, lxi0 b cells/ml) will be stimulated for 6 days in 96-well plates with allogeneic PBMCs previously blocked by mitomycin (blocked, stimulator ceils, 0.5xl0 6 cells/ml), ratio 2: 1, to perform one-way MLR. After 6 days, cells from the oneway allogeneic MLR will be collected, washed, and CD8aa Treg will be isolated by sorting to evaluate their suppressor properties in a secondar ' MLR.
  • MLR mixed lymphocyte reaction
  • PBMCs freshly isolated PBMCs will be labeled with CFSE (CFSE-labeled, responder cells) and mixed with freshly isolated non-labeled allogeneic PBMCs (stimulator cells) and CD8aa Treg (isolated from the primary one-way MLR), ratio 2: 1 :0.5.
  • CFSE CFSE-labeled, responder cells
  • CD8aa Treg isolated from the primary one-way MLR
  • CD8aa Treg-mediated suppression involves cytotoxicity to the target cells using a flow cytometry-based cytotoxicity assay that simultaneously measures expression of the degranulation marker CD 107a by effector cells (CD8aa Treg) and the apoptosis marker annexin V binding to target cells.
  • Sorted CD4 " CD25 " T cells (Target cells) pulsed with anti-CD3/anti-CD28 beads or non-pulsed, will be labeled with PKH 67 green fluorescent cell linker (Sigma-Aidrich) and mixed with sorted CD8cece Treg (Effector cells) at an E:T ratio of 2: 1 to 10: 1.
  • the % of CD 107a-expressing CD8 ⁇ T cells and annexin V binding to PKH 67-labeled target cells will be measured by FACS. We anticipate that an increase in both effector cell degranulation and target cell death will be observed only in the presence of CD8aa Tregs and activated target cells. In addition, phenotypic characterization of effector CD8aa Treg will be carried out using different antibodies, including anti -perform and anti-granzyme B.
  • CD8aa Treg A summary of exemplary modalities by which CD8aa Treg can be targeted for their activation/expansion in vivo and subsequent protection from autoimmune diseases.
  • CD8aa Treg can be_sorted and adoptive transfer protects from autoimmunity.
  • a peptide-based modality targets induction of CD8aa Treg which in turns protects from disease.
  • Anti-4-lbb protects from autoimmunity, e.g., from EAE.
  • CD8aa+TCRafH Treg ceils are increased following anti- 4-1 BB administration.
  • Groups of C57BL/6 mice were either administered with PBS or 4-1BB antibody on day 0. Three days later mice were sacrificed and liver mononuclear cells were isolated and stained with various fluorochrome labeled antibodies to determine the number of CD8aa Treg. EAE is ameliorated in WT B6 mice following anti -4- IBB antibody injection.
  • mice Groups of female B6 mice (7- 8 mice in each) were immunized subcutaneously on day 0 with 100 ⁇ g MOG33- 55 peptide emulsified in an equal volume of CPA. On the same day animals were injected intraperitoneally with 25 fig of anti-4-lBB antibody diluted in PBS.
  • Anti-CD3 protects from autoimmune disease.
  • Induction of CD8aa Treg following administration of anti-CD3 (4c 11) antibody and subsequent protection from EAE, CD8cux+TCRaP+ Treg cells are increased following anti-CD3 administration.
  • Groups of C57BL/6 mice were either administered with PBS or CDS antibody on day one and day three.
  • mice were sacrificed and liver mononuclear cells were isolated and stained with various fluorochrome labeled antibodies to detect induction of CD8aa Treg.
  • EAE is ameliorated in WT B6 mice following anti-CD3 injection.
  • mice Groups of female B6 mice (6 in each) were injected intraperitoneally with 200 ⁇ g of anti-CD3 antibody per mouse on day 5, EAE was induced on day 0 by injecting 100 g MOG33 -55 peptide emulsified in an equal volume of CPA, subcutaneously.
  • CD8aa Treg Adoptive transfer of sorted CD8aa Treg protects mice from MOG- induced EAE (a model for multiple sclerosis) and from CD45Rb hlgh CD4+ T cell-induced colitis (a model for IBD) in a perforin -dependent manner.
  • CD8+ Treg-inducing peptide protects mice from MOG-induced EAE as well as from DSS-induced colitis in a Qa-1 -dependent fashion.
  • Peptide-induced induction of CD8+ Treg protects WT mice but not
  • CD8+Treg-deficient mice from EAE CD8+Treg-deficient mice from EAE.
  • Peptide-induced induction of CD8+ Treg protects mice from DSS-induced colitis.
  • Circulating CD8aa Treg are increased in SLE patients.
  • T cells The function of T cells is controlled by both intrinsic (e.g., PDi and exhaustion) and extrinsic (regulatory T cells or Treg) cell-based mechanisms that prevent them from causing excessive tissue damage.
  • intrinsic e.g., PDi and exhaustion
  • extrinsic regulatory T cells or Treg
  • CDS Treg PLZF transcription factor in a population of PLZF " TCR + CD8aa " T cells (hereafter referred as CDS Treg) in both mice and humans distinguishes them from CD8 ⁇ nv .
  • CDS Treg are innate-like cells that are enriched in liver of naive mice and a large proportion of them are CDl lc "1" CD244 + NKG2D + NKl . l + .
  • CDS Treg are distinct from other innate-like T cells, including mucosal associated invariant T ( ⁇ ) cells (summarized in Table below).
  • CDS Treg target activated Qa-1 "1" T cells and not nai e Qa- T cells, thus, they can control an ongoing T cell response. Accordingly, our preliminary data in indicate that an ongoing autoimmune response may be required for their expansion.
  • CDS Treg expansion following autoimmune inflammation and their ability to control activated T cells qualifies them as part of a powerful negative feedback regulatory mechanism that protects tissues from excessive immune-mediated damage.
  • NK1.1 mostly ⁇ 20%
  • Cytokine secretion IL-2 ⁇ , IL- I 7.V . IFNy + , IL- IL-2 " , IL-17A ⁇ + ,
  • innate-like unconventional PLZF + CD8 Treg are enriched in the liver of naive mice because the liver provides a more suitable environment for their development (e.g. TL-15-dependency).
  • CD8 Treg with innate-like features offer a rapid mechanism for limiting any excessive immune stimulation to protect tissue against constant exposure to gut-derived antigens. Since this mechanism target only activated but not naive T cells, it allows effective immunity against microbial antigens to protect the organ.
  • Phenotype of CDS ' Treg is unique and can be distinguished from CD8 C om-: To our knowledge, we have identified for the first time that PLZF expression in combination with other cell surface markers (PLZF ⁇ TCRa D8aof) distinguishes CDS Treg from CDSamv similar to FoxP3expression that differentiates CD4 Treg from conventional CD4 ⁇ T cells. Furthermore, the presence of PLZF ⁇ CD8 Treg in germ-free mice, which lack MATT cells, and in CD Id-/- mice, which lack both NKT and innate-like CD8 + T ceils, clearly indicate their unique innate phenotype.
  • CD 11c and CD244 (2B4) co-expression of CD 11c and CD244 (2B4) on a substantial portion of CD 8 Treg further suggests uniqueness of PLZF + CD8 Treg in both mice and humans. Additional studies, including Single Cell RNA sequencing analysis, will uncover other ceil surface and functional markers for the identification, development and function of CDS Treg.
  • CDS Treg Single Cell RNA sequencing analysis of CD l ie " " ' and CD 11c " CDS Treg in both mouse and human will unravel the transcription program, gene signatures and TCR repertoire related to the regulatory properties.
  • the development of multiparameter flow cytometric analysis to identify CDS Treg in human PBMC is also highly innovative and important.
  • the expression of cell surface molecules, such as CD200 suggests that PLZF + CD8 Treg are not only capable of killing target T cells, but they may use negative signaling via CD200 to inhibit the function of APCs, including microglia.
  • 4- IBB expression on CDS Treg and how an agonistic mAb can be used to preferentially activate them with important implications for potential intervention in human autoimmune disease.
  • CD8 + T cells can provide barriers to stem, cell engraftment, indicating the clinical relevance of CD8 Treg in humans (41 ).
  • a critical role for CD8 + T cells in IL-2-/- mice was also shown as these animals develop colitis with an accelerated kinetics (18).
  • Human disease CD8 + T cells also have been implicated in various conditions in humans, e.g. transplant survival (19), prevention of inflammatory bowel disease (20) and after treatment of multiple sclerosis with either glatiramer acetate (GA) or vaccination with irradiated, autoreactive C I .
  • PLZF ⁇ CD8 Treg are also present in bone marrow (-3.0%), spleen (-0.5%), blood (0.3%) and lungs (0.3%) of naive B6 mice as well as in the neonatal thymus (0.5%) until day 5 after birth.
  • CDS Treg were undetectable confirming their thymic origin (data not shown). Since innate-like features in T cells are driven by the expression of PLZF (42-48), we examined its expression in this novel population.
  • CDS Treg have lower PLZF expression than iNKT cells (CD4 " CDld- aGalCer tetramer " ).
  • PLZF is not expressed by either CDSconv or CD4 T cells. Accordingly, PLZF mRNA expression was found exclusively in sorted CDS Treg but not in CDSconv.
  • PLZF-Cre x 26T mice in which PLZF- expressing cells are permanently labeled tdTomato (50). Most CDS Treg in liver of PCre x R26T mice were tdTomato" while CDSconv showed only background level of expression.
  • CDS Treg Adoptive transfer of CDS Treg protects B6 mice from EAE.
  • CDS Treg do not express Foxp3 but express glucocorticoid-induced tumor necrosis factor-related receptor (GITR), a marker of active Treg cells (data not shown) (28).
  • GITR glucocorticoid-induced tumor necrosis factor-related receptor
  • naive B6 recipients were adoptively transferred i.v. with lxlO 5 sorted CDS Treg or CDSconv isolated from liver of naive B6 mice before induction of EAE with MOG35--55/CFA/PTX as described before (51 ).
  • CDS Treg but not CDSconv significantly protected mice from EAE.
  • CD 8 Treg co-express CD244 and CD 11c.
  • CD244 2B4
  • CDS Treg also express CD 11c, suggested earlier as a marker on CDS suppressor T cells (53).
  • Co-staining of CDS Treg show that all CD1 lc + CD8 Treg are also CD244 " while none of the CD8 «mv express CD1 lc or CD244.
  • CD244 + CDl lc + CD8 Treg are dependent on PLZF.
  • CDS Treg Both percentage and numbers of CD244 + CDl lc ⁇ CDS Treg were significantly reduced in PLZF-/- mice compared to PLZF+/+ mice. Since CDS T cells co-expressing CD 11c with suppressive functions have been reported (53, 54), we investigated whether CD1 lc expression on CDS Treg play a role in their regulatory capacity. Thus, CDS Treg depleted of CD 11c (CDSaofCDHc " ) (bead-depletion) were adoptively transferred into naive B6 recipients, and EAE was induced next day. CD l ie depletion of CDS Treg resulted in loss of their ability to control EAE.
  • CDS Treg are CD 122*, displayed a memory/activated phenotype (CD44 hi s h CD62L !ow CD69 + ) and express only NK inhibitory receptors (Ly49A, Ly49E/F, Ly49G2 and Ly49I) but not activating receptors (Ly49D and Ly49H) (data not shown).
  • CDS Treg A significant reduced frequency of CDS Treg in Qa-P- /- mice indicate that a large number of CDS Treg are restricted by Qa-l b molecules.
  • these CDS Treg can be distinguish from gut-resident CD8aofTCRaP + T cells in that they are CD 103 " , PLZF* and are significantly reduced in Qa-l b -/- mice while gut-resident cells are CD103 + , PLZF " and their frequency do not change in Qal b -/- mice (data not shown).
  • Cytokine secretion We have also determined the cytokine secretion profile of sorted CDS Treg vs. CDSconv after in vitro stimulation with anti-CD3 mAbs.
  • CDS Treg do not secrete IL-10 or ⁇ , but secrete typical cytokines produced by cytotoxic T cells (TNFa, IL-17A) as well as PLZF-driven secretion of both IL-4 and IFNy, similar to that in NKT cells (data not shown). It is noteworthy that these cells very rapidly secrete large amounts of IL-2 as well In the future, we will be investigating whether IL-2 secreted by CDS Treg may engage Foxp3 Treg that do not secrete IL-2. CDS Treg also express high levels of perform and granzyme B (data not shown).
  • CDS Treg Gene expression profile of murine CD8 Treg using single cell RNA sequencing.
  • Our preliminary data show that the TCRa repertoire of CDS Treg is polyclonal based on FACS, RT-PCR and high- throughput sequencing (47), indicating their distinctiveness from either MAIT cells that use an invariant TCR Vol 9 with biased usage of ⁇ 8/ ⁇ 6 in mice (42) or NKT cells.
  • CDl lcf CDS Treg also polyclonal? Since the Single Cell RNA (scRNA) sequencing technology allows the simultaneous analysis of the TCRo$ in single cells, we will also examine the TCRaP repertoire of CD 11 c ⁇ CDS Treg and compare it with the bulk CDS Treg population as well as CD 11c " CDS Treg. If the TCR repertoire of CD l ie 4" CDS Treg is oligoclonal, we plan identify the most common TCR for generation of TCR transgenic or retrogenic mice for their developmental studies as well as for the identification of antigenic peptides recognized by CDS Treg.
  • scRNA Single Cell RNA
  • MAIT cells which are also PLZF + CD8 + , represent a small population in mice
  • MAIT cells which are also PLZF + CD8 +
  • MR1-5-OP-R.U tetramers 55) (received from the NIH tetramer facility) for the RNASeq analysis of CDS Treg similar to our gating strategy in humans.
  • liver MNCs from 5-10 naive PEG mice will be stained and sorted using a FACSAria 111 into 4 subsets: PLZFTCRap + CD8aP% P LZ FTC RaP + CD 8 aof, PLZFTCRap D8acf CD1 lc + and PLZFTCRaP + CD8aofCDl lc " ⁇ The recommended starting point will be 10, 000 sorted cells to generate Gel Bead-In-EMulsions (GEMs).
  • GEMs Gel Bead-In-EMulsions
  • the ceils are delivered at a limiting dilution, such that the majority (-90-99%) of generated GEMs contains no cell, while the remainder largely contains a single cell. Approximately, 5,000 cells will be loaded into each Single Cell 3' Chip. After dissolution of the Single Cell 3' Gel Bead in a GEM, primers containing (i) an lliumina Rl sequence (read 1 sequencing primer), (ii) a 16 bp lOx Barcode, (iii) a 10 bp randomer and (iv) a poly-dT primer sequence will be released and mixed with cell lysate and Master Mix.
  • an lliumina Rl sequence read 1 sequencing primer
  • a 16 bp lOx Barcode a 16 bp lOx Barcode
  • a 10 bp randomer a poly-dT primer sequence
  • the l xTM GemCodeTM Technology will sample a pool of - 750,000 barcodes to separately index each cell's transcriptome by partitioning thousands of cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs) and will produce full-length, barcoded cDNA that will be amplified by PCR to generate Single Cell 3' libraries for sequencing and analysis.
  • Single cell RNA sequencing data will be analyzed in collaboration with the Bioinformatics Core at LJI.
  • complementary methods of single-ceil differential gene expression (SCDE) and model-based analysis of single-cell transcriptomics (MAST) analysis will be used to compare the full-length transcriptome between the populations.
  • GSEA Gene set enrichment analysis
  • IPA ingenuity pathway analysis
  • CD8 Treg are significantly expanded during EAE in the periphery and that they also infiltrate into the CNS during EAE. It would be important to analyze the TCR repertoire and transcription profile of the CNS-mfiltratmg CD8 Treg and compare that to CDSconv as well as hepatic CD8 Treg. Since regulatory activity is associated with CD 11c expression, it is likely that CDl lc " CD8 Treg infiltrate into CNS and negatively signal microglia. Therefore, we will examine and compare the gene expression and TCR repertoire of sorted CD8 Treg and CD8 C onv isolated from CNS of PEG mice in the recovery phase of EAE. These data should provide additional important information about the nature of CDS Treg infiltrating the target tissue.
  • CDl lc expression as a phenotypic marker for CD8 Treg
  • CDl lc CD8 T cells have been suggested to be suppressor T cells earlier (53).
  • CDl lc is also expressed by NK cells, activated T cells, ⁇ cells and certain macrophage populations (Immunological Genome Project).
  • Dr. Jonathan Ashwell's laboratory reported another TCRetp ⁇ CD8 ⁇ T cell subset with DC properties (57).
  • our CDS Treg are distinct from these others in several cell surface markers, including CD44, CD69, CD25, IL-7R, CD 122 and PLZF expression.
  • CDS Treg do not express other DC markers, including MHC class II, CDl lb, F4/80 and FcRg (data not shown).
  • our preliminary data also suggest that around 58% of CD8 Treg express CDl lc and that adoptive transfer of CDS " T cells depleted of CDl lc "1" cells resulted in loss of protection from EAE. Since CDl lc expression can potentially be used as a phenotypic marker for CDS Treg in both mice and in humans, it is crucial to address whether CDl lc expression on CD 8 Treg is regulated by transcriptional mechanisms, which require prior protein synthesis, or acquired through intercellular transfer (58).
  • CDl lc-EYFP transgenic reporter mice B6.Cg-Tg(Itgax- Venus)lMnz J
  • YFP yellow fluorescent protein
  • CD1 lc + CDS Treg expression of PLZF in CD1 lc + CDS Treg in these reporter mice will confirm their transcription regulation rather than their acquisition by intercellular transfer upon chronic activation. It is also clear from our data that CD8 Treg require thymus for development as they are absent in athymic and RAG1-/- mice (data not shown). Also, CDS Treg express all other markers of classical cytotoxic T cells, including perforin and granzyme B. Thus, a detailed transcriptome RNA sequencing analysis of CDl lc" CDS Treg (above) will further clarify whether a transcription signature of DC lineage or T cell lineage.
  • PLZF+ CDS Treg have a specific transcriptional development program and and control control of autoimmunity in a negative feedback regulation. Our hypothesis is that PLZF expression is crucial for the development of CDS Treg and that chronic autoimmune inflammation ultimately leads to their expansion and physiological control of autoimmunity.
  • CDS Treg express PLZF, although their expression levels are lower than iNKT cells. Accordingly, CDS Treg are significantly reduced in PLZF-/- mice.
  • PLZF-/-, RORa-/- or Id3-/- BM transfer may not be able to fully reconstitute B6 host. If this were the case, B6 mice will be reconstituted with a mixture of BM cells isolated from CD45.2 PLZF-/--, RORa-/- or Id3-/- mice and congenic CD45.1 B6 mice at a ratio of 50:50 and the development of CDS Treg analyzed 12 to 16 weeks post- transfer. These studies should give us important clues regarding the role of these related key transcription factors in the biology of CD8 Treg.
  • CDS Treg are primed/expanded physiologically to control autoimmunity:
  • Our preliminary data using adoptive transfer of sorted CDS Treg clearly indicate that CDS Treg can control autoimmunity.
  • CDS Treg are physiologically expanded during the course of EAE and play an important role in the control of disease. Consistent with this hypothesis and the Qa-l b restriction of a large number of CDS Treg, it has been shown that the recovery as well as susceptibility to re-induction of EAE is compromised in Qa- !-/- mice (30).
  • our preliminary data suggest that CDS Treg are expanded physiologically during the recover ⁇ ' phase of EAE.
  • B6 mice were immunized with either MOG35-55 for EAE induction as above or an irrelevant peptide derived from hen egg iysozyme (HEL) and hepatic CDS Treg were analyzed by FACS on day 10 at the onset of disease (EAE dlO) or at day 25 during the recovery phase of the disease (EAE d25).
  • CD 8 Treg transitorily decreased at the onset of disease (day 10) but significantly expanded during the recovery phase (day 25) of EAE.
  • CDS Treg were not altered in non- diseased mice immunized with an irrelevant HEL peptide.
  • mice that are deficient in PLZF + T cells, including CD8 Treg, using floxed genes in a 2-step process by inserting two loxP sites simultaneously to ensure deletion of the intervening D A (two double stranded cuts in the DNA followed by repair of the gap by the cell).
  • D A double stranded cuts in the DNA followed by repair of the gap by the cell.
  • CD4-Cre mice we will first establish that they are deficient in PLZF " T cells. Based upon our data in PLZF-/- mice, CD4-Cre PLZFfiox/flox (PLZF F F ) mice should be deficient in PLZF + CD8 Treg cells.
  • mice and negative littermates will be immunized with PLP172-183/CFA/PTX to induce EAE and to be able to address re-induction of disease. Mice will be monitored every day for clinical score and disease onset, severity, incidence and recovery analyzed. Next, we will examine whether susceptibility to re-induction of EAE with PLP172-183/CFA is altered in PLZF f/fl mice.
  • mice and littermates will be immunized with PLPi72-i83/ ' CFA and 30 days later mice will be challenged with PLP/CFA PTX and disease monitored.
  • CD 8 Treg induced initially in CD8 Treg ⁇ mice (littermates) will protect them from disease whereas their absence in PLZF*'* mice should allow induction of clinical disease.
  • CDS Treg CDS Treg from negative littermates and determine whether regulation can be reconstituted in PLZF F F mice.
  • CDS Treg are enriched in liver, we will also investigate whether PLZF*'* mice develop sterile inflammatory liver injury. We will determine whether inflammation and induction of related genes occur in livers of naive PLZF F/F mice using Nanostring Technology (UCSD Core). These studies will conclusively prove that CD8 Treg are able to not only control physiologically autoimmune disease but also control sterile liver inflammation.
  • UCSD Core Nanostring Technology
  • 4-1BB (CD137, tafrsf ) is a member of the TNF receptor superfaroily that is not expressed in naive T cells but only in activated/memory CDScoav T cells and is involved in their survival and activation (66, 67). Accordingly, agonistic anti-4-lBB Abs injected system icaJ.lv result in expansion of primarily memory CD8 + T cells (68, 69). However, several studies have shown paradoxical outcomes depending upon the timing and doses of anti-4-lBB Abs administration. For example, agonistic anti-4-lBB Ab can enhance some antiviral and anti-tumor T cell responses while inhibiting CD4 + T cell-dependent autoimmunity, including EAE (54, 67).
