WO2018224506A2 - Diagnostic auto-immun à l'aide de populations de lymphocytes t - Google Patents

Diagnostic auto-immun à l'aide de populations de lymphocytes t Download PDF

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WO2018224506A2
WO2018224506A2 PCT/EP2018/064789 EP2018064789W WO2018224506A2 WO 2018224506 A2 WO2018224506 A2 WO 2018224506A2 EP 2018064789 W EP2018064789 W EP 2018064789W WO 2018224506 A2 WO2018224506 A2 WO 2018224506A2
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cells
tfr
cxcr5
tfh
blood
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PCT/EP2018/064789
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WO2018224506A3 (fr
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Luis GRAÇA
Válter FONSECA
Vasco Madeira CRISPIM ROMÃO
João Eurico Cortez CABRAL DA FONSECA
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Instituto De Medicina Molecular
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2812—Immunoglobulins [IGs], 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 against CD4
    • 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
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • the invention relates to the diagnosis of autoimmune disorders, and pertains to methods, uses, products, biomarkers and kits for diagnosing, identifying or stratifying patients with an autoimmune disorder.
  • the invention relates to a kit, product, biomarker, use or method for diagnosing or stratifying Sjogren Syndrome.
  • Autoimmune diseases occur when the immune system mounts an undesired response to a self-antigen. These diseases are characterized by the presence of autoreactive antibodies that arise spontaneously. Autoantibodies are generated as a result of dysregulation of the immune system, in particular the germinal centres.
  • Germinal centres are the main sites of generation of high affinity, antibody-secreting plasma cells and Ig class- switched memory B-cells during T-cell-dependent immune responses.
  • Tfh T follicular helper
  • GC reactions are orchestrated mainly by follicular helper T (Tfh) cells and T follicular regulatory (Tfr) cells.
  • Tfh cells provide cognate help to B cells, thus promoting their clonal selection and affinity maturation (7).
  • Tfr T follicular regulatory
  • Tfr cells are believed to have the potential to regulate and limit the GC reaction assuring antigen-specific antibodies are produced while preventing the generation of auto-antibodies (8).
  • Tfr cells are generally defined by Bcl-6 + CXCR5 + PD-l + ICOS + Foxp3 + and are a distinct subset of thymic Foxp3+ regulatory T cells (Tregs) present in lymphoid tissues.
  • Tfr cell-commitment requires both dendritic cell and B cell interactions, as well as CD28, SAP, ICOS, and PD-1 signaling (10, 11, 14).
  • Tfr cells have specialized functions in controlling the magnitude of GC responses and in limiting the outgrowth of non-antigen- specific B cell clones (9, 10).
  • CTLA-4 and regulation of metabolic pathways seem to play a key role (15-17).
  • Tfh and Tfr cells are characterized by their location in lymphoid tissues, an increasing number of studies have described putative circulating counterparts of these cells in peripheral blood. This is particularly relevant for studying the biology of these cells in humans, as access to secondary lymphoid tissues can be limiting.
  • Human blood CXCR5 + T cells have been established as memory Tfh-like cells, based on the their ability to recapitulate bona fide Tfh cell functions: human blood CXCR5 + T cells can promote plasmablast differentiation, AID expression and class switch recombination by naive B cells. However, they are phenotypically distinct from tissue Tfh cells and do not express the transcriptional repressor Bcl-6 (18-20).
  • CXCR5 -expressing Tregs and GC Foxp3 -expressing T cells have been found in humans (11, 24, 25), so far, no study have addressed the biological significance of these putative circulating Tfr-like cells in humans.
  • Human tonsil CD25 CD69- T cells have been shown to directly suppress B cell responses, but the relationship of these putative Tregs to Bcl-6 + CXCR5 + PD-l + ICOS + Foxp3 + Tfr cells is unclear (26, 27).
  • Peripheral blood CXCR5 + Tregs are being studied as circulating Tfr cells in many different human diseases, despite the biological relevance of these cells being unclear (28-32).
  • Tfh and Tfr cells are therefore of limited utility as access to secondary and tertiary lymphoid tissues can be limiting.
  • CXCR5 -expressing Tregs and GC Foxp3 -expressing T cells have been found in humans, no studies have addressed the biological significance of these putative circulating Tfr-like cells.
  • Tfr cells are thought to play a role in the regulation of GCs, but to date their significance in autoimmune disease remains unclear.
  • human blood Tfr cells defined as CXCR5+ Foxp3+ T cells
  • CXCR5+ Foxp3+ T cells are generated in peripheral lymphoid tissues as humoral immune responses are established.
  • tissue Tfr cells and conventional CXCR5- Tregs circulating Tfr cells have a na ' ive-like phenotype.
  • the present inventors have demonstrated that blood Tfr cells are generated following the initial steps, that lead to germinal centre responses being distinct from tissue Tfr cells. Unexpectedly, they have also found a striking increase in the level of circulating Tfr cells in subjects with autoimmune diseases as compared to age- matched healthy donors.
  • the present invention therefore stems from the inventors' surprising discovery that T follicular regulatory (Tfr) cells, particularly a population identified as CD4 + CXCR5 Foxp3 T, are useful as biomarkers for autoimmune diseases.
  • the invention therefore provides methods of diagnosing autoimmune diseases, biomarkers of autoimmune diseases and kits for the detection of autoimmune diseases.
  • the invention provides a method of diagnosing an autoimmune disease in a subject.
  • said method comprises: obtaining a sample from a subject; detecting the presence or absence of an elevated level of CD4 + CXCR5+ Foxp3+ T cells in the sample; and diagnosing the patient with an autoimmune disease when the presence of an elevated level of CD4 + CXCR5 + Foxp3 + T cells in the sample is detected.
  • an elevated level of CD4 + CXCR5+ Foxp3+ T cells in the sample is a level of CD4 + CXCR5 + Foxp3 + T cells in the sample from a subject that is at least 1.5 times greater than in a sample from a matched control subject, for example an age- matched healthy donor.
  • the elevated level may be 2, 2.5, 5, 10 or more times greater than in a matched control subject.
  • a matched control may be matched for the species, gender, age and/or any other relevant factor.
  • the elevated level may also be in reference to a reference or standard level of CXCR5 + Foxp3 + T cells.
  • the elevated level may be at least 1.5 times greater than a reference level for the subject.
  • the elevated level may be 2, 2.5, 5, 10 or more times greater than the reference level.
  • An elevated level may also be determined by reference to an absolute cut-off value.
  • a level of blood CD4 + CXCR5 + Foxp3 + T cells in excess of 15,000/ml, 20,000/ml, 30,000/ml, 50,000/ml or higher may be considered to be an elevated level.
  • the invention provides a method of diagnosing an autoimmune disease in a subject, said method comprising: obtaining a sample from a subject; determining the level of circulating Tfr cells in the blood; and diagnosing the patient with an autoimmune disease when the level of circulating Tfr cells in the blood is elevated.
  • Example 1 demonstrates the unexpected finding that there is an increased frequency of circulating Tfr cells in the exemplary autoimmune disease Sjogren Syndrome (SS) compared to age-matched healthy donors.
  • SS Sjogren Syndrome
  • an elevated level of Tfr cells in the sample is a level of Tfr cells in the sample from a subject that is at least 1.5 times greater than in a sample from a matched control subject, for example an age-matched healthy donor.
  • the elevated level may be 2, 2.5, 5, 10 or more times greater than in a matched control subject.
  • a matched control may be matched for the species, gender, age and/or any other relevant factor.
  • the elevated level may also be in reference to a reference or standard level of Tfr cells.
  • the elevated level may be at least 1.5 times greater than a reference level for the subject.
  • the elevated level may be 2, 2.5, 5, 10 or more times greater than the reference level.
  • An elevated level may also be determined by reference to an absolute cut-off value.
  • an elevated level of Tfr cells in the sample may be present when greater than 20%, 25%, 30%, 35%, 40% or 50% of Tregs in the sample are CD4 + CXCR5 + Foxp3 + Tfr cells.