  • CDS Treg As B6 mice but not Qa-i-/- mice are protected from EAE. It further indicates that under these conditions CD4 Treg that can also express 4-1BB do not play a significant role. Interestingly, a high dose of anti-4- !BB Ab (200 ⁇ ig/mouse) does not protect mice from EAE, but rather potentiates disease due to overwhelming activation of encephaiitogenic T cells and APCs.
  • Our preliminary data further suggest a significant absence of CDS Treg (4- lBB " CD lc + CD244 " ) in 4- I BB-/- mice in comparison to B6 mice (data not shown). Therefore, we will investigate the role of 4- IBB expression on CDS Treg in the control of EAE and. hopefully, provide an explanation related to its paradoxical effects.
  • CD244 (also known as 2B4, SLAMF4) is a member of the Ig superfamily and is expressed predominantly on the surface of MK cells and ⁇ T cells. Unlike the other members of the SLAM family receptors that can engage in homotypic interactions, 2B4 interacts with a high affinity receptor SLAMF2 or CD48, which is expressed on a number of hematopoietic cells.
  • the CD244- CD48 interactions can have dual functions either activation or inhibition of the immune response depending upon the degree of receptor expression, extent of ligation and level of adaptor molecules (70-72).
  • An K-independent regulatory role of CD244 has also been shown in the control of experimental lupus (73).
  • CD244 a splice variant of CD244 has also been reported to be preferentially expressed in SLE patients and is associated with defective regulation in SLE (74). Since CD244 interactions can control both activation and lysis of target cells, we believe that is important to study the role of CD244 expression in CDS Treg-mediated immune regulation.
  • CD200 or OX-2 is a member of Ig superfamily and is expressed in lymphoid cells, including B cells and activated T cells, in both mice and humans. It interacts with its cognate ligand CD200R that is expressed on granulocytes, monocytes, DC and macrophages (75). The interaction of CD200-CD200R inhibits activation of macrophages and microglia and, accordingly, CD200-/- mice develop a rapid and severe form of EAE with enhanced axonal damage (75, 76).
  • CD200-CD200R interactions are also involved in regulation of Thl/Thl7 immune responses in arthritis, IBD, transplantation and cancer (77- 79), Since CDS Treg infiltrate into CNS during EAE, we beleive that binding of CD200 on CDS Treg to CD200R expressed on microglia, astrocytes, and oligodendrocytes could suppress axonal damage that is mostly mediated by activated microglia thus protecting m ice from EAE.
  • NKG2D Similar to NKT cells, CDS Treg express NK receptors, such as NKl . l and NKG2D, and these receptors may be involved in fine-tuning CD 8 Treg function. Since the in vivo function of NKl. l is poorly understood, we will focus here on the role of NKG2D in the function of CDS Treg. NKG2D is encoded by the KLRK1 gene expressed in both mice and humans and binds to cell surface MHC class I-related proteins, MICA MICB (human) and Rae (mice) (80, 81). In CDS T cells and NKT ceils, NKG2D plays an important role as a co- stimulatory molecule and promotes Thl cytokine production (82).
  • NKG2D expression is enhanced in inflammatory tissues in autoimmune diseases, including arthritis, lupus and diabetes (80, 81, 84).
  • CDS Treg We have developed a simple in vitro assay to examine the regulator ⁇ ' activity of CDS Treg. Specifically, sorted CDS Treg from B6 or deficient mice will be co-cultured with either CFSE-labeled OVA -reactive OT-II CD4 + T cells or sorted CD4 " T cells from B6 mice in the presence of OVA peptide or anti- CD3/anti-CD28 beads, respectively. CFSE-diiution and staining with Annexin and PI will be measured by FACS as an indication of response. The conventional CD8aP T cells will be used as controls. Preliminary data suggest that in addition to the killing of the target CD4 + T cells, CDS Treg appear to inhibit the proliferation of the target CD4 ⁇ T cells. In some cases, we will use titrated concentration of blocking mAbs, for example anti-CD200 (OX2, Bioiegend), in in vitro assays to directly examine effect on regulatory activity. Isotype control antibodies will be used as controls
  • CDS Tree in human also have a similar phenotype, gene signature profile and regulatory function.
  • CDS Treg are also present in humans and, potentially, play an important role in the regulation of autoimmunity. Consistent with our data showing increased frequency of CDS Treg only during the recovery phase of ongoing EAE,we have also found an increased frequency of CD8 Treg in PBMCs from arthritis and lupus patients (data not shown).
  • CDS Treg are PLZF ⁇ TCRa "CD8aa ⁇ T cells that express intermediate to very low- levels of CD161 (CD161 +/_ ).
  • CDS Treg are present in PBL of healthy individuals and represent -12% in human PBMC, ranging from 0.4 to 35%.
  • the expression of CD244 and CD 11c was significantly higher in human CDS Treg than in both MAIT and CDSconv.
  • human CD8 Treg Similar to murine CD 8 Treg, a substantial portion of human CD8 Treg also co-express CD244 and CD 11c (CD244 ⁇ CDl lc + ) that is almost absent in MAIT cells (-16% vs -1%). Furthermore, the expression of several markers known to be up-regulated in MAIT cells, including IL-18 receptor a (IL ⁇ 18Rc ), RORyt, CXCR6 and CCR6, was significantly reduced in human CD 8 Treg (data not shown). Notably, human CD8 Treg also secreted higher levels of Granzyme B and perforin similar to murine CD8 Treg and also secreted IFNy, IL-17 and 1L-4 (data not shown). In preliminary in vitro suppression assays, sorted human CDS ' Treg also inhibits CD4 + T cell proliferation significantly (data not shown).
  • CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages.
  • CDl lc+CD8+ T cells two-faced adaptive immune regulators.
  • Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue. Nat Immunol 16: 85-95
  • Chlewicki LK Velikovsky CA, Balakrishnan V, Mariuzza RA, Kumar
  • NKG2D performs two functions in invariant NKT cells: direct TCR-independent activation of NK-like cvtolysis and co-stimulation of activation by CDld. Eur J Immunol 41 : 1913-23
  • T cell antigen receptor TCR

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Abstract

Isolated immune cells having TCRαβ+, CD8αα+, Nk1.1+, PLZF+, CD161+, and optionally having one or more of CD11c+, CD137+ CD244+ or one or more of NK-inhibitory receptors, and methods of obtaining and using those cells, as well as methods to inhibit or enhance those cells in a mammal are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. application Serial No. 62/575,714, filed on October 23, 2017, and U.S. application Serial No. 62/576,493, filed on October 24, 2017, the disclosures of which are incorporated by reference herein.
BACKGROUND
The understanding of the process of immune tolerance is crucial for preventing autoimmunity as well as for the generation of effective cancer immunity. Conventional MHC-restricted TCRap+ T ceils are involved in the development of several autoimmune disorders. A detailed knowledge of the cellular and molecular mechanism(s) controlling pathogenic self-reactive T cells is important for treatment of autoimmune diseases. T cells are controlled by both T cell-intrinsic and extrinsic cell-based mechanisms that prevent them from causing excessive tissue damage: immune ignorance, anergy, exhaustion, phenotype skewing and antigen-induced apoptosis are part of the intrinsic mechanisms whereas regulatory T cells (Treg), including Foxp3" CD4" and Trl, natural killer T (NKT) cells and CD8 " T cells comprise cell-based mechanisms.
Since 1970s, it has been suggested that CD8+ T cells play an important role in immune regulation of autoimmune diseases, transplant tolerance and in homeostasis of cellular and humoral immune responses. Initially, using mice genetically deficient in or depleted of CD8÷ T cells by treatment with anti-CD8 mAb, an important role for CD8 T cells in regulation of EAE and arthritis has been shown. Similarly, a critical regulatory role of CD122÷CD8÷ T cells involving a cytolytic mechanism, has been shown in IL-2-/- and IL-2RP-/- mice, interestingly, IL-2/IL-15Rp-deficiency in humans also leads to a severe combined immunodeficiency syndrome as observed for IL-2Ra. Also,
CD122 "CD8 " T ceils can provide barriers to stem cell engraftment, indicating the clinical relevance of CD8 Treg in humans. A critical role for CD8+ T cells in IL-2-/- mice lacking CD8+ T cells was shown as these animals develop colitis with an accelerated kinetics. CD8+ T ceils also have been implicated in various conditions in humans, e.g. transplant survival, prevention of inflammatory bowel disease, and the treatment of multiple sclerosis with either glatiramer acetate (GA) or vaccination with irradiated, myelin basic protein-activated CD4+ T cells. Recently, GA-induced CD8+ Treg have been shown to eliminate CD4+ T cells in a HLA-E-restricted manner. In a recent clinical trial with anti-CD3 mAb
(teplizumab), increased frequency of memor - CDS'"" T cells with regulatory gene expression was found to be associated with a positive clinical response in type 1 diabetes. Despite their critical role in control of autoimmunity, the biology of CDS'"" Treg has not sufficiently advanced because of our inability to distinguish them from conventional CD8+ T cells based on either the ceil surface phenotype or a specific transcription program similar to that described for Foxp3+CD4+ Treg.
CD8+ T regulatory ceils have been a subject of study for many years, but this cell population has been difficult to define and identify. These
immunosuppressive cells can be very important in the initiation and/or the maintenance of diseases when their numbers are skewed (high or low), such as in cancer, 1BD, lupus, rheumatoid arthritis and various autoimmune diseases. The hardest part so far has been to identify and target these cells efficiently to
Figure imgf000003_0001
An immunosuppressor type of unconventional CD8+ T regulator ' cell, which is more prevalent in the liver and in the gut, is identified. Adoptive transfer of these CD8+ T regulatory ceils protect mice from developing antigen- induced EAE (a model for multiple sclerosis) and also protect mice from developing T cell- induced-colitis (a model for IBD). In other experiments, administration of anti- 4-iBB antibody increases the number of these CDS" T regulatory in the liver. Not only that, when the anti-4-lBB antibody was administered in mice with antigen -induce EAE, these mice show lesser symptoms than the negative controls. Similar results were obtained when anti- CD3 was administered in these EAE mice. Thus, innate-like PLZF+CD8aa Treg enriched in liver and in intestine play an important role in the homeostasis of immunity. The disclosure provides for the use of antibodies to identify a CD8+ T regulatory cell population (e.g., by flow cytometry) in a subject with the following minimal phenotype : TC a(3+ CD 8 axr NK 1.1+ PLZF* CD 161 ÷ in humans, CD1 lc+, though other markers are usually present too, for example one or more of CD137+ ('1)244 ' . or one or more of the NK-inhibitory receptors.
The disclosure provides for a method of treating cancer patients by administering monoclonal antibodies that bind to this CD8+ T reg cell population with the goal of lowering the number of this T cell population or eliminating these altogether in patients, allowing them to mount a proper immune response to the tumor.
The disclosure provides for a method of treating IBD, lupus, RA patients and patients suffering from other autoimmune diseases by administering Qa- l/HLA-E binding peptides that will stimulate CD8T regs.
The disclosure provides for a method of treating IBD, lupus, RA patients and patients suffering from other autoimmune diseases by administering anti- CD3 in patients.
The disclosure provides for a method of treating IBD, lupus, RA patients and patients suffering from other autoimmune diseases by administering anti- CD137 (also known as anti-4-lBB).
The disclosure provides for sorting and expanding patient ceils with cytokines IL-2/IL-15 and a combination of antibodies mentioned above.
In one embodiment, the cells are identified using monoclonal antibodies specific for CDSota, NKl . l, PLZF, or CD161 , and optionally antibodies specific for one or more of CD I lc, CD 137, CD244, TCRctfl or one or more of NK- inhibitory receptors or a combination thereof. In one embodiment, the cells are human cells. In one embodiment, the method includes isolating the identified cells and optionally expanding the isolated cells. In one embodiment, the cells are from a patient with an autoimmune disease. In one embodiment, the cells are cultured with IL-2, IL-15, Qa-l/HLA-E binding peptides, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof. In one embodiment, the composition is systemicaiiy administered. In one embodiment, the composition is locally administered.
Further provided is a method to prevent, inhibit or treat cancer in a mammal. The method includes administering to the mammal an effective amount of a composition comprising one or more antibodies specific for CD8aa, specific for NK 1.1, specific for PLZF, or specific for CD 161, or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD 11c, specific for CD 137, specific for CD244, specific for TCRaP, or specific for one or more of NK -inhibitory receptors, or a combination thereof. In one embodiment, the cancer is neck cancer. In one embodiment, the cancer is melanoma, in one embodiment, the mammal is a human. In one embodiment, the composition is systemicailv administered. In one embodiment, the composition is locally administered.
A method to pre vent, inhibit or treat autoimmune disease in a mammal is provided. The method includes administering to the mammal a composition comprising one or more Qa-l/HLA-E binding peptides, anti-CD3 antibodies or anti-CD137 antibodies in an amount effective to stimulate CDSa+T regs. In one embodiment, the disease is IBD, colitis, lupus or RA. In one embodiment, the mammal is a human. In one embodiment, the composition is systemicailv administered. In one embodiment, the composition is locally administered.
BRIEF DESCRIPTION OF FIGURES
Figures 1A-H. Liver enriched CD 8 Treg are PLZF positive and utilize an mnate-like PLZF-driven transcription program. (A) Representative flow cytometry plots showing gate strategy for murine CDS Treg in liver
mononuclear cells (MNCs) from naive B6 mice. CDS Treg were identified as B220~CD4~TCRp+CD8a+P~ ceils. Scatter graphs show cumulative data of percentage and number of hepatic CD 8 Treg among TCR ÷ T cells in naive B6 mice (n=21). Each dot represents an individual mouse. Results shown as mean ± SEM. (B) Histograms show CD69, NK 1.1, CD44 and CD62L expression by CDS Treg (top) and CDScmw (bottom) compared with isotypes (filled gray) as determined by flow cytometry. Numbers in histograms indicate the percentage of positive cells. (C) Representative Dot plots showing CD8 Treg and CD8Conv (top) as well as iNKT cells and CD4 T cells (bottom). Histograms show expression of PLZF or GFP (reporting PLZF) by CD 8 Treg and CD8Conv (top) as well by iNKT cells (CD4~aGaiCer/CDld tetramer+) and CD4+ T cells
(CD4÷aGalCer/CDl d tetramef") (bottom) from naive B6 mice or PLZF-eGFP reporter (PEG) mice. CDS Treg and iNKT cells (open); CD8Conv and CD4j" T cells (filled gray). (D) Quantitative RT-PCR analysis of PLZF expression on sorted CDS Treg and CDSconv following normalization using β actin gene. (E) Expression of TdTomato in CDS Treg and CDSconv from livers of B6 (top) and Pcre x R2.6T (26T) (bottom) mice. CDS Treg and CDSconv were stimulated for 3 days with anti-CD3/anti-CD28 mAbs and TdTomato expression was measured by FACS on sorted cells. Numbers indicate percentage. (F) Quantitative RT- PCR analysis of selected transcription factors expression on sorted CDS Treg. The expression of each transcription factor is represented by mRNA fold change to that on CDSconv. (G) Numbers of CDS Treg in liver and spleen of homozygous PLZF-/- mice and littermates PLZF+/+ and PLZF+/- mice as determined by FACS. Data represent mean ± 2SD. *p < 0.05, **p < 0.01, impaired t test. (H) Numbers of CDS Treg in livers of homozygous RORa-/- or Id3-/- mice and B6 mice (RORa+/+ or Id3+/+) as determined by FACS. Data represent mean ± 2SD. **p < 0.01, ***p < 0.001, unpaired t test. Data representative of three independent experiments.
Figures 2A-I. Hepatic CDS Treg are innate-like and a substantial portion of CDS Treg co-express CD244 and CD 11c. (A) Representative dot plot showing CD244 and CD 11c expression by hepatic CDS Treg (left) and CDSconv (right) from naive B6 mice following gate strategy. Numbers indicate % of CDS Treg or CDSconv that were CD244+ or CD244÷CDl lc+. (B) Bar graphs sho percentage (right) and numbers (left) of CD244÷CDl lc+ CDS Treg in liver MNCs from PLZF-/- mice (n=3) and littermates PLZF+/+ and PLZF+/- mice (n=4). Data are represented as mean ± 2SD. **p< 0.01, unpaired t test. (C) Sorted hepatic CDSaofCD l e" T cells (2xl05/mouse) from naive B6 mice were adoptively transferred i.v. into naive B6 mice one day prior to EAE induction. EAE was also induced in non-transferred naive B6 mice (control, 3-5 mice). Mean disease scores ± SEM are shown on the y-axis versus days post- immunization on the x-axis. (D) Representative dot plot showing NK1.1, NKG2D, CD137 (4- IBB) and CD200 expression by hepatic CD244"CDl le+ CD 8 Treg from naive B6 mice. (E) CDS Treg are not Foxp3 " and represent a distinct population of Treg. Histograms show Foxp3 and GITR expression by CDS Treg (black line) and CDSconv (gray line) in liver MNCs from naive B6 mice. (F) Histograms show CD25, CD 122, PD-1, CD28, CD27, OX40, CD200 and 4-lBB expression by CDS Treg (top) and CDSconv (bottom) compared with isotypes (filled gray). Numbers indicate percentage of positive cells. (G) Histograms show ICOS, CXCR5, CD 127, Eomes and CD 103 expression by CD8 Treg (top) and CDSconv (bottom) compared with isotypes (filled gray). Numbers indicate percentage of positive cells. (H) Histograms show the expression of NK cell surface markers, including Ly49A, Ly49E/F, Ly49G, Ly49G2, Ly49L Ly49D, Ly49H and NKG2D, by CDS Treg (top) or CDSconv (bottom) compared with isotypes (filled gray). Numbers indicate percentage of positive cells. (I) Quantitative RT-PCR. analysis of the expression of selected genes on sorted CDS Treg from liver of naive B6 mice. The expression of each gene is represented by mRNA fold change to that on CDH..,.m . Data representative of three independent experiments.
Figures 3A-B. CDS Treg are also present in other lymphoid
compartments and originated in the thymus. (A) Percentage of CDS Treg in bone marrow, thymus, liver, lung, spleen and peripheral blood of naive B6 mice (n= 3-4). Each dot represents data from an individual mouse. Horizontal line identified mean. (B) Percentage of CDS Treg in liver MNCs from athymic mice (n= 3) and euthymic heterozygous littermate (n= 3). Data represented as mean ± SEM. Data representative of two independent experiments.
Figures 4A-G. Hepatic CDS Treg have immune regulatory properties and are physiologically expanded during EAE. (A) Adoptive transfer of sorted CDS Treg protects mice from MOG -induced EAE (a model for multiple sclerosis). Sorted hepatic CDS Treg or CD8∞uv (lxl05/mouse) from naive B6 mice were adoptively transferred i.v. into groups of naive B6 mice one day prior to EAE induction with MOG35--55/CFA/PT. As a positive control, EAE was also induced in non-transferred naive B6 mice. EAE clinical severity (disease scores) was monitored daily in all groups. Mean disease scores ± SEM are shown on the y- axis versus days post-immunization on the x-axis. *p < 0.05, Student t test.Data representative of 3-5 mice analyzed in each group. (B) Sorted hepatic CDS Treg or CDSconv (2xl0-Vmouse) from naive CDld-/- mice were adoptively transferred i.v. into groups of naive B6 mice one day prior to EAE induction as described in A. Mean disease scores ± SEM are shown. *p < 0.05, Student t test. Data representative of 3-5 mice analyzed in each group. (C) CDS Treg are increased during the recovery phase of EAE. EAE was induced in groups of naive B6 mice with either MOG35--55 or hen egg iysozyme (HEL) peptide along with CFA/PT and the clinical disease scores were evaluated daily. Recipient MOG35-55- immunized B6 mice were sacrificed at day 10 (n = 3, EAE dlO) or day 25 (n = 9, EAE d25) while HEL-immunized mice were sacrificed at day 25 (n :=: 3, HEL d25). The percentage of hepatic CDS Treg was determined by FACS and compared to that in naive B6 mice (n = 5). Data are represented as mean ± SEM. *p < 0.05, unpaired t test. (D) Percentage of CDS Treg (y-axis) versus EAE clinical disease scores (x-axis) in spleens of MOG35 --55/CF A/PT-immunized B6 mice (n = 16) at day 30 during the recovery' phase of EAE are shown. Data are represented as mean ± SEM. **p < 0.01, unpaired t test. Data representative of three independent experiments. (E) Adoptive transfer of sorted CDS Treg protects mice from CD45Rbh!ghCD4 " T cell-induced colitis (a model for IBD) in a perforin-dependent manner. Groups of Rag 1-/- mice (n := 4) were reconstituted i.v. with sorted 4x105 CD4+CD25~CD45RBhi h T ceils from spleen of naive B6 mice alone (control) or co-transfer with 2xl05 sorted CDS Treg from liver of naive B6 (perforin+/+) or perforin-/- mice. Body weight change was monitored weekly for 4 weeks and expressed as ratio of the weight before cell transfer. Representative colon histopathology examination by H&E staining in the indicated group of Rag 1-/- recipients 4 weeks after cell transfer. Magnifications: l Ox. Results are representative of two independent experiments. (F) Numbers of CFSE-labeled OT-II CD4÷ T-cells recovered from B6 recipients following co- transfer of OT-II CD4+ T cells (1 x 106) with either positive selected CDS"1" T cells or liver MNCs (8 x 106 cells/mouse) from B6 (perf+/+) or perforin-/- mice. Transferred B6 mice were injected with OVA323-339 peptide (50 .ug/mouse, ip) 24 hours after adoptive transfer. As control, transferred B6 mice were injected with either PBS or OVA peptide. After 3 days, splenocytes were analyzed for expression of CFSE-labeled OT-II CD4+ T-cells by FACS. Results represented as mean ± SEM. *p < 0.5, **p < 0.01, one way ANOVA with Tukey' s multiple comparisons test. (G) Percentage of CFSE-labeled CD4+ T ceils recovered following in vitro stimulation. CFSE-labeled sorted CD4+ T cells from spleen of B6 mice were cultured alone or co-cultured with either sorted CDS Treg or
CDScoiiv followed by in vitro stimulation with plate-bound anti-CD3 mAb (100 μ§Λνβ11) and irradiated APC. After 4 days, CFSE-labeled cells were analyzed by FACS. Results represented as mean ± SEM. **p < 0.01, t test. Figures 5A-E. Hepatic CDS Treg secrete typical PLZF-driven cytokines, are dependent on IL-15/IL-2R[¾ for their development and most of them are Qa- ib-restrieted. (A) Production of IFNy, IL-2, TNFa, IL-4, IL-17A, IL-6 and IL-10 by CDS Treg or CDSconv (IxlO5 cells/well) sorted from liver of B6 mice in response to in vitro stimulation with plate-bound anti-CD3 (1 ug/ml) niAb. Cytokine production was examined at various time points in supernatants from cultured cells by BD™ Cytometric Bead Array (CBA). Values are expressed in pg/ml, **p < 0.01, unpaired t test. (B) Percentage of CDS Treg in liver MNCs from CD25-/-, CD 122-/-, IL-15-/-, IL-7-/- IL-6-/- IFNy-/- and T-bet-/- mice compared with B6 mice as determined by FACS. Each dot represents an individual mouse. Data representative of 3-4 mice per group. Data are represented as mean ± SEM. ***p < 0.001, npaired t test. (C) Percentage of CDS Treg in liver MNCs from CD8a-/-, CD8p-/-, μΜΤ-/-, CD Id-/-, Jal8-/-, Qa- \ -l- and TAP1-/- mice compared with B6 mice as determined by FACS. Each dot represents an individual mouse. Data representative of 3-5 mice per group. Data are represented as mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001, unpaired t test. (D) Serum ALT levels from groups of B6 (n=6) and Qa-1-/- (m=5) sacrificed 24 hours after i.v. injection with ConA (170 ,iLg/20 g body weight). **p < 0.01, unpaired t test. E. H&E staining of liver sections of B6 and Qa-1-/- mice following ConA injection as before. Magnification X 100. Data representative of three independent experiments.