  • the invention provides a method of diagnosing an autoimmune disease in a subject, said method comprising: obtaining a sample from a subject; determining the ratio of Tfr/Tfh cells in the sample; and diagnosing the patient with an autoimmune disease when the ratio of Tfr/Tfh cells is greater than 0.2.
  • the ratio of Tfr/Tfh cells may be greater than 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 or higher.
  • the ratio of Tfr/Tfh cells in the sample can depend on the method of measurement of the Tfr and Tfh cells. Different gating strategies can give rise to different values for the ratio of Tfr/Tfh ratio.
  • the gating strategy used when determining the Tfr/Tfh ratio in the Examples is as shown in Figure 4. Briefly, cells were gated on CD4 + T cells, wherein the CD4 + cells were stained for CD25 and Foxp3. Cells that were CD25 + Foxp3 + (double positive) and CD25- Foxp3- (double negative) were gated. Double positive cells were stained for CD4 and CXCR5. CD4 + CXCR5 + cells were gated to isolate CXCR5 + CD25 + Foxp3 + CD4 + Tfr cells. Double negative cells were stained for CD45RO and CXCR5. CD45RO + CXCR5 + cells were gated to isolate CXCR5 + CD45RO + CD25-Foxp3-CD4 + cells Tfh cells.
  • the threshold for diagnosing a patient with an autoimmune disease is 0.1.
  • the different method of measurement returns a different value for the ratio of Tfr/Tfh cells when compared to the methods used in the Examples section of the present invention.
  • the invention therefore also provides a method of identifying subjects with serum autoantibodies comprising obtaining a sample from a subject; determining the ratio of Tfr/Tfh cells in the sample; and diagnosing the patient with an autoimmune disease when the ratio of Tfr/Tfh cells is greater than 0.2.
  • the ratio of Tfr/Tfh cells may be greater than 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 or higher.
  • the invention therefore also provides a method for stratification of subjects with the same underlying autoimmune disease into groups, wherein the groups are characterized by presence of histopathological changes.
  • the autoimmune disease is Sjogren Syndrome (SS)
  • the histopathological changes may include focal sialedenitis in salivary gland biopsies, or ectopic lymphoid structures in inflamed tissues.
  • the invention provides method for stratification of subjects with the same underlying autoimmune disease into groups comprising: a obtaining a sample from a subject; determining the ratio of Tfr/Tfh cells in the sample; and stratifying the subject with an autoimmune disease in different groups based on the ratio of Tfr/Tfh cells.
  • the subjects may be stratified into groups based on whether the ratio of Tfr/Tfh cells is greater than 0.2 or less than 0.2.
  • the subjects may also be stratified into bands based on the ratio of Tfr/Tfh cells.
  • subjects may be stratified into groups that have a ratio of Tfr/Tfh cells from 0.2 to 0.3, 0.3 to 0.4, 0.4 to 0.5, 0.5 to 0.6 or greater than 0.6.
  • the groups may be 0.2 to 0.5, 0.5 to 0.8 and greater than 0.8. The greater the ratio of Tfr/Tfh cells, the greater the likelihood of the presence of histopathological changes.
  • This embodiment of the invention provides a clinician with the ability to stratify subjects using a blood test, thereby avoiding the need to use biopsies, which carry associated risks.
  • the present invention therefore provides a less risky and minimally invasive test for the stratification of subjects with autoimmune disorders.
  • the invention provides a clinician with the ability to stratify subjects with Sjogren Syndrome using a blood test, thereby avoiding the need for salivary gland biopsies which is the conventional method currently used to stratify Sjogren Syndrome.
  • This aspect of the invention also provides a "companion diagnostic" test to identify a subset of subjects that are most likely to require a particular therapeutic treatment following diagnosis.
  • therapeutic agents that are likely to be of use in subsets of subjects who have a higher ratio of Tfr/Tfh cells and/or active disease include therapies targeting T cell - B cell interactions, such as, therapies targeting T cells, B cells, co-stimulation/immune checkpoints, antigen presentation, or cytokines affecting T or B cell function.