Figures 6A-F. Hepatic CDS Treg are polyclonal. (A) Bar graph showing TCR νβ chain expression on hepatic CDS Treg from naive B6 mice (n=3) by FACS. Liver MNCs were stained with nnti-TCRp. anti-CDSa, anti-CD8p.2 and distinct anti-TCR νβ mAbs. Data are represented as mean ± SEM. (B)
Frequency distribution of TRBV gene segment usage by ail unique clonotypes from sorted CD8 Treg (n = 31, left) and CDSconv (n = 7256, right) isolated from liver MNCs from naive B6 mice (n = 10) and analyzed by high-throughput sequencing of the CDR3fi regions. Each TRBV gene segment is represented by a slice proportional to its average frequency. (C) Frequency distribution of TRBJ gene usage by all unique clonotypes from sorted CDS Treg (white bar) and CDSconv (black bar). (D) CDR3 length distribution by all unique clonotypes from sorted CDS Treg (white bar) and CDSconv (black bar). (E) Bar graph showing TCR. Va chain expression on hepatic CD8 Treg from naive B6 mice. Liver MNCs were stained with anti-TCR , anti-CD8a, anti-CD8 .2 and available anti- TCR Va mAbs. (F) RT-PCR analysis of TCR Va gene segments expression in CD8 Treg and CD8Conv. Total R A was extracted from sorted CDS Treg and CDSconv from liver MNCs of naive B6 mice. The TCR Va chain was amplified using specific primers for each Va gene and a common Ca primer. The PCR products were electrophoresed in 1.2% agarose gel and visualized by ethidium bromide staining.
Figures 7A-B. CD8aa Treg are enriched in colonic IEL and colonic IEL CDS Treg express NK-inhibitory receptors. A. Contour plots represent live CD8+ T cells (gating: CD45"fTCR *'CD4-B22Cr) from different tissues in gut and Gut-associated lymphoid tissue (GALT) in WT B6 mice. Numbers indicate % of CD 8 Treg (left) and Οίδαβ T cells (right). Histogram overlay shows intracellular PLZF expression in colonic CDS Treg (red histogram) using an anti-PLZF mAb (left) or as GFP expression in PLZF-GFP mice (right). Blue histograms show control staining in non CDSaa T cells (left.) or in non-GFP mice (right). Plots are representative of at least 2 independent experiments for each tissue. B. CD45 TCRp' CD8aa/aP live cells from colonic lELs were gated for lymphocyte activation marker, CD44 and IL-2 receptor β chain, CD 122. The expression of NK-inhibitory receptors was analyzed on CD44+CD122+ colonic IEL CDS Treg and IEL CD8o$ T cells. Square gates within contour plots show percentage of Ly49 C/l/F/FL cells in B6 (WT) colonic IEL CDS Treg (first panel), WT colonic IEL CD8 β T cells (second panel), Qa-l -/- colonic IEL CDS Treg (third panel) and Batf3-/- colonic IEL CDS Treg (fourd panel).
Numbers indicate the frequency of events.
Figures 8A-C. Spontaneous inflammation in Qa-Ib-/- mice genetically deticient in CDS Treg. A. Inflammation in liver of Qa-lb-/- mice. Representative H&E and Sirius-red staining of liver sections from naive Qa-lb-/- mice.
Magnification X100. B. Infiltration of infiammatorv' CD4 T cells into liver of Qa-lV- mice. Representative H&E, anti-CD4 and anti-CD 8 staining of liver sections from inflamed livers of naive Qa-lb-/- mice. Magnification X200. C. Spontaneous inflammation in colonic tissue in Qa-lb-/- mice. H&E staining of colonic sections showing big patch of cellular infiltrates in Qa~ lb-/~ mice (Magnification X4). Bar graph shows IL-6 secretion from colon explant culture from B6 (WT) and Qa-lb-/- mice as measured by ELISA. Figures 9A-H. Activation/expansion of CDS Treg by TCR-derived peptides protect mice from MOG-induced EAE as well as from DSS-induced colitis in a Qa-1 -dependent fashion. A. Proliferative response of splenocytes from B6 (Qa-lb+/+) (white bar) and Qa-lb-/- (black bar) mice against in vitro stimulation with individual TCR-derived peptides. Proliferation was measured by incorporation of pH] -thymidine in triplicate cultures. Bar graph depicts mean ± 2SD of stimulation index. *p < 0.05, paired t test. B. Percentage of CFSE- labeled CD4+ and CD8a+ T cells in response to in vitro stimulation with individual TCR-derived peptides. Proliferation by CFSE dilution analysis was examined in splenocytes of B6 mice. Gating was performed on CD4+ (white bar) and CD8a+ (gray bar) T cells. Data plotted are mean ± 2SD. *p < 0.05, paired t test. C. EAE incidence in B6 mice after vaccination with specific TCR- derived peptides. Groups of B6 mice were vaccinated i.p. with 50 x.g of one of the following peptides: p4L, p5L, p6L, p8.2L, pi 1L, p5C and p42-50 in IFA or with PBS in IFA as control. One week later, EAE was actively induced following immunization with MOGss-ss/CFA/PTx. The clinical symptoms of EAE were monitored and scored daily until day 30. Results represent the mean ± SD. *p < 0.05, Student t test. D. Cytokine response after vaccination with peptide p8.2L. B6 mice were vaccinated with either peptide p8.2L or control peptide p6L. One week after, EAE was induced by injection of MOG35-
55/CFA/PTx. On day 20, splenocytes from vaccinated mice were cultivated and challenged in vitro with MOG35-55 (40 g/ml) for 72 hours. Supernatants were collected and analyzed by CBA (BD). Results in graphs shown as mean ± SEM of pg/ml cytokine secretion. *p < 0.05, npaired t test. E. CD8 Treg infiltrate into CNS during EAE. Representative flow cytometry dot plots showing percentage of CD8 Treg (left) and ( )8αβ (right) T cells in liver (top) and CNS (bottom) of B6 mice following vaccination with peptide p8.2L on day 12 after EAE induction in comparison with control mice. Bar graphs showing percentage of CDS Treg in liver (top) and CNS (bottom) of p8.2L-vaccinated B6 mice. **p < 0.01, unpaired t test. F. Bar graphs showing percentage of CD4+ and CD8+ T cells in liver and CNS of p8.2L-vaccinated B6 mice and control mice. G. (Left) EAE incidence in B6 (Qa-lb+/+) and Qa-P-/- mice after vaccination with peptide 8.2L. B6 and Qa-P-/- mice were vaccinated i.p. with 50 g of peptide 8.2L in IFA or PBS/IFA (control). One week later, EAE was induced with MOG35 -55/CFA/PTX, (Right). EAE incidence in SJL mice after vaccination with peptide 8.2L. SJL mice were vaccinated i.p. with 50 μg of peptide 8.2L in IFA or PBS/IF A (control). One week later, EAE was induced with PLPi39-isi/CFA/PTx. All mice were monitored for disease symptoms until day 25. Results represent the mean ± SD. These data are representative of three independent experiments. H. Treatment with a CD 8 Treg -inducing peptide confers protection from DSS- induced colitis in WT but not in Qa-lb-/- mice. Groups of B6 (WT) (upper panels) or Qa-lb-/- (lower panels) mice were supplied with 2.5% DSS containing water for 7 days following prophylactic (3 days prior) i.p. injection of the Qa-l - binding peptide p8.2L (50 μ /ηιοιιβε) as indicated (+P) or control (-P). The relative reduction in colon length (gross morphology) as well as patches of cellular infiltrates in epithelial damage (white arrows) using H&E staining of colonic section are shown. Bar graphs (middle panels) indicate cumulative clinical scores in one representative experiment (each dot represents indiv idual mouse). Cumulative clinical scores were calculated on the day of sacrifice (peak disease by body weight) by the following: i) grading loss in body weight (0 to 4% as 0: 4 to 10% as 1; 10 to 15% as 2; 15 to 20% as 3 and a loss of >20% as 4); is) grading colon length decrease (0.5 to 1 cm as 1 ; 1.1 to 1.5 cm. as 2; 1.6 to 2 cm as 3 and > 2 cm as 4); iii) average colon thickness of proximal and distal colon in mm; iv) stool softness on a scale of 0 to 3 with 0 for regular hard stool and 3 for liquid stool. Bar graphs (right panels) show cytokine secretion in supernatants from colon explant culture (1 cm tissue from distal end) using CBA kit. MFI values for each pro-inflammatory cytokine were compared between peptide treated and untreated groups. *p<0.05, **p<0.01, * **= pO.001 (unpaired t test).
Figure 10. Requirement of migratory DC in CD 8 Treg-mediated protection from DSS-induced colitis. Treatment with peptide p8.2L (50 μ§;/ηιοι 8ε) fails to prevent colitis in BatB-/- mice deficient in migrator}- CD 103 " DC but not in other DC populations. Age matched WT and Batf3-/~ male mice were treated with peptide prophyiactically and challenged with 2.5% DSS in drinking water. Gross photography (left) and cumulative clinical disease scores (right) are shown. Each dot in the bar graphs represents one mouse. ****p< 0.0001, Student's t test. Figures 11A-F. Agonistic anti-4-l BB Ab administration leads to expansion/activation of CDS Treg and protection from EAE in a Qa-1 dependent manner. A. Groups of B6 mice were administered i.v. with 25 μg of 4- IBB Ab and 72 hr later BrdU" CDS Treg were quantified in liver as shown. B. In parallel, frequency of hepatic CD ! I c 2 4 ' CDS Treg was determined as shown. * *p<0.01. C. Groups of B6 mice (n=5) were injected i.p. with either 25 μg or 200 ,ug/mouse of anti-4-lBB Ab or an isotype matched control Ah and the next day challenged with MOG35-55/CFA/PTX for the induction of EAE. Clinical scores were recorded daily as shown. D. Groups of Qa-1-/- mice (n=4-5) were injected with 25 g/mouse of anti-4-lBB Ab and immunized for the induction of EAE as in C. Clinical scores are shown. E. Reduced number of CDS Treg and CDl lc+2B4+ CDS Treg in 4-1BB-/- mice genetically deficient in 4- IBB molecule. F. Induction of CDS Treg following administration of anti-4-lBB Ab and subsequent protection from EAE.
Figures 12A-E. Induction of CDS Treg following administration of anti-
CD3 mAb and subsequent protection from EAE. A. Representative dot plots and cumulative data of CDS Treg in spleen and liver of B6 mice after anti-CD3 mAb treatment. Female B6 mice were immunized with anti-CD 3 mAb i.p. on day I and day 3. On day 7, mice were sacrificed and spleen and liver mononuclear cells were isolated and stained for FACS. B. The frequency of CDS Treg in liver mononuclear cells was monitored daily by FACS after immunization with 200 μg of anti-CD3 mAb. C. Groups of female B6 mice were injected i.p. with 200 μ-g of anti-CD3 mAb and five days later challenged with MOG35-55/CFA/PTX for the induction of EAE. Clinical scores were recorded daily as shown .
*p<0.05, Student t test. D. Female B6 mice were immunized with anti-CD3 mAb i.p. on day 1 and day 3. On day 7, mice were sacrificed and spleen cells were isolated and stained with various fluorochrome labeled antibodies. CDS Treg and CDap T cell proliferation was measured by BrdU incorporation. E. Anti- CD3 mAb is the best anti-T cell surface antibody for induction of CDS Treg. Anti-CD3 (clone 2C11) mAb activates/expands CDS Treg in liver. Naive female B6 mice were administered i.p. with various antibodies (200 μg) or just with PBS (control). Five days later, liver mononuclear cells were isolated and stained with CDSa, CDSp and TCRp fluorochrome labeled antibodies. H57 (anti- TCRP), GK1.5 (anti-CD4), 2.43 (anti-CD8), YTS177 and YTS 05 are also different anti-CD4 mAbs.
Figure 13. Low frequency of CDS Tregs in liver mononuclear cells from Yaa lupus mice. Representative dot plot showing hepatic CDS Treg in two 10 weeks old BXSB-Yaa mice (right) and one age-matched B6 mouse (right). Numbers indicate % of CDS Treg.
Figure 14. Upregulation of several proinflammatory genes in liver of Qa- l -/- mice deficient in CDS Treg. Volcano plot displaying each gene's -log 10 (p- value) and log2 fold change with the selected covariate. Highly statistically significant genes fall at the top of die plot above the horizontal lines, and highly differentially expressed genes fall to either side. Horizontal lines indicate various False Disco very Rate (FDR) thresholds or p-value thresholds if there is no adjustment to the p-values. Genes are colored if the resulting p-value is below the given FDR. or p-value threshold. 'The 40 most statistically significant genes are labeled m the plot.
Figure 15. We have successfully generated several founders (mice) in which the allele of Zbtb l6 (PLZF) containing loxp sites flank the exon that encodes the BTB domain and the zinc fingers. The place where the loxp oligos were inserted and the sequencing scans showing the successful insertion of the loxp sites are shown. These founders are currently being bred with CD4-Cre to generate mice lacking PLZF+ T cells, including CDS Treg.
Figures 16A-E. CDS Treg are also present in human peripheral blood and express similar cell surface markers. A. Representative flow cytometiy plots showing gating strategy for the identification of circulating CDS Treg in human PBMC. The total CDS1" gate was identified using anti-CD8a and anti-CDSp mAbs within the TCRaP+ gate: after intracellular staining for PLZF,
CD8TPLZF÷ T cells were gated within the CD8÷ population; using anti-CD8ce and anti-CDSp mAbs, CD8a+p~ T cells were identified within the CD8+PLZF+ gate; finally, based on expression of CD 161 and the invariant TCR Va7.2, CDS Treg were defined as PLZF+TCR.aP+CD8aa+ T cells negative for TCR
Va7.2/Ja33 expression and with intermediate or low expression of CD161 (CD 16 ! ' ) while MAT!" cells were identified as CD161 ++TCR Va7.2/Ja33 ". B. B. Cumulative data for the frequency of CDS Treg in PBMC from, healthy donors (n=25) following gate strategy. Each dot represents an individual donor. Results shown as mean ± SEM. C. Histogram profile and cumulative data of Geo MFI of CD244, CD 11c and Granzyme B expression by circulating CDS Treg compared with both CDSconv and MA1T cells. Each dot represents individual donors. Data were obtained from 13 to 16 healthy donors. Results shown as mean ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA with Bonferroni's multiple comparisons test. D. (Top) Representative flow cytometry plots showing co-expression of CD244 and CD1 ic by circulating CD8 Treg and MATT cells. Gate indicates CD244+CD 1 lc+ cells and numbers indicate percentage of CDS Treg or MAIT cells that are CD244OD 1 lc+. Scatter graph shows percentage of CDS Treg or MAIT cells that are CD244+CD 1 lc÷ in PBMC from healthy donors (n=9). **p < 0.0 , Wilcoxon matched-pairs signed rank test. (Bottom) Representative flow cytometry plots showing co-expression of Granzyme B and perforin gated on CD244+CD 1 lc+ circulating CDS Treg or MAIT cells. Numbers in dot plots show the percentage of events in each quadrant. Scatter graph shows percentage of CD244 D 1 lc+ CDS Treg or MAIT cells that are Granzyme B Peribs in ' in PBMC from healthy donors (n=2). **p< 0.01, unpaired t test. E. Frequency distribution of TRBV gene segment usage by all unique clonotypes from, sorted circulating CD 8 Treg (n = 794, left) and CDSconv (n = 12,244, right) isolated from PBMC of one healthy donor and analyzed by high-throughput sequencing of the CDR3P regions. Each TRBV gene segment is represented by a slice proportional to its average frequency. The CDR3 was defined as starting at the last cysteine encoded by the 3' portion of the Υβ gene segment and ending at the phenylalanine in the conserved TRB J segment motif FGXG.
Figures 17A-B . A. Cumulative data of Geo MFI of IFNy, IL- i 7A, TNFa and IL-4 (top) as well as IL-18R i, RORyt, CXCR.6 and CCR6 (bottom) expression by circulating CD8 Treg compared with both CDSconv and MAIT cells. Each dot represents individual donors. Data were obtained from 6-12 healthy donors. Results shown as mean ± SEM. *p < 0.5, * *p < 0.01, ***p < 0.001, one-way ANO VA with Bonferroni's multiple comparisons test. B.
Frequency distribution of TRBJ gene usage (top) and CDR3 lengths (bottom) by all unique clonotypes from sorted CD8 Treg (white bar) and CDScon (black bar) from human PBMC. Both CDS Treg and CDSconv were sorted from peripheral blood of one healthy donor and subjected to high-throughput sequencing of the€'ΒΚ3β regions. The CDR3 was defined as starting at the last cysteine encoded by the 3' portion of the νβ gene segment and ending at the phenylalanine in the conserved TRB J segment motif FGXG.
Figures 18A-E. Circulating CDS Treg are increased in PBMCs from chronic Rheumatoid Arthritis (RA) patients. Cumulative data of (A) percentage of CDS T cells in total TCRap^ T cells, (B) percentage of CDS Treg cells in the TCRaP+PLZF+CD8aa+Va7.2"CD 16 gate and (C) percentage of
CD244O31 lc+ CDS Treg in PBMC of healthy individuals and RA patients, (D) CD8 Treg from RA patients significantly secrete more Granzyme B than healthy controls in i O- l 1) (**p< 0.01, Mann Whitney test). Scatter plots show Geo MFI values for Granzyme B, IL-17A, Perforin, CD244 and CDl lc expression. (E) High frequency of circulating Granzyme B+ and CD244 " CDS Treg in RA patients. Scatter plots show percentage of CDS Treg that were positive for Granzyme B, 1L-17A, Perforin and CD 11c. PBMC were analyzed by multi- parameter flow cytometry. Each dot represents an individual donor. Results shown as mean ± SEM.
Figures 19A-D. The frequency of CDS Treg is increased in PBMCs from systemic lupus erythematosus (SLE) patients. Cumulative data of (A) percentage of CDS T cells in total TCRap" T cells and (B) percentage of CD8 Treg cells in the TCRap÷PLZF D8aa^ Va7.2~CD161+/~ gate. (C) Circulating CDS Treg from SLE patients (n=4) significantly secrete more IFNy, Granzyme B and TNFa than healthy controls. Scatter plots show Geo MFI values in CDS Treg for IFNy, Granzyme B, TNFa, IL-17A and IL-4. (D) CDS Treg from SLE patients have significantly high expression of RORyt. No differences were found between groups regarding to IL-lSRa, CCR6 and CXCR6 expression. Scatter plots show Geo MFI values in CDS Treg for IL-18Ra, CCR6, CXCR6 and RORyt. PBMC were analyzed by multi-parameter flow cytometry Each dot represents an individual donor. Results shown as mean ± SEM. (E) High frequency of circulating TNFa" CDS Treg in SLE patients. Scatter plots depict percentage of CDS Treg are positive for the corresponding cytokine. PBMC were analyzed by multi-parameter flow cytometry. All data are represented as mean ± SEM. *p<0.05, Mann -Whitney test.
Figures 20A-C. No significant alterations in the frequency of CDS Treg in PBMCs from head and neck squamous cell carcinoma patients. Cumulative data of (A) percentage of CD 8 T cells in total TCRc$+ T cells, (B) percentage of CDS Treg cells in TCRap+PLZF+CD8aaVa7.2-CD161÷ - gate and (C) percentage of CD244+CD1 lcT CDS Treg from PBMC of healthy individuals and head and neck squamous ceil carcinoma (HNSCC) patients (n=5). Each dot represents an individual donor. Results shown as mean ± SEM.
Figures 21A-D. A significant increase in the frequency of circulating CDS Treg in PBMCs from chronic melanoma patients. Cumulative data of (A) percentage of CDS T cells in total TCRaP<+ T cells, (B) percentage of CDS Treg cells in TC aP^PLZF^CDSaaVaJ^CDiei^gate and (C) percentage of (1)244 ( D i lc+ CD8 Treg from PBMC of healthy individuals and melanoma patients. The frequency of CD8 Treg in PBMC from melanoma patients (n;=13) was significantly increased compared to healthy controls. PBMC were analyzed by multi -parameter flow cytometry. Each dot represents an individual donor. Results shown as mean ± SEM. (D) Identification of infiltrating CDS Treg into human melanoma tumor by flow cytometry.
Figure 22. A working model of the negative feedback regulatory mechanism mediated by a novel innate-like PLZF÷CD8aa+TCRc$+ Treg population (CDS Treg) enriched in liver and colon of naive mice, (a) During chronic inflammation, CDS Treg recognize Qa-1 -expressing activated CD4+ T cells; (b) the CDS Treg TCR bind to the non-classical Qa-1 -bound peptides on the surface of activated CD4+ T cells; (c) following recognition, CDS Treg mediate release of granzyme B and perforin and induce apoptosis in the target activated CD4+ T cells. Since naive CD4+ T cells do not express Qa-1 molecules on the surface, they are not susceptible to CDS Treg-mediated apoptosis allowing the immune response to proceed.
DETAILED DESCRIPTION
Definitions
One of ordinary skill in the art will appreciate that an antibody consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (Vi-i) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C -terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody.
An antibody, or antigen-binding fragment thereof, can be obtained by any means, including via in vitro sources (e.g., a hybridoma or a cell line producing an antibody recombinam y) and in vivo sources (e.g., rodents).
Metliods for generating antibodies are laiown in the art and are described in, for example, Kohler and Milstein, Eur. J. Immunol.. 5:511 (1976); Harlow and Lane (eds.). Antibodies: A Laboratory Manual. CSH Press (1988); and C.A . Janeway et al. (eds.), Immunobiology. 5th Ed., Garland Publishing, New York, NY (2001)). In certain embodiments, a human antibody or a chimeric antibody can be generated using a transgenic animal (e.g., a mouse) wherein one or more endogenous immunoglobulin genes are replaced with one or more human immunoglobulin genes.
The term, "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of antigen-binding fragments include but are not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody.
Routes and Formulations
Administration of compositions can be via any of suitable route of administration, particularly parente rally, for example, intravenously, intra- arterially, intraperitoneally, intrathecally, intraventricularJy, intraureth rally, intrasternally, intracranially, intramuscularly, or subcutaneously. Such administration may be as a single bolus injection, multiple injections, or as a short- or long-duration infusion. Implantable devices (e.g., implantable infusion pumps) may also be employed for the periodic parenteral delivery over time of equivalent or varying dosages of the particular formulation. For such parenteral administration, the compounds (a conjugate or other active agent) may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances such as salts, sugars (particularly glucose or mannitoi), to make the solution isotonic with blood, buffering agents such as acetic, critric, and/or phosphoric acids and their sodium salts, and preservatives.
The compositions alone or in combination with other active agents can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
Thus, the compositions alone or in combination with another active agent may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, maybe compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the composition optionally in combination with an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of conjugate and optionally other active compound in such useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should he pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the phospholipid conjugate optionally in combination with another active compound may be incorporated into sustained-release preparations and devices.
The composition optionally in combination with another active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or li uid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms during storage can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, buffers or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating compound(s) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, one method of preparation includes vacuum dr ing and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the antigen(s) and adjuvant(s) optionally in combination with another active compound may be applied in pure form, e.g., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
In addition, in one embodiment, the invention provides various dosage formulations optionally in combination with another active compound for inhalation delivery. For example, formulations may be designed for aerosol use in devices such as metered-dose inhalers, dry powder inhalers and nebulizers.
Useful dosages can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
Generally, the concentration of the active compound in a liquid composition, such as a lotion, will be from about 0.1-25 wt-%, e.g., from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, e.g., about 0.5-2.5 wt-%. The active ingredient may be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 μΜ, e.g., about 1 to 50 μΜ, such as about 2 to about 30 μΜ. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.0 -5.0 mg/kg/lir or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).
The amount of the active compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. In general, however, a suitable dose will be in the range of from, about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, for instance in the range of 6 to 90 mg/kg/day, e.g., in the range of 15 to 60 mg/kg/day.
The active compound may be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. The dose, and perhaps the dose frequency, will also vary according to the age, body weight, condition, and response of the individual patient. In general, the total daily dose range for an active agent for the conditions described herein, may be from about 50 mg to about 5000 mg, in single or divided doses. In one embodiment, a daily dose range should be about 100 mg to about 4000 mg, e.g., about 1000-3000 mg, in single or divided doses, e.g., 750 mg every 6 hr of orally administered compound. This can achieve plasma levels of about 500-750 uM, which can be effective to kill cancer cells. In managing the patient, the therapy should be initiated at a lower dose and increased depending on the patient's global response.
Exemplary Embodiments
In one embodiment, a method to identify or detect immune cells having
Τα αβ+, CD8CKX+, Nkl. l t, PLZF+, CD161+, and optionally having one or more of CD1 lc+, CD 137+ CD244+, or one or more of NK-inhibitory receptors is provided. The method includes contacting a sample having mammalian immune cells with a ligand that binds CD8aa, a ligand that binds NK 1.1, a ligand that binds PLZF, and a ligand that binds CD 161, and optionally a ligand that binds CD 11c, a ligand that binds CD137, a ligand that binds CD244, a ligand that binds ΤΟΕΙαβ, or a ligand that binds NK-inhibitory receptors; and identifying or detecting an amount of a population of cells CD8aa+, Nkl . B-, PLZF+, and CD 161+, and optionally having one or more of CD1 lc+, CD137+ CD244+ TCRaP+, or one or more of NK-inhibitory receptors. In one embodiment, the cells are identified using antibodies specific for
TCRa[3,CD8 ct, Nkl . l, PLZF, or CD161, and optionally antibodies specific for one or more of CD1 lc, CD 137, CD244, or one or more of NK-inhibitory receptors. In one embodiment, the cells are human cells. In one embodiment, the method further comprises isolating the identified cells. In one embodiment, the method further compr ses expanding the isolated cells. In one embodiment, the cells are from a patient with an autoimmune disease. In one embodiment, the isolated cells ae cultured with IL-2, IL-15, Qa-l HLA-E binding peptides, anti-CD 3 antibodies or anti-CD 137 antibodies, or any combination thereof.
In one embodiment, a method to decrease the number of immune cells having ΤΟΙαβ, CDSotot, Nkl . l , PLZF, and CD 161, and optionally having one or more of CD1 lc, CD 137, CD244, or one or more of NK-inhibitory receptors in a mammal, is provided. The method includes adm inistering to the mammal an effective amount of one or more antibodies specific for CD8aa, specific for Nk 1.1 , specific for PLZF, or specific for CD 161 , or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD1 lc, specific for CD137, specific for CD244, specific for TCRa , or one or more of NK-inhibitory receptors, or a combination thereof. Also provided is a.method to prevent inhibit or treat cancer in a mammal, comprising: administering to the mammal an effective amount of one or more antibodies specific for CD8aa, specific for Nk 1.1, specific for PLZF, or specific for CD161, or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD1 lc, specific for CD 137, specific for CD244, specific for ΊΌ αβ, or specific for one or more of NK- inhibitory receptors, or a combination thereof.In one embodiment, the cancer is neck or head cancer. In one embodiment, the cancer is melanoma.
Further provided is a method to prevent, inhibit or treat autoimmune disease in a mammal, comprising: administering to the mammal one or more Qa- 1/HLA-E binding peptides, anti-CD 1 lc antibodies, anti-CD3 antibodies or anti- CD137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a+T regs. In one embodiment, the one or more antibodithat are specific for CD1 lc and CD137, CD 1 lc and CD3, or CD3 and CD137. In one embodiment, the disease is IBD, colitis, lupus or RA. In one embodiment, the disease is an autoimmune liver disease. In one embodiment, the disease is autoimmune hepatitis or primary biliary cirrhosis. In one embodiment, the T or B cells in the mammal with the disease are increased relative to a mammal without the disease.
In one embodiment, a method to prevent, inhibit or treat orgn or graft rejection in a mammal is provided. In one embodiment, comprising:
administering to the method includes administering to a mm ami in need theref one or more Qa-l/HLA-E binding peptides, anti-CD 1 lc antibodies, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a.+T regs.
In one embodiment, the mammal is a human. In one embodiment, the peptide(s), antibodies, or
composition is systemically administered.
The invention will be described by the following non-limiting example.
Example 1
The liver has a central role not only in the metabolism and clearance of the diet and intestinal microbe-derived toxins but also mount immunity against infectious agents and cancer. Chronic infections in the liver indicate that several tolerance mechanisms are evolved to protect the hepatic tissue from excessive immune stimulation (1-3). Liver has several residential T cells, B cells and macrophages or Kupffer cells and in steady state immune cells are
predominantly in the sinusoidal blood vessels and do not infiltrate into parenchyma. Among T lymphocytes, liver uniquely harbors both conventional T cells and several unconventional T cells (4). The unconventional T cells are comprised of NKT cells, MATT cells, and gd T cells in the liver. While adaptive T cells are class la MHC-restricted, innate-like unconventional T cells are reactive to non-classical MHC-Ib molecules and recognize a class of antigens. Thus, NKT cells and MAIT cells recognize both self and microbial antigens in the context of CD id and in the context of the MR-1 molecules, respectively and could provide immunity against microbes (5-7). A detailed understanding of cellular and molecular interactions in liver among different innate and conventional T cells involved in the maintenance of immune tolerance is lacking.
T cells are controlled by both intrinsic (e.g., PD1, anergy and exhaustion) and extrinsic cell-based (regulatory T cells or Treg) mechanisms that prevent them, from causing excessive tissue damage. An important role for FoxP3+CD4+ Treg has been well-defined, a phenotypic characterization of CD8+ cells with regulatory activity is poorly studied. Although immune regulatory nature of CD8~ T cells in homeostasis of cellular and humoral immune responses has been suggested (8-10) (1 1 -13), ability to distinguish them (CD8 Treg) from non- regulatory conventional CD8÷ T cells (CDSconv) has not sufficiently advanced as happened for CD4 Treg following the discovery of Foxp3 (14). Similar to CD4+Treg, an important role for IL-2 signaling for CD8+ Treg has been shown in mice deficient in IL-2 and IL-2Rb chain (15-17) (18). Regulatory CD8÷ T cells also have been implicated in various conditions in humans, e.g. transplant survival (19), inflammatory bowel disease (20) and in multiple sclerosis (21-25). In a clinical trial with anti-CD3 mAb (Teplizumab), increased frequency of memory CD8+ T cells with regulatory gene expression was found to be associated with a positive clinical response in type i diabetes patients (26). Interestingly, IL~2/IL-15Rp~deficiency in humans also leads to a severe combined immunodeficiency syndrome as observed for IL-2Rcx (27). In this study, for the first time, we have identified that the expression of the promyelocytic leukemia zinc finger (PLZF) transcription factor in a novel population of PLZF"TCRap+CD8aa+ Treg (hereafter referred as CDS"1" Treg) enriched in liver of naive mice and in human peripheral blood distinguishes them from conventional CD8+ T cells. CD8+ Treg have a unique ceil surface phenotype in that they are CD I lc-\- 2B4-\-NKG2D+ and, despite having innate- like features, they are distinct from other innate-like T cells, including NKT cells or mucosal associated invariant T (MAIT) cells, CD8+ Treg do not express Foxp3 but they express glucocorticoid-induced tumor necrosis factor-related receptor (GITR), a marker of active Tregs cells (28). A large number of them are dependent upon Qa-lb molecules as a significant reduced frequency is foind in Qa-P-/- mice. They are TAP-independent but dependent upon IL15 signaling for their development. CD8+ Treg do not secrete IL-10 or TGF[3 but secrete typical cytokines produced by cytotoxic T cells (TNFa, IL-17A) as well as PLZF -driven secretion of both IL-4 and IFNy similar to that in NKT cells. It is noteworthy that these cells very rapidly secrete large amounts of IL-2 as well . CD8 Treg shares several features with oilier unconventional T cells, including expression of memoiy/activation markers, a common transcription program imprinted by the expression of PLZF and their enrichment in non-lymphoid organs.
Results
Liver enriched CD8 Treg are PLZF+ and dependent upon PLZF and related transcription factors
We have identified a subset of TCRafi÷CD8÷ T cells with immune regulatory properties enriched in liver of naive C57BL/6 (B6) mice that express the homodimer CD8 a and hereafter referred to as CD8 Treg and different from the conventional TCRaP+CD8+ T cells (CD8conv) that do not express PLZF and are CD8ab+. CD 8 Treg represent -3.25% of TCRap+ T cells (within the B220-CD4- CD8b- gate) in liver mononuclear cells (MNCs) in naive B6 mice. They are also present in other lymphoid compartments, including bone marrow, spleen, peripheral blood and lung. Although, CDS Treg are not readily detectable in adult thymus, they are present in the neonatal period (~ 0.5%) until day 5 after birth (data not shown), Athymic nude mice are deficient in CD8 Treg, indicating their thymic origin. In contrast to hepatic CD8∞nv, the majority of CD8 Treg are CD69+ and N K ί I . suggesting an activated profile similar to other innate-like T cells, including NKT cells. Additionally, CDS Treg are mostly CD44h,gh and CD62Liow compared to CD8Conv, suggesting a memory phenotype.
Since the promyelocytic leukemia zinc finger (PLZF) transcription factor controls the development of several innate-like T cells, including NKT cells, γδ T cells, MATT cells, and memory-like CD8+ T cells (Savage AK, Immunity 2008; Wemreich MA, Nat Immunol 2010; Gordon SM, JI 201 1 ; Eidson M, PLoS ONE 201 1 ; Fergusson JR, Cell Reports 2014; Marrero I, Front Immunol 2015) we investigated whether CDS Treg also express PLZF and whether it is involved in their development. We used anti-PLZF mAb for intracellular staining and GFP expression in liver MNCs from B6 and PLZF-eGFP reporter (PEG) mice (Zhang S, Sci Rep 2 15), respectively, and found that CD8 Treg are PLZF1" but express lower levels PLZF in comparison to iNKT cells. In contrast, CD8conv or CD4 T cells did not express PLZF. Accordingly, PLZF mRNA expression was found exclusively in sorted CD 8 Treg but not in CD8∞nv.
Next, we investigated whether low level of PLZF expression in hepatic CDS Treg is similar to that recently described adipose tissue-resident iNKT cells (Lynch L, Nat Immunol 2015). We used the fate-mapping experiment with PLZF-Cre x R26T mice, in which ceils that express PLZF are permanently labeled for tdTomato (Zhang S, Sci Rep 2015) and found that most CDS Treg in the liver of PCre x R26T mice were positive for tdTomato while CDSconv showed only background level of expression.
Next, we examined whether other related transcription factors, which are associated with innate and/or activated/memory T cells, are involved in the development of CDS Treg. A semi-quantitative analysis of sorted CDS Treg vs. CDSConv showed relative upregulation of the retinoic acid-receptor-related orphan receptor alpha (RORa) (Dzhagaiov I, JI 2004; Leppkes M,
Gastroenterology 2009), DNA-bmding protein inhibitor ID-3 (M3) (Ji Y, Nat Immunol 2011) and immunoglobulin enhancer-binding factors E12/E47 (E2A) (Mjosberg J, Eur J Immunol 2012) in CDS Treg whereas the DNA-binding protein inhibitor ID -2 (Id2), the T-box transcription factor Eomes and HeLa E- box binding protein (HEB) were down-regulated in comparison to CD8Conv. However, T-bet and Kruppel-like factor 2 (KLF2) were not significantly different between CD8 Treg and CDSconv. A critical role oiPLZF, RORa and Id 3 was confirmed in nuce genetically deficient in these transcription factors. PLZF- /-, RORa-/- and Id3-/- mice showed a significant reduction in the number of CD8 Treg. These results indicate that CDS Treg are PLZF+ and are dependent upon PLZF and related transcription factors.
Altogether, we have identified a novel subset of hepatic
PLZF+CD8aa+TCRa " Treg that express innate-like activated-memory markers in naive mice. Hepatic CD8 Treg have immune regulatory properties and are physiologically expanded during an experimental autoimmunity
Next, we determined whether CDS Treg have regulator}' potential to control autoimmunity. An equal number of sorted hepatic CDS Treg from naive B6 mice were transferred into B6 recipients that were immunized next day with myelin-oligodendrocyte glycoprotein (MQG)35-JJ peptide in completed Freund's adjuvant (CFA) plus pertussis toxin (PTx) (MOGss-ss/CFA/PTx) for the induction of experimental autoimmune encephalomyelitis (EAE). Mice that received CDS Treg showed a significant suppression
Figure imgf000028_0001
those receiving l_,Doconv or PBS control . To exclude the role of both
PLZF+CD ld-restricted NKT cells as well as innate-like memory CDS" T cells that depend on IL-4 secreted by NKT cells, hepatic CDS Treg or CDSconv were sorted from CD Id-/- mice deficient in both populations and used in adoptive transfer experiment as above. Mice that received CDS Treg but not CDSconv from CD Id-/- mice showed significant protection from EAE. These data indicate regulatory property of CDS Treg independent of NKT ceils or innate-like memory CD 8 T cells.
Next, we investigated whether CDS Treg are physiologically altered in liver during the course of EAE. Thus, B6 mice were immunized with either MOG35-55 for the EAE induction or an irrelevant peptide derived from hen egg iysozyme (HEL) and hepatic CDS Treg were analyzed on day 10 at the onset of disease (EAE dlO) or at day 25 during the recovery phase of the disease (EAE d25). CD8 Treg were significantly' expanded during the recovery phase (day 25) but not at the onset of EAE. In contrast, CDS Treg numbers were not altered in non-diseased mice immunized with HEL peptide. Next, we determined whether the CD8 Treg expansion inversely correlates with the severity of EAE.
Interestingly, a significantly higher expansion of CD8 Treg occurred in mice with Sow clinical scores (0-1) compared with those with severe clinical scores (3- 4). These data indicate CD8 Treg are physiologically expanded during recovery phase of EAE and their higher numbers correlating with less svere disease suggest their physiological role in spontaneous recovery as suggested earlier for the CD8 T cells .
To further investigate whether the immune regulation mediated by CD 8 Treg requires other lymphocytes and whetlier a cytotoxic mechanism is involved in this regulation, we used the CD4"CD25~CD45RBiilg!i T cells mediated colitis into Rag 17- mice and co-transfered CDS Treg from B6 or perforin-/- mice, ARa l-/- recipients that received CD 8 Treg from perforin-/- mice developed colitis similar to the control mice whereas mice that received CD8 Treg from perforin+/+ mice were significantly protected as indicated by increase in body weight ratio and histological analysis of colon using H&E staining. These results indicate that CDS Treg use a perforin-dependent mechanism to control autoimmunity mediated by activated CD4+ T cells.
To further examine the mechanism of regulation, we used both in vivo and in vitro assays to determine the fate of CFSE-labeled ΟΤ-Π CD4+ T cells specific for a chicken ovalbumin (OVA)323-339 peptide. Thus, CFSE-labeled OT- II CD4+ T cells were transferred to naive B6 mice alone or co-transferred with positive-selected CD8+ T cells or liver MNCs from B6 or perforin-/- mice. Next day, recipients were i.p. challenged with OVA323-339 peptide or PBS and, 4 days later, proliferation of CFSE-labeled OT-I1 CD4+ T cells was analyzed by flow cytometry. Enriched CDS1" T cells and liver MNCs from B6 (perforin+/+) but not from perforin-/- mice significantly suppressed OVA proliferation of CD4+ T cells as indicated by reduced recovery of CFSE-labeled OT-II CD4÷ T cells. Next, we examined whetlier CDS Treg are able to inhibit proliferation of activated CD4+ T cells in vitro. Sorted CFSE-labeled CD4+ T cells alone or co- cultured with sorted hepatic CDS Treg or CD8eonv were in vitro stimulated with plate-bound anti-CD3 mAb. CDS Treg but not CDS conv were able to inhibit significantly the expansion of CD4 " T cells. Collectively, these results indicate that CDS Treg have regulatory activity and able to control activation/expansion of disease-causing CD4 T cells. Moreover, the addition to the culture of anti-Qa- lb rnAb but not anti-MHC class I rnAb significantly blocked the suppressive function of CDS Treg (data not shown), suggesting a dependence on Qa-lb molecules. Hepatic CDS Tree secrete typical PLZF-driven cytokines and are dependent on TL-15/IL-2R.P signaling for their development
To further understand the mechanism of regulation by CDS Treg, we determined the cytokine secretion profile of sorted hepatic CDS Treg and compared to the sorted CDSconv from naive B6 following in vitro stimulation with the plate-bound anti-CD3 rnAb for 96 hr and cytokine secretion measured in culture
supematants. As shown in Figure 42A, CDS Treg secreted typical proinflammatory cytokines produced by cytotoxic T cells (TNFa, IL-17A and IL-6) as well as PLZF-driven secretion of both IFNy and 1L-4. Notably, CDS Treg secreted significantly higher levels of IL-2 within 24 hr while CDSconv secreted only a minimal amount. Importantly, CDS Treg do not secrete the suppressive cytokine IL-10 and the expression of ΤΌΡβ was significantly down-regulated in these cells. Consistently, CDSconv showed a typical cytotoxic T cell profile, including secretion of IFNy and TNFa.
We next determined the cytokine signaling requirement for the survival and/or development of CDS Treg. Hepatic CDS Treg were significantly reduced in both CD 122-/- and IL-15-/- mice in comparison to B6 mice. Considering that CD 122, the β chain receptor for IL-2 and IL-15, is essential for CD8+ T cell response to IL-15, these results indicate that IL-15 signaling is necessary for CD8 Treg development. In contrast, CD 8 Treg are not affected in CD25-/-, IL-7- /-, IL-6-/-, and IFNy-/- mice. Surprisingly, CDS Treg are T-bet independent and are not reduced in T-bet-/- mice.