  • therapies targeting T cell - B cell interactions such as, therapies targeting T cells, B cells, co-stimulation/immune checkpoints, antigen presentation, or cytokines affecting T or B cell function.
  • Some suitable therapies are set out below.
  • the provision of this companion diagnostic allows for personalised medicine, wherein the patient is identified as likely to benefit from a particular therapy based on their ratio of Tfr/Tfh cells and/or disease state. In certain disclosed embodiments, the patient is treated following the diagnosis or stratification
  • the Tfr cells are CD4 + CXCR5 + Foxp3 + T cells.
  • the Tfr cells may be CD4 + CXCR5 + PD-1 +/- ICOS +/- Foxp3 +/- CD25 +/ T cells.
  • Tfr cells may also be CXCR5 + PD-1 +/- ICOS +/- CD 127- CD25 +/ CD4 + T cells.
  • the Tfh cells may be CXCR5 + CD45RO + Foxp3- CD25- CD4+ T cells.
  • Tfh cells may be CXCR5 + CD25 CD127 + CD4 + T cells.
  • the Tfh cells may be PD-1 + ICOS + and/or PD-1 + CXCR3 GC-like cells.
  • An elevated level of PD-l + ICOS + Tfh cells in a subject may also be used to stratify disease state.
  • an elevated level of PD-1 IC0S + Tfh cells in a subject is indicative of higher autoimmune disease activity.
  • An elevated level of PD-1 IC0S + Tfh cells in a subject can be defined as greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more CD4 + cells also being PD-1 IC0S + .
  • an elevated level of PD-1 IC0S + Tfh cells in a subject can be defined as greater than 100 cells/ml, for example greater than 150, 200, 250, 300, 400 or more PD-l ICOS + Tfh cells/ml in a sample.
  • the sample is a blood sample.
  • stratification may also refer to the identification of specific subject populations who will benefit from different approaches to the monitoring and treatment of their autoimmune disease.
  • stratification refers to the identification of subjects with an elevated Tfr/Tfh ratio. These subjects are more likely to have histopathological changes including tissue infiltration with ectopic lymphoid tissue, and are more likely to suffer from complications of Sjogren syndrome, including lymphoma, and are more likely to have high levels of autoantibodies.
  • Stratification of Sjogren syndrome also relates to the identification of subjects with more active disease. These subjects have an elevated level of PD-l + ICOS + Tfh cells.
  • ESSDAI scale a clinical index designed to measure disease activity in patients with primary Sjogren syndrome.
  • Moderately active disease is usually defined as an ESSDAI >5.
  • An exemplary schematic of the stratification of Sjogren Syndrome is provided in Figure 54.
  • the present invention also provides a method to identify different subsets of subjects with the same underlying autoimmune condition.
  • the invention provides a method of identifying subgroups of subjects with Sjogren Syndrome, comprising determining the Tfr/Tfh ratio in said subject and detecting presence or absence of an elevated level of PD-1 IC0S + Tfh cells in said subject.
  • a Tfr/Tfh ratio of greater than 0.2 and a level of PD-1 IC0S + Tfh cells greater than 100 cells/ml can identify different groups of subjects: (1) Group 1 subjects have an elevated Tfr/Tfh ratio and an elevated level of PD- 1 IC0S + Tfh cells; (2) Group 2 have a normal Tfr/Tfh ratio and an elevated level of PD- 1 IC0S + Tfh cells; (3) Group 3 have an elevated Tfr/Tfh ratio and a normal level if PD- l + ICOS + Tfh cells; and (4) Group 4 have both Tfr/Tfh ratio and a level of PD-l + ICOS + Tfh cells within the normal range. The identification of these groups of subjects is depicted in Figure 51.