We next determined the requirement of other immune molecules for CDS Treg development. As expected, hepatic CDS Treg were absent in CDSa-/- mice but their frequency was significantly increased in CD8p~/- mice, confirming the expression of the CDSa chain in CDS Treg. No significant changes in the frequency of hepatic CDS Treg was observed in CD Id-/-, JaiS-/- and μΜΤ-/- mice, indicating that NKT cells and B cells are not required for CDS Treg development. Importantly, a significantly reduced frequency of CDS Treg in Qa- lb-/- mice indicate that a proportion of CD8 Treg are restricted by Qa-lb molecules. Since Qa-1 -restricted T cells can be either TAP-dependent or TAP- independent, no differences in the frequency CDS Treg between TA 1-/- and B6 mice indicate that TAP-dependent antigen processing is not required for the development of CD 8 Treg.
The regulatory function hepatic CDS Treg and their dependence on Qa-1 expression was further confi rmed in a model of acute immune-mediated hepatitis using concanavalin A (ConA) (Maricic I et al, JI 2014; Heymann F et al. Lab Anim 2015)., ConA-induced hepatitis was significantly exacerbated in Qa-1-/- mice in comparison to B6 mice, as indicated by liver damage using ALT levels and H&.E staining.
Hepatic CD8 Tree are polyclonal and use diverse TCR a and β chains
Since PLZF-dependent innate-like T cells, including NKT and MAIT cells, often used limited TCR, we examined the TCR repertoire of CD8 Treg using available anti-TCR-Va or Υβ antibodies, RT-PCR and high-throughput sequencing., Flow cytometry-based TCR Υβ repertoire analysis of CD 8 Treg indicate a diverse Υβ chain usage with a bias toward the use of Υβ8.1/8.2, Υβ5.1/5.2, Υβ8.3 and Υβΐ ΐ chains. We further investigate the TCR repertoire of CDS Treg by high- throughput sequencing of the TCR β chain CDR3 region of sorted CD 8 Treg as described (Marrero I, PLoS ONE 2013; Marrero I, Mol Immunol 2016). This confirmed that CDS Treg expressed a diverse TCRp repertoire with great variations of TCR sequences and preferential usage of TRBV 13-3/13-2
(Υβ8.1/8.2), TRBV20 (Υβ15), TRBV12-1/12-2 (νβ5.1/5.2), TRBV5 (νβΐ) and TRBV4 (ΥβΙΟ). Additionally, TRBJ usage, another indicator of the
heterogeneity, was also diverse and almost similar among CDS Treg and CDSconv, except for an increase in 1β2.5 usage by CD8 Treg. CD8 Treg also showed a skewed bell-shaped distribution of CDR3 lengths with a peak at 39 nucleotides while CDScom- show a more typical Gaussian-like distribution with a peak at 42 nucleotides. Furthermore, TCR Va analysis of CDS Treg by FACS and RT-PCR revealed diverse Va usage similar to that in CD8Conv. Together, these data indicate that the TCR repertoire of the hepatic CDS Treg in nai e mice is polyclonal. Hepatic CD8 Treg are CD1 lc+ and CD244+ and express several markers typical of innate-like T cells
To further differentiate CD8 Treg from CD8Con», we determined the expression of different cell surface markers using flowcytometry and quantitative RT-PCR., A large proportion of CDS Treg but not CD8∞nv CD1 lc. In addition, the majority of CDS Treg express CD244 (2B4). Co-staining of CDS Treg indicates that all CD I lc+ CDS Treg are also CD244+ while none of the CD8COnv express CD1 lc or CD244. Next, we determined whether CDS Treg co-expressing CD244 and CD 11c (CD244+CD1 lc+) are dependent on PLZF., Both percentage and numbers of CD244 "CDl lc+ CDS Treg were significantly reduced in PLZF- /- mice. Consistent with the correlation of regulatory activity with CD1 l c expression in Treg, CD1 lc-bead depletion resulted in loss of CDS Treg ability to control EAE upon adoptive transfer (data not shown). Thus, CDS Treg have distinguishing features, they are NK1.1+PLZF+, CD2441" and CD I lc+.
Next we determined the expression of FoxP3 in CDS Treg. Similar to
CDSconv, CD8 Treg were negative for FoxP3 but do express glucocorticoid- induced tumor necrosis factor-related receptor (GITR), also present on CD4 Treg (Ronchetti S, J Immunol Res 2015). Consistent with the role of IL-15 signaling, CDS Treg were CD122(lL-2Rp)hi8h CD25(IL-2Ra)low, similar to innate-like CD8+ T cells (Walzer T, JI 2002), and most of them were CD27 sh, marker associated with memory CDS'1" T cells. Interestingly, CDS Treg were CD28+, PD-l÷and also express another regulatory signaling molecule CD200. CDS Treg were negative for OX-40. Notably, CDS Treg also expressed low levels of 4- IBB (CD137), a co-stimulatory molecule belonging to the TNF- receptor superfamiiv 9 and expressed only on activated T cells. In addition, CDS Treg did not express ICOS, CXCR5, Eomes as well as CD103, the a-chain of the αΕβ7 integrin that characterize tissue-resident memory (TRM) T cells (Ariotti S, Adv Immunol 2012). CDS Treg are not typical memory cells as they do not express CD 127 (IL-7Ra), which is required for memory CDS T cells.
Since CDS Treg are PLZF ", we determined the expression of other markers associated with innate cells. As shown in Figure 5F, CDS Treg express only NK inhibitory receptors (Ly49E/F>Ly49G2>Ly49A>Ly49I>Lv49G), but not activating receptors (Ly49D and Ly49H). Consistent with an innate-like behavior, CDS Treg also express NKG2D. To further confirm their phenotype, we analyzed the expression of a set of selected genes by semiquantitative RT-PCR on sorted hepatic CDS Treg vs. CDSeonv. Consistent with the flow cytometry analysis, CDS Treg showed significant down-regulation of€08β and up-regulation of CD8a, CD244 (2B4), CD200, Ly49A, Granzyme A (Gzma), 4-1BB, CD25, CD 122 and CD28.
Interestingly, Fibrinogen-like 2 (Fgl2), which is a negative regulator of the immune response was up-regulated in CDS Treg. Interestingly, other genes associated with CD4 Treg, including TGF-βΙ and Lymphocyte-activation gene 3 (LAGS), were down-regulated in CDS Treg in comparison to Cl)H,„m .
PLZF+ CDS Treg are also present in human peripheral blood and have several features similar to the murine counterpart
To determine whether PLZF+ CDS Treg are also present in humans, we examined peripheral blood mononuclear cells (PBMC) from 23 healthy individuals by multiparameter flow cytometry using a gating strategy based on PLZF expression and exclusion of circulating mucosal-associated invariant T (M AIT) cells, which have been defined by usage of the semi -invariant canonical TCRa chain Va7.2/Ja33 and high expression of CD161 (CD 161^) (Porcelli S, JEM 1993). Following this strategy, in human PBMC, CDS Treg were defined as Pl FTCRaP+CD8a+CD8p- T cells that express low to intermediate levels of CD161 ί(1) ! 61 ) Human circulating CD 8 Treg represent -0.24% (± SEM 0.06) of the total TCRc$+' T cells and -0,87% (± 0.21) of the total CDS'" T cells.
Next we used flowcytometry to analyze human CD8 Treg directly ex vivo without stimulation using combination of niAbs specific for cell surface and intracellular molecules as well as cytokines. Similar to murine CDS Treg,
CD244 (2B4) and CD l ie expression was significantly higher in circulating CD8 Treg than in both CD8∞nv. Notably, circulating CDS Treg also secrete higher levels of Granzyme B than both CD8Com- and MAIT cells. Similar to murine CDS Treg, human CDS Treg also secrete IFNy, TNFcc, IL-4 and IL-17A. Furthermore, the expression of IL-18 receptor a subunit (IL-18Ral ), RORyt, CXCR6 and CCR6, known to be up-regulated in MAIT cells were significantly reduced in CDS Treg. Similar to murine CD 8 Treg, a subset of human CD8 Treg also co- express CD244 and CD1 lc (CD244"1"CD1 lc+) that was significantly higher in CDS Treg than in MA IT cells (15.7% ± 3.0 vs 1 .4% ± 0.7). Interestingly, -69 % of CD244"1"CD 1 lc+ CDS Treg secrete both Granzyme B and perforin .
Next we determined the TCRp repertoire of circulating CDS Treg by high-throughput sequencing and compared it to the repertoire of CDSconv, We obtained 794 productive unique sequences from sorted CDS Treg and 12,244 productive unique sequences from sorted CDSconv . Similar to murine CDS Treg, circulating CDS Treg exhibited a non -restricted usage of multiple TRBV gene segments that resemble the TCRft repertoire of CD8Conv. Several TRBV gene segments, including TRBV6 (VfU3), TRBV7(), TRBV5Q, TRBV27Q and
TRBV20 (V 2), were expressed at almost similar frequencies by both subsets. Both CDS Treg and CDSconv do not show any preferential usage of specific TRBJ gene segment and showed a Gaussian-like CDR3 length distribution profiles. Collectively, these results indicate that circulating human CDS Treg express a diverse and polyclonal TCRJ3 repertoire. The proportion of circulating CD 8 Treg within total PLZF DSacc Diei^ T cells was -12.7% (± 2.2) that represent ~2xIQ3 (± 0.6x103) CDS Treg. In contrast, MAIT cells represent
-2.4% (± 0.58) of the total TCRa[¾÷ T ceils and -8.2% (± 2.0) of the total CD8+ T ceils (data not shown) Example 2
We have identified an innate-like memory PLZF+
CD8alphaalpha "TCRalphabeta+ T'reg enri ched in liver and colon of naive mice, hereafter referred to CD 8 Treg, expressing the promyelocyte leukemia zinc finger (PLZF) transcription factor that distinguishes it from conventional CDS" T ceils. Our long-term objective is to define the unique phenot pe and the regulatory mechamsm(s) mediated by CDS Treg that target activated, but not naive T cells in a negative feedback regulation mechanism. We propose to characterize the phenotvpe and mechanism of regulation mediated by CDS Treg in murine models of dextran sodium sulfate (DSS)-induced colitis in B6 mice and CD45RbHlgbCD4+ T cells-induced colitis in Ragi-/- mice.
These investigations are supported by key preliminary data
demonstrating that CDS Treg: 1) are enriched within the colonic
CDSacfTCRap* IEL population and are memory-like (CD44h!¾hCD62Liow), activated (CD 122+CD25+CD69+), innate-like (ΝΚ1. Γ) cells that express NK- inhibitory (Ly49C/1/F/H) but not NK-activating receptors; 2) are distinct from MAIT or NKT cells; 3) do not secrete IL-10 and TGF-b but express features of cytotoxic CDS T ceils with enhanced perforin/granzyme B expression; 4) protect Ragl-/- mice from CD4÷ T cells-induced colitis; and 5) are Qa-lb restricted and can be induced with a Qa-lb-binding TCR leader peptide and protect DSS- mduced colitis in a Qa- lb-dependent manner. We propose the central hypothesis that PLZF+CD8 Treg, displaying a unique phenotype, play a critical role in the control of inflammation in gut in a negative feedback regulatory mechanism. The following specific aims are designed to test this hypothesis in ongoing investigations:
To characterize cell surface markers and gene expression profile of colonic CDS Treg: Our hypothesis is that colonic CD 8 Treg
(PLZF÷TCRaiphabeta+NKl .1+CD44 D 122+ expressing NK-mhibitory receptors) are PLZF -dependent and have a transcriptional signature similar to CDS Treg in the liver.
To determine regulatory properties, induction and mechanism of regulation of colitis by colonic CDS Treg. We will test the hypothesis that CDS Treg use cytolytic mechanism to control colitis in a Qa- lb-dependent manner and that CDS Treg can be activated/expanded following immunization with Qa- lb-binding peptides. Also CDS Treg can inactivate or kill key dendritic cell populations involved in colitis.
The proposed studies will be important for a greater understanding of the phenotype and function of this newly discovered CDS Treg population in colon and will have important implications for novel experimental therapeutics in 1BD.
Colonic CDS Treg have a similar phenotype and function: Consistent with earlier data, CD8aTCRap+ 1 cells are present in both the small intestine (SI) and colon. It also has been shown that SI CDSaaTCRotP T cells and not conventional CDS TCR T cells are regulatory and maintain g t homeostasis. We propose that colonic CDS Treg with a phenotype similar to liver (PLZF+, NK1.1+, expressing NK-inhibitory receptors and memory phenotype,
CD44 "CD 122+) are present within the CDSaaTCRcxp" intraepithelial lymphocytes (IEL) population.
Interestingly, although total CDSaaTCRap" T cells are not affected in Qa-lb-/- mice, the frequency of CDS Treg (Ly49C I/F/H+) is significantly reduced similar to that in the liver, indicating their Qa-lb-MHC restriction. Next, we have determined whether CDS Treg are able to protect from CD4+ T cell- induced colitis in Ragl-/- mice. Adoptive transfer of CDS Treg protect Ragl-/- miee from CD4+CD45Rbb¾u T cell-induced colitis. Thus, if CDS Treg are important in the maintenance of gut homeostasis, we predict that Qa-lb-/- mice, lacking CD8aa Treg, should become more susceptible or sensitive to dextran sulfate sodium (DSS)-induced colitis.
A Qa-lb-binding peptide induces CDS Treg and protect DSS colitis: Since CDS Treg are Qa- lb-restricted, we have synthesized several peptides with the potential Qa-lb-binding motif derived from, the conserved regions of the TCR νβ chains, heat shock proteins (HSP), and QDM. Next, we have determined their potential to induce Qa-1 -dependent CD8+ T ceil response in naive B6 mice. We found 6 peptides that are able to bind to the Qa-1 molecule (in in vitro Qa-lb folding assays) and induce a CD8+ T cell proliferative response (CFSE-dilution) in naive B6 mice. Among these peptides, we found that one peptide, p8.2L, derived from the leader sequence of the TCR V[38.2 chain, is able to induce CDS Treg and significantly protect mice from DSS- induced colitis as shown by the cumulative colitis scores and histology. In peptide -treated mice, the secretion of pro-inflammatory cytokines, like !L-6,
TNF-αΠ LVJIFNy and IL-17A, decreased significantly in comparison to the control mice. Next, we determined whether the peptide-induced protection is dependent upon the presence of Qa-1 molecules. While peptide treatment significantly protected WT B6 mice, Qalb-/- mice are not protected from colitis. Collectively, our data suggest that CDS Tregs are involved in a dominant immune regulatory mechanism able to control both DSS-induced colitis, which is primarily mediated by innate cells, and CD45RbhlghCD4+ T cell-induced colitis, which is mediated by adaptive Thl/Thl? cells. These preliminary findings are quite significant as CDS Treg are also present in humans. Using a multi parameter flow cytometry analysis, we have also defined a CDS Treg population in PBMCs from healthy donors that are PLZF+TCR "t'CD8 "t'CD161÷<iQt) with a memory phenotype expressing NK-inhibrtory receptors similar to the phenotype in mice.
Our hypothesis is that colonic CDS Treg
(PLZFTCRa +NKl .1+CD44 D122+ expressing NK-inhibitory receptors) are PLZF -dependent and have a transcriptional signature similar to CDS Treg in the liver.
To determine the cell surface phenot pe of colonic CDS Treg:
Rationale: We will identify whether colonic CDS Treg share common cell surface markers with the newly discovered PLZF+CD8aa Treg enriched in liver (Preliminary data & manuscript in preparation). Our preliminary data show the presence of CDS Treg in colon and enrichment in the IEL compartment in B6 mice. As observed for hepatic CDS Treg, we will investigate whether colonic CDSaa T cells express characteristic innate-like and memory markers, including CD122, CD1 lc, NK1.1, NK-inhibitory receptors, etc. Most importantly, we will determine whether colonic CDS Treg are PLZF4", which is found to be crucial in the development of hepatic CDS Treg and their innate-like features.
Experimental Strategy: We have standardized the isolation of IEL and LP fractions from mouse colon using the isolation kit from Miltenyi Biotech.
Briefly, following removal of mesenteric lymph nodes and stool, colon will be cut longitudinally and transversely into small pieces and washed twice in HBSS solution without calcium and magnesium containing 10 mM HEPES, 5 mM EDTA, 5% FBS, lmM DTT using the MACSmix tube rotator at 37°C. The washed fluid is the IEL fraction. Following another wash with only HBSS without calcium and magnesium containing 10 mM HEPES, colon pieces will be treated with an enzyme mixture following manufacturer's protocol at 37°C for 30 min. Finally, the enzyme-treated colonic pieces will be dissociated into single cell suspension using the gentleMacs tissue dissociator. This is the LP fraction. The IEL and the LP fractions will be labeled with different fluorescent labeled mAbs to identify CDS Treg within the TCRp+CD4-B220"CD45+ gate and their CDS counterpart using flow cytometry. We will include staining for several activation markers, NK cell-specific markers, memory T cell markers as well as CD1 lc expression and compare these profiles to conventional 038αβ T cells as well as to CD8 Treg derived from liver.
To determine the gene transcript signature of colonic CDS Treg for the identification of cell-surface markers and junctional pathways.
Rationale: We hypothesize that a distinct gene expression profile in this novel CD 8 Treg will further help to define not only their cell-surface markers but also the molecular pathways for their identification, development and function. Using cluster analysis program, we will classify these genes into functional molecular classes, such as cell surface molecules and molecules involved in transcription signaling, cell death, metabolism, etc. Since memory program also encompasses changes in a broad set of cellular pathways, including signal transduction, survival, apoptosis, cell cycle regulation, metabolism, nuclear function, and cytoskeietal organization, we will compare these gene signatures between gut- and liver-deri ved CD8aa Treg as well as ti ssue-resident memory T cells and virtual memory T cells that are also responsive to IL-15,
Experimental Strategy: We will perform RNA sequencing for transcriptome profiling of CD8 Treg and conventional CD8ap T cells sorted from colon tissues of naive B6 mice . It is likely that both populations should share many genes associated with CDS T cells, but they will have distinct global gene expression patterns owing to the innate-like properties of CDS Treg. We will define shared genes and those differentially expressed either up-regulated or down-regulated in CDS Treg in comparison to CD8 T cells under stringent statistical analysis (p value <0.05). The candidate genes will be validated by either real-time PCR with specific primers designed using web-based programs as before or at the protein level using available antibodies for western blotting and flow cytometry. Sorted CD 8 Treg and CD8o$ T cells will be used for the extraction of total RNA using the RNAeasy Micro Kit (Qiagen) followed by its integrity analysis using Agilent RNA 600 Nano Kit (Agilent). Poly-A mRNA enrichment will be used for library preparation and 50 bp-reads will be generated by sequencing on an Alumina HiSeq4000 analyzer using the TruSeq v3 Cluster kit at the UCSD Institute for Genomic Medicine core facility. RNA sequence data will be analyzed by the Bioinformatics core facility at UCSD and the log2 RPKM (Read per kilobase per million) will be quantile normalized. The R statistical software will be used to calculate differentially expressed genes between CD8aa Treg and CD8ap T cells. Genes satisfying the following criteria will be chosen for analysis: first, the average count is more than 1 0 in at least one sample group, and second, the global False Discovery Rate (FDR) is controlled at p values of 0.05 with a minimum fold-change of 2. This should generate a CD8aa Treg vs. CD8ap T cells "signature gene set" for further analysis. To determine PLZF expression and its role in the development of colonic CD8aa Treg.
Rationale: Our preliminary data suggest lower le vels of PLZF expression in both hepatic and colonic CD8oa Treg compared to NKT cells. Also, PLZF plays an important role as hepatic CD8aa Treg are significantly reduced in PLZF-/- mice. First, we will determine the expression of PLZF in CD8aa Treg using the fate mapping strategy as well as bone marrow chimeric mice using PLZF-/- mice as described earlier. The absence of CD8aa Treg in nude mice (data not shown) indicate their thymic origin and we hypothesize that following their migration from thymus CDSoux Treg lose PLZF expression similar to the recently described mechanism, for adipose tissue-resident NKT cells. Since PLZF*' NKT and MAIT cells are also dependent upon IL-15/iL-2R|3 signaling, we will use several gene-deficient mice to determine by flow cytometry whether similar pathways are also crucial for the development of colonic CD8a Treg.
Experimental Strategy: First, we will determine the frequency and absolute numbers of both CD8aa Treg and CD8a T cells in colonic IEL and LP of WT B6 and se veral gene deficient mice, including PLZF-/-, Qa-1-/-, CD 122-/- and CD25-/- mice. To determine expression of PLZF using a fate-mapping strategy, we will cross PLZF-Cre mice (which express a bacterial artificial chromosome transgene in which the gene encoding Cre recombinase is knocked into gene encoding PLZF) with Rosa26 fl/fl mice (which express the fluorescent marker tdTomato and cany a lox -flanked stop codon at the ubiquitous Rosa26 locus). Therefore, in the resultant PLZF-Cre X Rosa26 fl/fl mice, cells that express PLZF (and therefore Cre) are permanently tdTomato+. We will examine whether CD8aa Treg from gut, liver and spleen are tdTomato+. We will also use CD8 T cells as negative and type I NKT cells as positive controls in these
experiments. These mice are available from Dr. Derek Sant'Angelo who will help us with these experiments. A clear pronounced tdTomato staining will confirm that colonic CD8aa Treg also had expressed PLZF during development. Next, since the conventional T cell development is not perturbed in the absence of PLZF as shown in PLZF-/- mice, chimeric mice using CD45.1 and CD45.2 markers will be generated to study CD8aa Treg. First, we will determine whether the requirement for PLZF is intrinsic to CD8aa Treg by transferring PLZF-deficient bone marrow depleted of T cells and B cells into irradiated B6 host and monitoring the presence of CD8aa Treg using FACS. In parallel, we will also transfer hone marrow from WT litterrnates into B6 host for comparison. If PLZF plays a cell-intrinsic role, PLZF-/- bone marrow transfer may not be able to fully reconstitute B6 host. To further demonstrate the intrinsic role of PLZF, WT B6 mice will be reconstituted with a 50:50 mix of bone marrow cells from PLZF-/- and WT mice and the development of CDScux Treg analyzed 12 to 16 weeks after transfer by cell surface markers, including CD45.1 vs. CD45.2 to differentiate WT from PLZF-/- cells.
Alternate strategies: As mentioned before, all colonic CD8acr ΤΟ αβ" T cells do not have regulatory properties. Our preliminary data suggest that around 35-50% of CD8aa+TCRaP+ T cells express NK-inhibitory receptors (and are
CD44+CD 122÷ and CD 1 lc~, a marker thought to be present on suppressor T cells (29). In addition, the depletion of CD1 lc+ cells from bulk CD8+ T ceils results in loss of protection from EAE in adoptive transfer experiments (data not shown). Therefore, attempts will be made to sort and compare gene expression between CD1 lc+ Treg and CD1 lc" populations. If there is a difference it would indicate that within CD8aa T cells only those that are also CD1 lc+ are CDSoux Treg. It is possible that PLZF requirement is not intrinsic to CD8aa Treg. In this case, CD8aa Treg could still develop in chimeric mice generated with bone marro cells from PLZF-/- mice. However, CD8cece Treg in these chimeric mice may not have fully developed their innate-like properties, including cytokine secretion, NKi . l expression or expression of other cytotoxicity genes, like perforin and granzyme B. Therefore, we will use flow cytometry to analyze all relevant markers after in vitro stimulation with plate-bound anti-CD3/CD28 mAbs as shown.
To determine regulatory properties, induction and mechanism of regulation of colitis by CD8aa Treg. In this Aim, we design a series of experiments to test the hypothesis that CD8aa Treg use cytolytic mechanism to control colitis in a Qa- lb-dependent manner and that CD8aa Treg can be activated/expanded following immunization with Qa-lb-binding peptides.
To determine the role of perforin/ granzyme B in the regulation of colitis.
Ration ale: Our preliminary data indicate a regulatory role of CD8aa Treg in both CD4-induced and DSS-induced colitis. We hypothesize that CD8aa Treg utilize cytolytic mechanism to kill activated target CD4+ T cells based upon the following observations: (a) they do not secrete detectable levels of suppressive cytokines, such as IL-10 and ΤΟΡβ; but produce cytokines typical of cytotoxic CDS T cells and NKT cells; (b) RT-PCR analysis show that CD8aa Treg express enhanced levels of perforin and granzyme B, but not granzyme A; (c) earlier data using bulk CD 122+CD8+ T cells or cloned TCR-peptide-reactive CDS'"" T cells show killing of activated target cells. Since there is similarity between hepatic CD8aa Treg and colonic CD8aa Treg in all the features analyzed so far, we propose to compare whether both Treg populations use similar regulatory mechanisms involving perforin/granzyme B.
Experimental Strategy: We will use sorted colonic CD8aa Treg to investigate protective efficacy and mechanism using the CD4~CD45RBtH8h T cell adoptive transfer model of colitis. The CD4÷CD45 Bhlgh pathogenic T cells will be sorted from naive B6 mice and co-adoptively transferred into nai e Rag l-/- mice with sorted CD8aa Treg or CDS T cells from colonic tissues. Comparative body weight loss will be monitored following adoptive transfer and mice will be sacrificed to measure colonic inflammation by histopathology and cytokine analysis as shown in Fig 9. Liver CD8aa Treg will be used as a positive control in these experiments. If colonic CD8aa Treg are able to provide protection in Rag l-/- mice, then in next experiments, colonic CD8aa Treg will be isolated from WT, Perforin-/- and Granzyme B-/- mice and use in adoptive transfer experiments into Rag l-/- mice. Next, we will use sorted colonic CD8aa Treg from PLZF-GFP mice to determine their ability to homing into gut to protect colitis.
To determine the role of Qa-1 in CD8aa Treg-mediated regulation ofCD4 T cell-mediated colitis.
Rationale: Experiments using Qa-P knockout and knock -in mice or anti-Qa-1 Ab have indicated that the Qa-1 molecules play an important role in the function of CD8 Treg (30-35). Qa-1 in mice (I f i .A-i - in humans), a on-classical MHC class lb molecule, forms a heterodimer with β2 -microglobulin and can present peptides from both self and foreign antigens. Studies in H-2U mice also indicated Qa- la-dependent apoptotic depletion of only activated but not naive MBP- reactive \'138.2+CD4+ T cells. Furthermore, Qa- la-restricted CDS Treg are expanded in H-211 mice following capture of apoptotic T cells by conventional DCs that mediate cross-presentation of TCRp-cham-derived peptides to the CDS Treg, We will determine whether Qa-l is required for the CD8aa Treg -mediated regulation of colitis.
Experimental Strategy: First, we will determine whether Qa-l expression on target CD4 " T cells is required for CD8aa Treg-mediated regulation of colitis in Ragl-/- mice. To determine this, we will adoptively co-transfer colonic CD8aa Treg from WT mice with CD4+CD45Rbhish T cells from B6 mice or Qa-lb-/- mice into Ragl-/- recipients and monitor colitis. In this case, if Qa-l expression on target cells is required for suppression, Ragl -/- mice co-transfer with pathogenic CD4÷ T cells from Qa-lb-/- should develop colitis even in the presence of CD8aa Treg. If we find a Qa- lb-dependent suppression, we will further explore whether there is a Qa-l allelic difference in regulation. We will use CD8(xa Treg from B6 (Qa-lb) or (B6 X B6.tia) Fl mice (Qa-la/b) in these co-transfer experiments. This will clarify whether Qa-Ib-restricted CD8aa Treg can suppress Qa-ia+ effector T cells (Teff) or not.
To further examine whether CD8aa Treg-mediated apoptosis of Teff is cell- cell contact dependent both Teff and CD8aa Treg will be co-cultured in vitro. Sorted CD45.1 *'CD4*' Teff and CD45.2+ CD8aa Treg will be co-cultured (1 : 1 to 10: 1 ratio) for 72-96 hr in the presence of plate-bound anti-CD3 mAb and the degree of apoptosis (by number of surviving Teff) determined by FACS using staining with PI/Annexm V. To distinguish between anti-proliferative vs.
apoptotic mechanisms, we will examine suppression or killing of CFSE-labeled Teff counterstamed with PI. It is possible that, in addition to induction of death, the proliferation of PI -negative Teff may also be inhibited indicating that other mechanisms may be involved. We will then use CD8aa Treg from perform-/- or granzyme B-/~ mice to further examine their anti-proliferative or apoptotic role in regulation. Similarly, Teff derived from Qa-lb-/- mice will be used to investigate Qa- 1 -dependency of immune regulation. Ceil contact will be further confirmed using Transwell plates to separate CD8aa Treg and ( 1) 1 Teff populations.
To determine the role of Qa-l -restricted CDSaa Treg in acute and chronic DSS- induced colitis.
Rationale: Our preliminary data show a significant reduction in both colonic and liver CD8aa Treg in Qa-lb-/- mice. Consistent with the role of Qa-l -restricted CD8aa Treg in the maintenance of immune homeostasis in the gut, we found increases secretion of IL-6 and infiltration of (1) T cells in gut tissue in naive Qa-lb-/- mice in comparison to naive WT B6. These data suggest that in the absence of CD8aa Treg, Qa-1-/- mice may be more susceptible to the development of colitis. As mentioned earlier, we have identified several Qa-1- binding peptides derived from the TCR-Υβ chains, which activates splenic and hepatic CD8aa Treg both in vitro and in vivo (data not shown). Here we will use the most potent peptide, p8.2L, that significantly protects mice from. DSS- induced colitis in a Qa- 1 b-dependent manner. Therefore, we will determine the susceptibility of Qa-lb-/- mice to DSS-induced colitis and whether they are protected from colitis following immunization with the Qa-lb-bmding peptide p8.2L that induces CD8aa Treg.
Experimental Strategy: To detect increased sensitivity to colitis in Qa-1-/- mice, WT and Qa-1-/- mice will be exposed to a low dose of DSS (1.5% as opposed to 2.5%) in the acute model and to three doses of 0.5-1% DSS in the chronic model. Comparative colitis will be measured by parameters described in Figure 19. Next, we will determine whether the Qa-1 -binding peptide p8.2L that most potently activates CD8 Treg will be protective both prophylactically and therapeutically in Qa-1-/- mice. To examine efficacy prophylactically, we will administer the peptide p8.2L to WT and Qa-1-/- mice on day 0 and, after 3 days, DSS (2.5%) will be added to drinking water. On the 8th or 9th day following DSS treatment, mice will be sacrificed to measure colitis using parameters as described before. For therapeutic function, we will administer the peptide on day 5 after beginning of DSS treatment when the body weight loss start. Next, we will determine the priming or activation of CD8aa Treg following peptide- immunization by examining CD69 increase and cytokine expression by intracytoplasmic staining. An irrelevant peptide from hen-egg lysozyrne will be used as a negative control in these experiments.
Physiological induction of colonic CDSaa Treg in the regulation of colitis. Rationale: We will examine the hypothesis that the frequency of colonic CD8aa Treg may differ in different phases of colitis and may correlate with the disease severity. Since CD8cece Treg appear to be involved in the maintenance of homeostasis, we will determine whether the number CD8aa Treg in colon is significantly decreased during the peak of disease and whether it is restored to normal values during the relapsing phase indicating their physiological importance.
Experimental Strategy: We will investigate both acute and chronic models of DSS-induced colitis. In the acute model, we will add 2.5% DSS (Affymetrix) to the feeding water and expose the mice for 7 days. After that, mice will be put on regular water. In the chronic model, mice will be put to 3 cycles of 7 days with 1.5% DSS plus 14 days with regular water. We have verified that 1.5% DSS gives a minimal response in the acute model of the disease. Mice will be sacrificed and colonic (LP and IEL) and MLN tissue will be dissociated into single cell suspensions and analyzed by flow cytometry. For the acute model, days of sacrifice for phenotyping will be 6th, 9th, !6m and 25* and, in the chronic model, will be at the end of each cycle, i.e. days 21st, 42nd and 63rd
approximately. Following input in the chronic phase, we will observe mice and sacrifice after every 15 days for phenotyping of CD8aa T cells in colon until the time when body weights are restored. To measure colitis we will use body weight loss, gross colonic features and histopathologic score of colon and cytokine measurement in colon explant culture. In addition, we will use FITC- Dextran tracer to detect epithelial barrier malfunction and Myeloperoxidase (MPO) assay, whenever required.
Potential pitfalls and alternate strategies: There is a possibility that other molecules such as Granzvme A or TRAIL (36)(8)(10) that are not overexpressed in CD8oa Treg in steady state, become involved following activation. We will use other gene deficient mice to explore other pathways. Alternatively, Granzvme B or perform may have only partial effect, we may have to cross and generate dual perforin/Granzyme B-deficient mice for the isolation of CD8aa Treg and examine their suppressive role. We may alternatively use ILK)-/- mice in the C3H/HeJBir background in collaboration with our colleague Dr. Lars Eckmann to determine the role of CD8aa Treg in spontaneous colitis model . Since CD8aa Treg rapidly secrete IL-2, there is a possibility that they may recruit CD4 "CD25+Foxp3÷ Treg. Attempts will be made to examine this in DSS colitis model following peptide treatment and examining intestinal tissues by FACS on days 2, 4, 8 and 16 for the induction of CD4+ Treg and compare to the control peptide-treated mice. If there is paucity of colonic CD8aa Treg for adoptive trsanfer studies, we may use sorted liver derived CD8aa Treg and then compare their gene signature and other properties. Since DSS colitis is primarily mediated by innate cells, alternatively regulatory mechanism(s) may need to be explored (37)(9)( 11).
A detailed knowledge of the cellular and molecular mechanism(s) involved in maintaining immune tolerance in gut is cmciai in the development of novel strategies for treatment of 1BD. Our central hypothesis is that CD8 Treg are also enriched in colon and play an important role in limiting the intensity or duration of the inflammatory immune response in gut This mechanism is different but complimentary from the one mediated by Foxp3+CD4+ Treg that prevents a damaging response from occurring. Thus, CD8aa Treg-mediated regulation is a negative feedback response in the sense that it allows operation of the natural mechanisms of defense or healing but shuts down these mechanisms after a delay in order to avoid excessive tissue damage (29, 38). Studies proposed here are designed to characterize the colonic PLZF+CD8aa Treg and investigate the molecular mechanism(s) involved in the negative feedback immune regulation. Importantly, we have identified Qa- lb-binding peptides that activate CD8cca Treg and significantly protect mice from colitis. Since Qa-1 or HLA-E molecules in human are highly conserved and non-polymorphic, a screen can be setup to identify HLA-E-binding peptides that stimulate human CD8a Treg with key implications for clinical studies. These studies are highly significant as they will characterize a novel population of colonic CDScxa Treg and will have major implications in designing new strategies for the
prevention/and treatment of IBD. Example 3
A similar phenotype, gene signature and regulatory function of CD8aa Treg in human peripheral blood. We will test the hypothesis that circulating CDSotot Treg also play an important role in the regulation of autoimmunity in humans. Patients with active autoimmune disease may have altered frequency of CD8aa Treg in PBMC and this will be examined after their characterization, including their frequency, cell surface phenotype, cytokine and gene expression profile, MHC-restriction, TCR repertoire and their regulator}' function. It is important to investigate whether the CD8 a Treg equivalent to the mouse counterpart are also present in humans. Our preliminary data using multiparameter flow cytometry analysis suggest that in peripheral blood of healthy individuals, potential CD8aa Treg are present. In human PBMCs, CD8aa Treg are defined as PLZF+TCRaP+CD8aa+ T cells that do not express the TCRVa7.2/Ja33, which identifies MAIT cells, and have intermediate or very low expression of CD161, different from MAIT cells that are CD161!li !\ The frequency of CD8aa Treg was 0.20% ± 0,05 (mean ± sem) that correspond to 2 103 ± 0.5 x 103 in a million of PBMC. Notably, the expression of RORyt, the transcription factor of MAIT cells, as well as CXCR6 and CCR6 in CDSaa Treg was significantly lower than both in MAIT cells and in CD8ap T cells (data not shown). Similar to murine CD8aa Treg, CD244 or 2B4, and CD1 lc were also significantly high expressed in human CD8aa Treg. Though IF y, IL-17 and IL- 4 showed no differences between CD8aa Treg and either€Γ)8αβ T cells or MAIT cells, granzyme B, perforin and T Fa secretion by human CD8aa Treg were significantly increased similar to murine CD8aa Treg (data not shown).
Phenotype analysis: Following isolation of PBMC from EDTA or heparinized blood by Histopaque gradient centrifugation, CD8aa Treg will be identified within the TCRaP÷CD8÷PLZF+ gate as
CD8 +Va7.2/j a33negCD161n!ed''i0W and analyzed for the expression of different markers associated with the murine of CD8 D D Treg, including CD 122 (IL- 2Rp), CD28, CD 127, Foxp3, GITR, CTLA-4, CXCR3, CD45RC, CCR8 and TGF-β, as well as activation markers such as CD25 and CD69. Our preliminary data on murine CD8aa Treg show high expression of CD25, CD28, CD 122 and GITR, but not CD127 and FoxP3 in comparison with conventional CDScxp T cells. However, it is unknown whether human CD8aa Treg also express some of these markers. Also, we will examine the memory status of human CD8 a Tregs based on the expression profile of CCR7 and CD45RA. Previously, it has been described that D%+CD\6\÷ and CD8+CD122+ T ceils are memory-like exhibiting a TEM (CCR7-CD45RA-)/TEMRA (CCR7"CD45RAH-) and TCM
(CCR7+CD45RA") phenotype, respectively. Fluorescent monoclonal antibodies will be purchased from BD Bioscience, BioLegend or eBioscience.
Cytokine analysis: Our preliminary data indicate that CD8aa Treg in humans also constitutively secrete significantly more granzyme B, TNFa and perforin than MAIT cells and conventional€ϋ8αβ T cells (data not shown), however IFNy, IL-4 and IL17 secretion was similar between subsets. To investigate the full potential of CD8aa Treg to secrete different cytokines and chemokines, sorted CD8cece Treg and CD8a(3 T cells will be stimulated with phorbol 12-myristate 13 -acetate (PMA) and ionomycin for 4-6 hours with Monensin (BD GolgiStop). After stimulation, the frequencies of cytokine- producmg cells (IL-2, IFNy, IL-4, IL-10, IL-13, IL-17, IL-22, ΤΟΡβ, TNFa, granzyme B and perforin) will be measured by flow cytometric analysis of intracellular cytokines or by enzyme-linked immunospot (ELISPOT). The culture supernatant will be collected and analyzed using BD™ CBA Human Chemokine Kit: IL-8 (CXCL8/IL-8), RANTES (CCL5/RANTES), monokine induced by interferon-y (CXCL9/MIG), monocyte chemoattractant protein- 1 (CCL2/MCP-1), and interferon-y--mduced protein-10 (CXCLlO/IP-10).
Alternatively, cytokine gene transcripts will be analyzed by real-time PCR using RNA isolated from sorted CD8aa Treg and CD8ap T cells after stimulation with PMA and ionomycin.
Gene expression and the TCR repertoire analysis: To characterize the gene signature of human CD8aa Tregs, we will perform RNA sequencing (RNA-seq) of freshly sorted CD8aa Treg and conventional€'08αβ T cells from 3 different donors to compare their total RN A expression profile. RNA-seq data of sorted CD8CKX Treg and CD8ap T cells will be generated by an Alumina HiSeq4000 analyzer using the TruSeq v3 Cluster kit at the UCSD Institute for Genomic Medicine as in the case of murine CD8aa Treg. Similar to the analysis of murine ( 1)8 Treg, a high throughput sequencing of the TCRp genes from genomic DNA from sorted CD8aa Treg and CDS T cells will be carried out as described previously (Adaptive Biotechnologies, Seattle, WA) (39). The raw sequence data will be preprocessed to remove errors and to compress the data. TCRp sequences will be analyzed using their ImmunoSEQ Analyzer. For each unique sequence, the nucleotide and predicted amino acid sequence, V
(variable), D (diversity) and J (joining) genes and the number of sequencing reads will be determined. The data will be further sorted to exclude any sequence with an out-of-frame rearrangement or a stop codon in the CDR3, and the frequency will be determined for each of the remaining productive unique sequences (nucleotide clonotypes). Irnmimoregidatory properties : First, we will examine suppression of T cell proliferation as measured by the dilution of CFSE-labeled (CellTrace™ Thermo Fisher Scientific) sorted autologous CD4+CD25" T cells cultured either alone (3 x K /well) or with sorted CD8aa Treg (1 x 10 '/w ell) in the presence of anti-CD3/CD28 microbeads. After 72h of stimulation, cells will be labeled with fluorochrome -conjugated anti-CD8 mAb and intracellular staining with anti- human IFNy. We predict that CD8aa Treg will be able to suppress both T cell proliferation when co-cultured and the Thl cytokine secretion profile. To examine MHC restriction, we will add a purified anti -human HLA-E antibody (clone 3D 12) to the cultures to block antigen presentation by HLA-E and determine whether the suppressive activity is blocked. Furthermore, we will examine different mechanisms that potentially can be involved in suppression of CD4 T cell proliferation by CD8aa Treg, including neutralizing antibodies against specific soluble factors to reverse inhibition of proliferation and Trans- well experiments to examine whether cell-cell contact is required for apoptosis induction using annexm V staining.
The ability of CD8aa Treg to suppress allogeneic response in a mixed lymphocyte reaction (MLR) is determined. Briefly, PBMCs (responder cells, lxi0b cells/ml) will be stimulated for 6 days in 96-well plates with allogeneic PBMCs previously blocked by mitomycin (blocked, stimulator ceils, 0.5xl06 cells/ml), ratio 2: 1, to perform one-way MLR. After 6 days, cells from the oneway allogeneic MLR will be collected, washed, and CD8aa Treg will be isolated by sorting to evaluate their suppressor properties in a secondar ' MLR. Thus, freshly isolated PBMCs will be labeled with CFSE (CFSE-labeled, responder cells) and mixed with freshly isolated non-labeled allogeneic PBMCs (stimulator cells) and CD8aa Treg (isolated from the primary one-way MLR), ratio 2: 1 :0.5. After 4 days, the cells will be collected, washed, stained for CD4 and the proliferation of the CD4+ ceils will be determined by flow cytometry. We expect that CD8aa T'regs derived from the primary culture will reduce the proliferation of allo-reactive CD4+ T cells in the secondary culture.
Next, we will determine whether CD8aa Treg-mediated suppression involves cytotoxicity to the target cells using a flow cytometry-based cytotoxicity assay that simultaneously measures expression of the degranulation marker CD 107a by effector cells (CD8aa Treg) and the apoptosis marker annexin V binding to target cells. Sorted CD4"CD25" T cells (Target cells) pulsed with anti-CD3/anti-CD28 beads or non-pulsed, will be labeled with PKH 67 green fluorescent cell linker (Sigma-Aidrich) and mixed with sorted CD8cece Treg (Effector cells) at an E:T ratio of 2: 1 to 10: 1. The % of CD 107a-expressing CD8÷ T cells and annexin V binding to PKH 67-labeled target cells will be measured by FACS. We anticipate that an increase in both effector cell degranulation and target cell death will be observed only in the presence of CD8aa Tregs and activated target cells. In addition, phenotypic characterization of effector CD8aa Treg will be carried out using different antibodies, including anti -perform and anti-granzyme B.
Example 4
A summary of exemplary modalities by which CD8aa Treg can be targeted for their activation/expansion in vivo and subsequent protection from autoimmune diseases.
CD8aa Treg can be_sorted and adoptive transfer protects from autoimmunity.
A peptide-based modality targets induction of CD8aa Treg which in turns protects from disease.
There is a natural expansion of CD8aa Treg in patients with RA and lupus
Anti-4-lbb protects from autoimmunity, e.g., from EAE. Induction of CD8aa Treg following administration of anti-4-lbb antibody and subsequent protection from EAE. CD8aa+TCRafH Treg ceils are increased following anti- 4-1 BB administration. Groups of C57BL/6 mice were either administered with PBS or 4-1BB antibody on day 0. Three days later mice were sacrificed and liver mononuclear cells were isolated and stained with various fluorochrome labeled antibodies to determine the number of CD8aa Treg. EAE is ameliorated in WT B6 mice following anti -4- IBB antibody injection. Groups of female B6 mice (7- 8 mice in each) were immunized subcutaneously on day 0 with 100 μg MOG33- 55 peptide emulsified in an equal volume of CPA. On the same day animals were injected intraperitoneally with 25 fig of anti-4-lBB antibody diluted in PBS.
Anti-CD3 protects from autoimmune disease. Induction of CD8aa Treg following administration of anti-CD3 (4c 11) antibody and subsequent protection from EAE, CD8cux+TCRaP+ Treg cells are increased following anti-CD3 administration. Groups of C57BL/6 mice were either administered with PBS or CDS antibody on day one and day three. On day seven mice were sacrificed and liver mononuclear cells were isolated and stained with various fluorochrome labeled antibodies to detect induction of CD8aa Treg. EAE is ameliorated in WT B6 mice following anti-CD3 injection. Groups of female B6 mice (6 in each) were injected intraperitoneally with 200 μg of anti-CD3 antibody per mouse on day 5, EAE was induced on day 0 by injecting 100 g MOG33 -55 peptide emulsified in an equal volume of CPA, subcutaneously.
Adoptive transfer of sorted CD8aa Treg protects mice from MOG- induced EAE (a model for multiple sclerosis) and from CD45Rbhlgh CD4+ T cell-induced colitis (a model for IBD) in a perforin -dependent manner.
Administration with CD8+ Treg-inducing peptide protects mice from MOG-induced EAE as well as from DSS-induced colitis in a Qa-1 -dependent fashion. Peptide-induced induction of CD8+ Treg protects WT mice but not
CD8+Treg-deficient mice from EAE. Peptide-induced induction of CD8+ Treg protects mice from DSS-induced colitis.
Frequency of CD8 Treg is significantly increased in PBMCs derived from patients with ongoing rheumatoid arthritis (RA) and lupus (SLE).
Frequency of CD8aa Treg significantly increased in RA patients (n=3) compared to healthy controls (n 26) (*p< 0.05, **p< 0.01 , Mann Whitney test).
Circulating CD8aa Treg are increased in SLE patients. The frequency of CD8aa Tregs was significantly increased in PBMC from SLE patients (n=4) compared to healthy controls (n=26) (**p< 0.01, Mann Whitney test).
Example 5
The understanding of immune tolerance is crucial for intervention in autoimmunity and for the generation of an effective anti-tumor immunity. The function of T cells is controlled by both intrinsic (e.g., PDi and exhaustion) and extrinsic (regulatory T cells or Treg) cell-based mechanisms that prevent them from causing excessive tissue damage. Although earlier studies suggested an important regulatory role for CD8 T cells, a major caveat has hampered their characterization due to lack of molecular markers that differentiate them from conventional non-regulatory CD8 T cells (CDSconv). We have discovered that the expression of PLZF transcription factor in a population of PLZF"TCR +CD8aa" T cells (hereafter referred as CDS Treg) in both mice and humans distinguishes them from CD8∞nv . CDS Treg are innate-like cells that are enriched in liver of naive mice and a large proportion of them are CDl lc"1"CD244+NKG2D+NKl . l+. Despite having innate-like unconventional T cell features, CDS Treg are distinct from other innate-like T cells, including mucosal associated invariant T (ΜΑΠΓ) cells (summarized in Table below). Furthermore, their enrichment in liver of naive mice combined with the PLZF expression and specific cell surface markers differentiate these CDS Treg from others described earlier by Cantor's laboratory or by ourselves following CFA or TCR-peptide immunizations, respectively (24, 29, 40). The Qa-1 -dependence also suggests that CDS Treg target activated Qa-1"1" T cells and not nai e Qa- T cells, thus, they can control an ongoing T cell response. Accordingly, our preliminary data in indicate that an ongoing autoimmune response may be required for their expansion. Thus, CDS Treg expansion following autoimmune inflammation and their ability to control activated T cells qualifies them as part of a powerful negative feedback regulatory mechanism that protects tissues from excessive immune-mediated damage. Consistently, we have found a significant increase in the frequency of CDS Treg in PBMC from lupus and rheumatoid arthritis patients (data not shown) similar to the increase of CDS Treg during the recover - phase of murine EAE. Studies proposed here are designed to further characterize the biology of CDS Treg and the molecular mechanism(s) of immune regulation. Furthermore, the expression of 4-1BB on CD 8 Treg and the ability of an agonistic anti-4-l BB Ab to expand CDS Treg and the subsequent protection from EAE suggest that the proposed studies will provide a potentially novel approach to expand/activate CD8 Treg for intervention in humans using the available humanized anti-4-lBB Ab and also offers an explanation for the effects of ami -4- IBB treatment in different experimental conditions. Collectively, these studies are highly significant as they will characterize a novel population of CDS Treg in both mice and humans with major implications in designing and testing new strategies for the prevention and/or treatment of autoimmune diseases as well as in providing a potential novel checkpoint for effective anti-tumor immunity. Characteristics distinguishing PLZF+CD8÷ Treg from MAIT cells
Mice
PLZF+CD8 Treg MAIT cells
TCR repertoire Polyclonal TCR VD and V□ Semi-invariant Val9 usage and limited Υβ
Enrichment in B6 Liver Lung
mice
Liver phenotype CD8oa Double negative and
CD8CKX
Transcription factors PLZF!ow, T-ber7", Eomes+/" PLZFhi§h, T-bei .
Eomes'1" "
NK1.1 mostly < 20%
Surface markers: CD62L^,CD69^, ICOS", (1)621.· .CDiVr .
CD103-, CD 127-, CD218" ICOS^, CD 103^,
(1) 127 · . CD218+++
Cytokine secretion: IL-2^, IL- I 7.V . IFNy+, IL- IL-2", IL-17A^+,
10" IFNy+, IL-10+
Frequency in B cell- Present (same as WT) Absent
deficient (μΜΤ) mice
Frequency in germ Present (same as WT) Absent
free mice
Human
PLZF+CD8 Treg MAIT cells
TCR repertoire Polyclonal TCR usage Semi-invariant Va7.2 and limited Υβ
PLZF expression Low High
CD 161 expression Very low to intermediate High
Surface receptors: H.- I SRu . CXCR6+, CCR6+, IL-18R<r ÷+, CXCR6+++,
avUivyl
Cytokine secretion Granzyme B÷÷~ Granzyme B-
MHC-restriction Qa-1 (mice)/HLA-E (in MR- 1 (both mice and human) human)
Antigen reactivity Self-Ags peptides, TCR- Microbial Ags,
derived peptides riboflavin
As proposed above, innate-like unconventional PLZF+CD8 Treg are enriched in the liver of naive mice because the liver provides a more suitable environment for their development (e.g. TL-15-dependency). CD8 Treg with innate-like features offer a rapid mechanism for limiting any excessive immune stimulation to protect tissue against constant exposure to gut-derived antigens. Since this mechanism target only activated but not naive T cells, it allows effective immunity against microbial antigens to protect the organ.
Phenotype of CDS 'Treg is unique and can be distinguished from CD8Com-: To our knowledge, we have identified for the first time that PLZF expression in combination with other cell surface markers (PLZF÷TCRa D8aof) distinguishes CDS Treg from CDSamv similar to FoxP3expression that differentiates CD4 Treg from conventional CD4÷ T cells. Furthermore, the presence of PLZF÷CD8 Treg in germ-free mice, which lack MATT cells, and in CD Id-/- mice, which lack both NKT and innate-like CD8+ T ceils, clearly indicate their unique innate phenotype. Also, co-expression of CD 11c and CD244 (2B4) on a substantial portion of CD 8 Treg further suggests uniqueness of PLZF+CD8 Treg in both mice and humans. Additional studies, including Single Cell RNA sequencing analysis, will uncover other ceil surface and functional markers for the identification, development and function of CDS Treg.
Our approach bridges cellular and molecular approaches related to innate T cells Our preliminary data using the fate-mapping strategy (PLZF-Tdtomato expression) indicate that CDS Treg express PLZF similar to iNKT cells. We now have generated several PLZF-flox founders that will be backcrossed with CD4- Cre mice to generate mice deficient in T cells lacking PLZF to investigate the role of PLZF+CD8 Treg in the physiological control of autoimmunity. Identification of this unique population of CDS Treg (PLZF+TCRa ÷CD8ao ) enriched in liver of naive mice and in healthy humans is quite innovative. In collaboration with Dr. Vijayanand, Single Cell RNA sequencing analysis of CD l ie""' and CD 11c" CDS Treg in both mouse and human will unravel the transcription program, gene signatures and TCR repertoire related to the regulatory properties. In parallel, the development of multiparameter flow cytometric analysis to identify CDS Treg in human PBMC is also highly innovative and important. The expression of cell surface molecules, such as CD200, suggests that PLZF+CD8 Treg are not only capable of killing target T cells, but they may use negative signaling via CD200 to inhibit the function of APCs, including microglia. We will investigate the role of 4- IBB expression on CDS Treg and how an agonistic mAb can be used to preferentially activate them with important implications for potential intervention in human autoimmune disease.
Importance of regulatory role for CD8+ T cells. Murine models: Since 1970s the role of CD8÷ T cells in immune regulation of autoimmune diseases, transplant tolerance and homeostasis of cellular and humoral immune responses has been suggested (8-10), but has not sufficiently advanced as happened for Foxp3+ CD4 Treg (14). In mice genetically deficient or depleted of CD8"f T cells by treatment with anti-CD8 mAb, an important role for CD8 T cells in regulation of autoimmunity has been shown (11-13). Similarly, a critical regulatory role of CD!2,2 "CD8 " T cells involving a cytolytic mechanism has been shown in IL-2-/- and IL-2R "/- mice (15-17). Interestingly, IL-2/IL-15Rp-deficiency in humans also leads to a severe combined immunodeficiency syndrome as observed for IL- 2Ra (2,7). Also, CD122+CD8+ T cells can provide barriers to stem, cell engraftment, indicating the clinical relevance of CD8 Treg in humans (41 ). A critical role for CD8+ T cells in IL-2-/- mice was also shown as these animals develop colitis with an accelerated kinetics (18). Human disease: CD8+ T cells also have been implicated in various conditions in humans, e.g. transplant survival (19), prevention of inflammatory bowel disease (20) and after treatment of multiple sclerosis with either glatiramer acetate (GA) or vaccination with irradiated, autoreactive C I .) i T cells (21, 22). Recently, GA-induced regulatory CDS T cells have been shown to eliminate CD4+ T cells in an HLA-E-restricted manner (21, 23-25). In a clinical trial with anti-CD3 mAb (Teplizumab), increased frequency of memory-like CDS1" T cells with regulatory gene expression was found to be associated with a positive clinical response in type 1 diabetes patients (26). Collectively these studies indicate an important role of CD 8 T cells in control of autoimmunity.
A novel population of innate-like PLZF+CD8 Treg. enriched in liver of naive mice, can be distinguished from CD8Conv. We have identified a population of PLZFTCRaP+CD8aa+ Treg within the B220~CD4" gate expressing only the homodimer CD8aa that represents -3.9% (± 0.80 SEM) of liver MNCs in naive B6 mice. PLZF÷CD8 Treg are also present in bone marrow (-3.0%), spleen (-0.5%), blood (0.3%) and lungs (0.3%) of naive B6 mice as well as in the neonatal thymus (0.5%) until day 5 after birth. In athymic nude mice, which lack all T cells, CDS Treg were undetectable confirming their thymic origin (data not shown). Since innate-like features in T cells are driven by the expression of PLZF (42-48), we examined its expression in this novel population. CD8 Treg from both B6 and PLZF-eGFP reporter (PEG) mice, which was generated using modified bacterial artificial chromosome transgene expressing eGFP under the control of PLZF regulatory elements (49), express PLZF or GFP, respectively. However, CDS Treg have lower PLZF expression than iNKT cells (CD4 "CDld- aGalCer tetramer "). in contrast, PLZF is not expressed by either CDSconv or CD4 T cells. Accordingly, PLZF mRNA expression was found exclusively in sorted CDS Treg but not in CDSconv. Next, we used the fate-mapping experiment to investigate PLZF expression using PLZF-Cre x 26T mice, in which PLZF- expressing cells are permanently labeled tdTomato (50). Most CDS Treg in liver of PCre x R26T mice were tdTomato" while CDSconv showed only background level of expression. To further investigate PLZF requirement, we examined the frequency of CDS Treg in PLZF-deficient mice (PLZF-/-) and heterozygous PLZF+/- and PLZF+/+ littemiates (49). PLZF-/- mice had significantly reduced numbers of CDS Treg in liver compared to PLZF+/+ mice. Importantly, these results indicate that CDS Treg are PLZF+ and dependent on its expression.
Adoptive transfer of CDS Treg protects B6 mice from EAE. CDS Treg do not express Foxp3 but express glucocorticoid-induced tumor necrosis factor-related receptor (GITR), a marker of active Treg cells (data not shown) (28). To determine the in vivo regulatory- potential of CDS Treg, naive B6 recipients were adoptively transferred i.v. with lxlO5 sorted CDS Treg or CDSconv isolated from liver of naive B6 mice before induction of EAE with MOG35--55/CFA/PTX as described before (51 ). CDS Treg but not CDSconv significantly protected mice from EAE. In addition, the significant protection from EAE observed after adoptive transfer of 2x105 sorted CDS Treg but not CDScon from liver of CDld- /- mice eliminated any potential contribution from CDS NKT cells. These data demonstrate that CD8 Treg have regulatory properties.
A substantial portion of CD 8 Treg co-express CD244 and CD 11c. The majority of CDS Treg express CD244 (2B4) (>70%), a marker expressed on NK cells, but also implicated in the regulation of co-stimulation and function of CD8+ T cells (52). Notably, CDS Treg also express CD 11c, suggested earlier as a marker on CDS suppressor T cells (53). Co-staining of CDS Treg show that all CD1 lc+ CD8 Treg are also CD244 " while none of the CD8«mv express CD1 lc or CD244. Next, we examined whether CD244+CDl lc+ CD8 Treg are dependent on PLZF. Both percentage and numbers of CD244+CDl lc÷ CDS Treg were significantly reduced in PLZF-/- mice compared to PLZF+/+ mice. Since CDS T cells co-expressing CD 11c with suppressive functions have been reported (53, 54), we investigated whether CD1 lc expression on CDS Treg play a role in their regulatory capacity. Thus, CDS Treg depleted of CD 11c (CDSaofCDHc") (bead-depletion) were adoptively transferred into naive B6 recipients, and EAE was induced next day. CD l ie depletion of CDS Treg resulted in loss of their ability to control EAE. Consistent with innate-like phenotype, the majority of CD244+€D1 lc+ CDS Treg also express NK1.1 and NKG2D and also express the regulatory molecules CD137 (4- IBB) and CD200 that is absent from CDSconv. Expression of CD137, CD200 and other key immune molecules was also confirmed using RT-PCR (data not shown). Furthermore, CDS Treg are CD 122*, displayed a memory/activated phenotype (CD44hish CD62L!ow CD69+) and express only NK inhibitory receptors (Ly49A, Ly49E/F, Ly49G2 and Ly49I) but not activating receptors (Ly49D and Ly49H) (data not shown).
MHC-restriction: A significant reduced frequency of CDS Treg in Qa-P- /- mice indicate that a large number of CDS Treg are restricted by Qa-lb molecules. Notably, these CDS Treg can be distinguish from gut-resident CD8aofTCRaP+ T cells in that they are CD 103", PLZF* and are significantly reduced in Qa-lb-/- mice while gut-resident cells are CD103+, PLZF" and their frequency do not change in Qalb-/- mice (data not shown). Furthermore, no changes in their frequency in CD Id-/-, J lS-/- and μΜΤ-/- suggest that NKT cells and B cells are not required for CDS Treg, indicating their distinctiveness from NKT-dependent innate-like CD8÷ T cells. As expected, while CDS Treg are absent in CD8a-/- mice, their frequency is increased in CD8P-/- mice. Since Qa-l-restricted T cells can be either TAP-dependent or TAP-independent, the presence of CDS Treg in TAP1-/- mice suggests that TAP-dependent antigen processing is not required for the development of CD8 Treg. Importantly, CD8 Treg were significantly reduced in both CD 122-/- and IL-15-/- mice. Considering that CD122, the β chain receptor for IL-2 and 1L-I5, is essential for CDS T cell response to IL-15, these results indicate that IL-15 signaling is necessary for CD8 Treg development.
Cytokine secretion: We have also determined the cytokine secretion profile of sorted CDS Treg vs. CDSconv after in vitro stimulation with anti-CD3 mAbs. CDS Treg do not secrete IL-10 or ΤΟΡβ, but secrete typical cytokines produced by cytotoxic T cells (TNFa, IL-17A) as well as PLZF-driven secretion of both IL-4 and IFNy, similar to that in NKT cells (data not shown). It is noteworthy that these cells very rapidly secrete large amounts of IL-2 as well In the future, we will be investigating whether IL-2 secreted by CDS Treg may engage Foxp3 Treg that do not secrete IL-2. CDS Treg also express high levels of perform and granzyme B (data not shown).
Gene expression profile of murine CD8 Treg using single cell RNA sequencing. We will test the hypothesis that unique characteristics of CDS Treg, including enrichment in liver, self-reactivity, memory/activated phenotype and innate-like features driven by the PLZF transcription factor, are due to a unique molecular signature. Our preliminary data show that the TCRa repertoire of CDS Treg is polyclonal based on FACS, RT-PCR and high- throughput sequencing (47), indicating their distinctiveness from either MAIT cells that use an invariant TCR Vol 9 with biased usage of νβ8/Υβ6 in mice (42) or NKT cells. However, preliminary data suggest that CDS Treg are heterogeneous and a substantial portion of them are CDl lc÷. Are CDl lcf CDS Treg also polyclonal? Since the Single Cell RNA (scRNA) sequencing technology allows the simultaneous analysis of the TCRo$ in single cells, we will also examine the TCRaP repertoire of CD 11 c÷ CDS Treg and compare it with the bulk CDS Treg population as well as CD 11c" CDS Treg. If the TCR repertoire of CD l ie4" CDS Treg is oligoclonal, we plan identify the most common TCR for generation of TCR transgenic or retrogenic mice for their developmental studies as well as for the identification of antigenic peptides recognized by CDS Treg. Although MAIT cells, which are also PLZF+CD8+, represent a small population in mice, we will negatively sort out this population using MR1-5-OP-R.U tetramers (55) (received from the NIH tetramer facility) for the RNASeq analysis of CDS Treg similar to our gating strategy in humans. Single cell RNA sequencing analysis to identify gene signature of CDS Treg in mice:
Rationale: We will perform single-cell RNA sequencing of four sorted populations isolated from liver of naive PLZF-GFP reporter (PEG) mice: PLZFTCRap+CD8aP% P LZ FTC RaP+CD 8 aof, PLZFTCRap D8acf CD1 lc+ and PLZFTCRaP"CD8 ir CD 11c". The gene expression profiles will he compared among these 4 populations for shared genes as well as those that are predominant within each population. Single-cell transcnptome analysis will further help to define new cell-surface markers as well as their molecular profile and the molecular pathways for their identification, development and function.
We plan to perform scRNA sequencing using the Single Cell 3' Protocol and the lOx™ GemCode™ Technology (56). Thus, liver MNCs from 5-10 naive PEG mice will be stained and sorted using a FACSAria 111 into 4 subsets: PLZFTCRap+CD8aP% P LZ FTC RaP+CD 8 aof, PLZFTCRap D8acf CD1 lc+ and PLZFTCRaP+CD8aofCDl lc"\The recommended starting point will be 10, 000 sorted cells to generate Gel Bead-In-EMulsions (GEMs). To achieve single ceil resolution, the ceils are delivered at a limiting dilution, such that the majority (-90-99%) of generated GEMs contains no cell, while the remainder largely contains a single cell. Approximately, 5,000 cells will be loaded into each Single Cell 3' Chip. After dissolution of the Single Cell 3' Gel Bead in a GEM, primers containing (i) an lliumina Rl sequence (read 1 sequencing primer), (ii) a 16 bp lOx Barcode, (iii) a 10 bp randomer and (iv) a poly-dT primer sequence will be released and mixed with cell lysate and Master Mix. The l x™ GemCode™ Technology will sample a pool of - 750,000 barcodes to separately index each cell's transcriptome by partitioning thousands of cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs) and will produce full-length, barcoded cDNA that will be amplified by PCR to generate Single Cell 3' libraries for sequencing and analysis. Single cell RNA sequencing data will be analyzed in collaboration with the Bioinformatics Core at LJI. In addition to the standard analysis, such as demultiplexing, alignment, and gene counting, complementary methods of single-ceil differential gene expression (SCDE) and model-based analysis of single-cell transcriptomics (MAST) analysis will be used to compare the full-length transcriptome between the populations. The Benjamini Hochberg test with adjusted P < 0.05 and >2-fold change will be used to identify differentially expressed genes. Gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA) will be also included. We will define shared genes and those differentially expressed either up-reguiated or down- regulated between the four populations. The candidate genes will be validated by either real-time PGR with specific primers designed using web-based programs as before (46) or at the protein level using available antibodies for western blotting and/or flow cytometry.
Our preliminary data indicate that CD8 Treg are significantly expanded during EAE in the periphery and that they also infiltrate into the CNS during EAE. It would be important to analyze the TCR repertoire and transcription profile of the CNS-mfiltratmg CD8 Treg and compare that to CDSconv as well as hepatic CD8 Treg. Since regulatory activity is associated with CD 11c expression, it is likely that CDl lc" CD8 Treg infiltrate into CNS and negatively signal microglia. Therefore, we will examine and compare the gene expression and TCR repertoire of sorted CD8 Treg and CD8Conv isolated from CNS of PEG mice in the recovery phase of EAE. These data should provide additional important information about the nature of CDS Treg infiltrating the target tissue.
CDl lc expression as a phenotypic marker for CD8 Treg:
It is quite interesting that both murine and human CDS Treg express CDl lc. Notably, CDl lc "CD8 " T cells have been suggested to be suppressor T cells earlier (53). Several studies on gene expression profiles have suggested that CDl lc is also expressed by NK cells, activated T cells, γδΤ cells and certain macrophage populations (Immunological Genome Project). Also, recently Dr. Jonathan Ashwell's laboratory reported another TCRetp÷CD8÷ T cell subset with DC properties (57). But, our CDS Treg are distinct from these others in several cell surface markers, including CD44, CD69, CD25, IL-7R, CD 122 and PLZF expression. Our preliminary data suggest that CDS Treg do not express other DC markers, including MHC class II, CDl lb, F4/80 and FcRg (data not shown). However, our preliminary data also suggest that around 58% of CD8 Treg express CDl lc and that adoptive transfer of CDS" T cells depleted of CDl lc"1" cells resulted in loss of protection from EAE. Since CDl lc expression can potentially be used as a phenotypic marker for CDS Treg in both mice and in humans, it is crucial to address whether CDl lc expression on CD 8 Treg is regulated by transcriptional mechanisms, which require prior protein synthesis, or acquired through intercellular transfer (58).
We will use the CDl lc-EYFP transgenic reporter mice (B6.Cg-Tg(Itgax- Venus)lMnz J) in which yellow fluorescent protein (YFP) expression is driven by the CD l ie promoter. Considering that high CD 11c expression has been shown to be primarily in DC and that only a small portion of cells with intermediate CDl l c expression was CD3+ in these mice (59), we will characterize the expression of different markers (PLZF, CD244, CD200, NKG2D and 4-1BB) in CD3÷ cells with intermediate CDl lc expression. Therefore, expression of PLZF in CD1 lc+ CDS Treg in these reporter mice will confirm their transcription regulation rather than their acquisition by intercellular transfer upon chronic activation. It is also clear from our data that CD8 Treg require thymus for development as they are absent in athymic and RAG1-/- mice (data not shown). Also, CDS Treg express all other markers of classical cytotoxic T cells, including perforin and granzyme B. Thus, a detailed transcriptome RNA sequencing analysis of CDl lc" CDS Treg (above) will further clarify whether a transcription signature of DC lineage or T cell lineage.
PLZF+ CDS Treg have a specific transcriptional development program and and control control of autoimmunity in a negative feedback regulation. Our hypothesis is that PLZF expression is crucial for the development of CDS Treg and that chronic autoimmune inflammation ultimately leads to their expansion and physiological control of autoimmunity.
To determine the role of PLZF. RORq and M3 in the development and function of CDS Treg:
It is clear from our preliminary data using antibody staining, GFP reporter mice and fate-mapping strategy that CDS Treg express PLZF, although their expression levels are lower than iNKT cells. Accordingly, CDS Treg are significantly reduced in PLZF-/- mice. We will employ bone marrow chimeric mice using PLZF-/- mice as described earlier (49, 60) to further investigate the role of PLZF transcription factor in the development of CDS Treg. The absence of CDS Treg in nude mice (data not shown) indicates their thymic origin as well. Our latest preliminary real-time PCR data also suggest enhanced expression of two additional transcription factors, Retinoic acid receptor-related orphan receptor alpha (RQRa) and inhibitor-of-DNA-binding (Id)3 (Id3), in CDS Treg in comparison to CD8Conv (data not shown). The transcription factor RORa is a negative regulator of inflammation (61, 62) while M3 is essential for the generation of memor - CD8÷ T cells (63). Interestingly, M2 that is required for the development and functions of innate lymphoid cells (64) and Eomes, which is a critical regulator for CDS" T cell differentiation and effector function (65), are expressed at similar levels in CDS Treg and ( Η..·.!Λ . Since Id3-/- mice (from Dr. Goldrath at UCSD) and RORa-/- mice (from. Jackson Lab) are available, we will investigate their role in the development of CDS Treg.
Our latest preliminary data indicate a significant loss of CDS Treg in both RORa-/- and Id3-/- mice similar to that found in PLZF-/- mice. To determine whether the requirement for PLZF, RORa or M3 is cell-intrinsic to CD 8 Treg, irradiated congenic CD45.1 B6 mice will be injected i.v. with bone marrow (BM) ceils, depleted of T ceils and B ceils, isolated from CD45.2 PLZF- /-, RORa-/- or M3-/~ mice and the presence of CDS Treg monitored by FACS. For comparison, we will also transfer BM cells from wild type littermates into B6 host. If these transcription factors play a cell-intrinsic role, PLZF-/-, RORa-/- or Id3-/- BM transfer may not be able to fully reconstitute B6 host. If this were the case, B6 mice will be reconstituted with a mixture of BM cells isolated from CD45.2 PLZF-/--, RORa-/- or Id3-/- mice and congenic CD45.1 B6 mice at a ratio of 50:50 and the development of CDS Treg analyzed 12 to 16 weeks post- transfer. These studies should give us important clues regarding the role of these related key transcription factors in the biology of CD8 Treg.
CDS Treg are primed/expanded physiologically to control autoimmunity: Our preliminary data using adoptive transfer of sorted CDS Treg clearly indicate that CDS Treg can control autoimmunity. We propose the hypothesis that CDS Treg are physiologically expanded during the course of EAE and play an important role in the control of disease. Consistent with this hypothesis and the Qa-lb restriction of a large number of CDS Treg, it has been shown that the recovery as well as susceptibility to re-induction of EAE is compromised in Qa- !-/- mice (30). Notably, our preliminary data suggest that CDS Treg are expanded physiologically during the recover}' phase of EAE. Thus, B6 mice were immunized with either MOG35-55 for EAE induction as above or an irrelevant peptide derived from hen egg iysozyme (HEL) and hepatic CDS Treg were analyzed by FACS on day 10 at the onset of disease (EAE dlO) or at day 25 during the recovery phase of the disease (EAE d25). CD 8 Treg transitorily decreased at the onset of disease (day 10) but significantly expanded during the recovery phase (day 25) of EAE. In contrast, CDS Treg were not altered in non- diseased mice immunized with an irrelevant HEL peptide. These data indicate that the development of a pro-inflammatory, autoimmune T cell response is important for the expansion of CDS Treg that are able to control excessive immune response.
Next, we are generating mice that are deficient in PLZF+ T cells, including CD8 Treg, using floxed genes in a 2-step process by inserting two loxP sites simultaneously to ensure deletion of the intervening D A (two double stranded cuts in the DNA followed by repair of the gap by the cell). We have several founders confirmed by sequencing of the entire loci that are being bred now. After crossing with CD4-Cre mice, we will first establish that they are deficient in PLZF " T cells. Based upon our data in PLZF-/- mice, CD4-Cre PLZFfiox/flox (PLZFF F) mice should be deficient in PLZF+CD8 Treg cells. Since we are using CD4-Cre mice (CD8-specific Cre is not available), function of NKT cells and a minor population of MAIT cells may also be compromised in these mice. First, groups of PLZFF" mice and negative littermates will be immunized with PLP172-183/CFA/PTX to induce EAE and to be able to address re-induction of disease. Mice will be monitored every day for clinical score and disease onset, severity, incidence and recovery analyzed. Next, we will examine whether susceptibility to re-induction of EAE with PLP172-183/CFA is altered in PLZFf/fl mice. Thus, PLZF-'* mice and littermates will be immunized with PLPi72-i83/'CFA and 30 days later mice will be challenged with PLP/CFA PTX and disease monitored. We predict that CD 8 Treg induced initially in CD8 Treg^ mice (littermates) will protect them from disease whereas their absence in PLZF*'* mice should allow induction of clinical disease. To rale out the potential caveat that other dysfunctional innate-like T cells in these mice may contribute to the effect, we will adoptively transfer sorted CDS Treg from negative littermates and determine whether regulation can be reconstituted in PLZFF F mice. Since CDS Treg are enriched in liver, we will also investigate whether PLZF*'* mice develop sterile inflammatory liver injury. We will determine whether inflammation and induction of related genes occur in livers of naive PLZFF/F mice using Nanostring Technology (UCSD Core). These studies will conclusively prove that CD8 Treg are able to not only control physiologically autoimmune disease but also control sterile liver inflammation. Key cell surface molecules involved in CD8 Tree-mediated immune regulation: Our hypothesis is that CDS Treg through either cytotoxic mechanism or by engaging other cell surface molecules like CD200 to inhibit activation of APCs, control immune responses. Here, we will detennine the role of four different cell surface molecules, 4- IBB, CD244 (2B4), NKG2D and CD200 that are expressed on CD1 lc~CD244+ CDS Treg in both mice and humans.
4-1BB (CD137, tafrsf ) is a member of the TNF receptor superfaroily that is not expressed in naive T cells but only in activated/memory CDScoav T cells and is involved in their survival and activation (66, 67). Accordingly, agonistic anti-4-lBB Abs injected system icaJ.lv result in expansion of primarily memory CD8+ T cells (68, 69). However, several studies have shown paradoxical outcomes depending upon the timing and doses of anti-4-lBB Abs administration. For example, agonistic anti-4-lBB Ab can enhance some antiviral and anti-tumor T cell responses while inhibiting CD4+ T cell-dependent autoimmunity, including EAE (54, 67). Since 4- I BB is expressed in CDS Treg in naive mice, we believe that low dose administration of the agonistic anti-4- 1BB Ab in naive B6 mice leads to expansion/activation of CD 8 Treg and subsequent protection from autoimmunity. In contrast, high doses of anti-4-lBB Ab overwhelms the effects on other cells, including activated disease-causing T cells and DCs, and, consequently, the CDS Treg protective effect is masked. Accordingly, we found that naive B6 mice injected with a low7 dose of anti-4- ΓΒΒ Ab (25 ug/mouse) preferentially expands BrdiT CD8 Treg but not CD8∞«v and results in a significant protection from EAE. Notably, the protective effect is dependent upon CDS Treg as B6 mice but not Qa-i-/- mice are protected from EAE. It further indicates that under these conditions CD4 Treg that can also express 4-1BB do not play a significant role. Interestingly, a high dose of anti-4- !BB Ab (200 ^ig/mouse) does not protect mice from EAE, but rather potentiates disease due to overwhelming activation of encephaiitogenic T cells and APCs. Our preliminary data further suggest a significant absence of CDS Treg (4- lBB "CD lc+CD244 ") in 4- I BB-/- mice in comparison to B6 mice (data not shown). Therefore, we will investigate the role of 4- IBB expression on CDS Treg in the control of EAE and. hopefully, provide an explanation related to its paradoxical effects.
CD244 (also known as 2B4, SLAMF4) is a member of the Ig superfamily and is expressed predominantly on the surface of MK cells and γδ T cells. Unlike the other members of the SLAM family receptors that can engage in homotypic interactions, 2B4 interacts with a high affinity receptor SLAMF2 or CD48, which is expressed on a number of hematopoietic cells. The CD244- CD48 interactions can have dual functions either activation or inhibition of the immune response depending upon the degree of receptor expression, extent of ligation and level of adaptor molecules (70-72). An K-independent regulatory role of CD244 has also been shown in the control of experimental lupus (73). Consistently, a splice variant of CD244 has also been reported to be preferentially expressed in SLE patients and is associated with defective regulation in SLE (74). Since CD244 interactions can control both activation and lysis of target cells, we believe that is important to study the role of CD244 expression in CDS Treg-mediated immune regulation.
CD200 or OX-2 is a member of Ig superfamily and is expressed in lymphoid cells, including B cells and activated T cells, in both mice and humans. It interacts with its cognate ligand CD200R that is expressed on granulocytes, monocytes, DC and macrophages (75). The interaction of CD200-CD200R inhibits activation of macrophages and microglia and, accordingly, CD200-/- mice develop a rapid and severe form of EAE with enhanced axonal damage (75, 76). Furthermore, CD200-CD200R interactions are also involved in regulation of Thl/Thl7 immune responses in arthritis, IBD, transplantation and cancer (77- 79), Since CDS Treg infiltrate into CNS during EAE, we beleive that binding of CD200 on CDS Treg to CD200R expressed on microglia, astrocytes, and oligodendrocytes could suppress axonal damage that is mostly mediated by activated microglia thus protecting m ice from EAE.
NKG2D. Similar to NKT cells, CDS Treg express NK receptors, such as NKl . l and NKG2D, and these receptors may be involved in fine-tuning CD 8 Treg function. Since the in vivo function of NKl. l is poorly understood, we will focus here on the role of NKG2D in the function of CDS Treg. NKG2D is encoded by the KLRK1 gene expressed in both mice and humans and binds to cell surface MHC class I-related proteins, MICA MICB (human) and Rae (mice) (80, 81). In CDS T cells and NKT ceils, NKG2D plays an important role as a co- stimulatory molecule and promotes Thl cytokine production (82). It can also directly promote cytotoxic killing through excretion of granules containing granzyme B and perforin (83). Consistent with our hypothesis, NKG2D expression is enhanced in inflammatory tissues in autoimmune diseases, including arthritis, lupus and diabetes (80, 81, 84).
We have developed a simple in vitro assay to examine the regulator}' activity of CDS Treg. Specifically, sorted CDS Treg from B6 or deficient mice will be co-cultured with either CFSE-labeled OVA -reactive OT-II CD4+ T cells or sorted CD4 " T cells from B6 mice in the presence of OVA peptide or anti- CD3/anti-CD28 beads, respectively. CFSE-diiution and staining with Annexin and PI will be measured by FACS as an indication of response. The conventional CD8aP T cells will be used as controls. Preliminary data suggest that in addition to the killing of the target CD4+ T cells, CDS Treg appear to inhibit the proliferation of the target CD4~ T cells. In some cases, we will use titrated concentration of blocking mAbs, for example anti-CD200 (OX2, Bioiegend), in in vitro assays to directly examine effect on regulatory activity. Isotype control antibodies will be used as controls.
CDS Tree in human also have a similar phenotype, gene signature profile and regulatory function. Our hypothesis is that CDS Treg are also present in humans and, potentially, play an important role in the regulation of autoimmunity. Consistent with our data showing increased frequency of CDS Treg only during the recovery phase of ongoing EAE,we have also found an increased frequency of CD8 Treg in PBMCs from arthritis and lupus patients (data not shown).
We have developed a multiparameter flow cytometry analysis to identify CDS Treg in human PBMCs based on PLZF expression in CD8+ T cells and exclusion of Va7.2 Ja33+ CD10ihish (CD16r+) MAIT cells (85), Thus, CDS Treg are PLZF÷TCRa "CD8aa÷ T cells that express intermediate to very low- levels of CD161 (CD161+/_). Following this gating strategy, our preliminary data indicate that CDS Treg are present in PBL of healthy individuals and represent -12% in human PBMC, ranging from 0.4 to 35%. The expression of CD244 and CD 11c was significantly higher in human CDS Treg than in both MAIT and CDSconv. Similar to murine CD 8 Treg, a substantial portion of human CD8 Treg also co-express CD244 and CD 11c (CD244÷CDl lc+) that is almost absent in MAIT cells (-16% vs -1%). Furthermore, the expression of several markers known to be up-regulated in MAIT cells, including IL-18 receptor a (IL~18Rc ), RORyt, CXCR6 and CCR6, was significantly reduced in human CD 8 Treg (data not shown). Notably, human CD8 Treg also secreted higher levels of Granzyme B and perforin similar to murine CD8 Treg and also secreted IFNy, IL-17 and 1L-4 (data not shown). In preliminary in vitro suppression assays, sorted human CDS 'Treg also inhibits CD4+ T cell proliferation significantly (data not shown).
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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:
1. A method to identify or detect immune cells having TCRafH, CD8aa+, Nk 1.1 +, PLZF+, CD 161 +, and optionally having one or more of CD 1 1 c+, CD137+ CD244+, or one or more of NK-inhibitory receptors, comprising contacting a sample having mammalian immune ceils with a ligand that binds CDSacL a ligand that binds NK1.1, a ligand that binds PLZF, and a ligand that binds CD 161, and optionally a ligand that binds CD1 lc, a ligand that binds CD 137, a ligand that binds CD244, a ligand that binds TCRafl or a ligand that binds NK-inhibitory receptors; and identifying or detecting an amount of a population of cells CD8 +, Nkl . l+, PLZF+, and CD 161+, and optionally having one or more of CD1 lc+, CD137+ ( )244 TCRaP+, or one or more of NK-inhibitory receptors.
2. The method of claim 1 wherein the cells are identified using antibodies specific for TCRo$,CD8aa, Nkl . l, PLZF, or CD 161, and optionally antibodies specific for one or more of CD1 lc, CD 137, CD244, or one or more of NK~ inhibitory receptors.
3. The method of claim 1 or 2 wherein the ceils are human cells.
4. The method of any one of claims 1 to 3 further comprising isolating the identified cells.
5. The method of claim 4 further comprising expanding the isolated cells.
6. The method of claim 5 wherein the ceils are from a patient with an autoimmune disease.
7. The method of any one of claims 4 to 6 wherein the cells ae cultured with 1L-2, 1L-15, Qa-l HLA-E binding peptides, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof.
8. A method to decrease the number of immune cells having ΤΧ'Καβ, CDScto, Nkl.l, PLZF, and CD 161 , and optionally having one or more of CDl lc, CD 137, CD244, or one or more of NK-inhibitory receptors in a mammal, comprising: adm inistering to the mammal an effective amount of a composition comprising one or more antibodies specific for CD8aa, specific for Nkl . l, specific for PLZF, or specific for CD 161, or a combination thereof, and optionally a composition comprising one or more antibodies specific for CD l lc, specific for CDl 37, specific for CD244, specific for ΤΟ½,β, or one or more of NK-inhibitory receptors, or a combination thereof.
9. A method to decrease the number of immune cells having TCRap, CD8aa, Nkl . l, PLZF, and CD 161, and optionally having one or more of CDl lc, CD 137, CD244, or one or more of NK-inhibitory receptors in a mammal, comprising: administering to the mammal an effective amount of one or more antibodies specific for CD8aa, specific for Nk 1.1, specific for PLZF, or specific for CD 161, or a combination thereof, and optionally one or more antibodies specific for CDl lc, specific for CD 137, specific for CD244, specific for TCRccP, or one or more of NK-inhibitory receptors, or a combination thereof.
10. A method to prevent, inhibit or treat cancer in a mammal, comprising: administering to the mammal an effective amount of a composition comprising one or more antibodies specific for CD8aa, specific for Nkl .1 , specific for PLZF, or specific for CD 161, or a combination thereof, and optionally a composition comprising one or more antibodies specific for CDl lc, specific for CDl 37, specific for CD244, specific for TCR($, or specific for one or more of NK-inhibitory receptors, or a combination thereof.
11. A method to prevent, inhibit or treat cancer in a mammal, comprising: administering to the mammal an effective amount of one or more antibodies specific for CDSota, specific for Nkl .1, specific for PLZF, or specific for
CD 161, or a combination thereof, and optionally one or more antibodies specific for CDl l c, specific for CD137, specific for CD244, specific for TCRaft, or specific for one or more of NK-inhibitory receptors, or a combination thereof.
12. The method of any one of claims 10 to 1 1 wherein the cancer is neck cancer.
13. The method of any one of claims 10 to 12 wherein the cancer is melanoma.
14. The method of any one of claims 10 to 13 wherein the cancer is head cancer.
15. A method to pre vent, inhibit or treat autoimmune disease in a mammal, comprising: administering to the mammal a composition comprising one or more Qa-l HLA-E binding peptides, anti-CDl lc antibodies, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a+T regs.
16. A method to prevent, inhibit or treat autoimmune disease in a mammal, comprising: administering to the mammal one or more Qa-l/HLA-E binding peptides, anti-CDl lc antibodies, anti~CD3 antibodies or anti-CD 137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a+T regs.
17. The method of claim. 15 or 16 wherein one or more bi- or tri -specific antibodies are administered.
18. The method of any one of claims 15 to 17 wherein the one or more antibodithat are specific for CD1 lc and CD 137, CD1 l c and CD3, or CD3 and CD 137.
19. The method of any one of claims 15 to 18 wherein the disease is IBD, colitis, lupus or RA .
20. The method of any one of claims 15 to 19 wherein the disease is an autoimmune liver disease.
21. The method of claim 20 wherein die disease is autoimmune hepatitis or primary biliary cirrhosis.
22. The method of any one of claims 15 to 21 wherein the T or B cells in the mammal with the disease are increased relative to a mammal without the disease.
23. A method to prevent, inhibit or treat orgn or graft rejection in a mammal, comprising: administering to the mammal a composition comprising one or more Qa-l/HLA-E binding peptides, anti-CD 1 lc antibodies, anti-CD3 antibodies or anti-CD 137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a+T regs.
2 . A method to prevent, inhibit or treat orgn or graft rejection in a mammal, comprising: administering to the mammal one or more Qa-l/HLA-E binding peptides, anti-CD 1 lc antibodies, anti-CD3 antibodies or anti-CD137 antibodies, or any combination thereof, in an amount effective to stimulate CD8a+T regs.
25. The method of any one of claims 8 to 24 wherein the mammal is a human.
26. The method of any one of claims 8 to 25 wherein the peptide(s), antibodies, or a combination thereof, or the composition, is systemicaliy administered.
27. The method of any one of claims 8 to 26 wherein the peptide(s), antibodies, or a combinantion thereof, or the composition, is locally
administered.
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