  • the ratio of Tfr/Tfh cells in the sample refers to one of: the ratio of CXCR5 + CD25 + Foxp3 + CD4 + T cells to CXCR5 + CD45RO + CD25 Foxp3-CD4 + T cells; the ratio of CD4 + CXCR5 + Foxp3 + T cells to CXCR5 + Foxp3- CD4 + T cells; the ratio of CXCR5 + PD-1 +/- ICOS +/ - Foxp3 + CD25+ 7- CD4 + T cells to CXCR5 + CD45RO + PD-1 +/- ICOS +/- Foxp3- CD25 CD4 + T cells; or the ratio of CXCR5 + CD127 CD25 + CD4 + T cells to CXCR5 + CD45RO + CD127 + CD25-CD4 + T cells.
  • the sample may be any suitable tissue sample. Suitable samples include blood, plasma, lymphoid tissues, and other tissues with tertiary lymphoid organs. In all embodiments of the invention, the sample may be an ex vivo sample and all methods may be carried out ex vivo, for example in vitro.
  • the subject may be an animal subject, preferably a mammalian subject. For example, in certain embodiments, the subject is a mouse, a rat, a guinea pig, ovine, bovine, porcine, or primate. Typically the subject is a human.
  • diagnosis of an autoimmune condition using methods according to the invention may optionally be followed by a step of treating the subject with a suitable therapy for ameliorating the autoimmune disease.
  • therapeutics that target the T-B interaction are envisaged for use in the invention.
  • Specific therapeutic agents include: B-cell depleting therapies (e.g., rituximab); anti-CD22 antibodies (e.g. Epratuzumab); LTbetaR-Ig (e.g. Baminercept); Rebamipide; glucocorticoids; anti-BAFF mAb (e.g.
  • Belimumab anti-CD40 mAb; anti-CD28; CTLA4-Fc Ig (e.g abatacept and belatacept); low-dose IL-2; anti IL-2, IL-2 immune complexes, or modified IL-2 molecules; Iguratimod; JAK inhibitors; anti-TNF mAb; anti-IL6/IL-6R; cathepsin S antagonists; cyclophosphamide; azathioprine; cyclosporine; mycophenolate mofetil; cyclophosphamide; hydroxychloroquine or methotrexate.
  • the invention also provides biomarkers for autoimmune diseases.
  • a bio marker is a measurable indicator of the severity or presence of the disease state.
  • the invention provides the use of CD4+ CXCR5+ Foxp3+ T cells as a biomarker for humoral activity and/or a biomarker for autoimmune disease.
  • the invention also provides the use of the ratio of Tfr/Tfh cells as a biomarker of increased germinal centre activity and/or an autoimmune disease and/or autoimmune disease activity or severity.
  • the invention further provides the use of PD-1 IC0S + Tfh cells as a biomarker of autoimmune disease and/or autoimmune disease activity or severity.
  • the invention provides the use of CD4 + CXCR5 + Foxp3 + T cells as a biomarker for autoimmune disease.
  • the level of CD4 + CXCR5 + Foxp3 + T cells is elevated.
  • An elevated level of CD4+ CXCR5+ Foxp3+ T cells in the sample is a level of CD4 + CXCR5 + Foxp3 + T cells in the sample from a subject that is at least 1.5 times greater than in a sample from a matched control subject, for example an age-matched healthy donor.
  • the elevated level may be 2, 2.5, 5, 10 or more times greater than in a matched control subject.
  • a matched control may be matched for the species, gender, age and/or any other relevant factor.
  • the elevated level may also be in reference to a reference or standard level of CXCR5 + Foxp3 + T cells.
  • the elevated level may be at least 1.5 times greater than a reference level for the subject.
  • the elevated level may be 2, 2.5, 5, 10 or more times greater than the reference level.
  • An elevated level may also be determined by reference to an absolute cut-off value.
  • an elevated level of CD4 + CXCR5 + Foxp3 + T cells in the sample may be present when greater than 20%, 25%, 30%>, 35%, 40% or 50% of Tregs in the sample are CXCR5 + Foxp3 + Tregs.
  • the present invention provides the use of the ratio of Tfr/Tfh cells as a biomarker to identify subgroups of disease state in subjects with autoimmune diseases, and in particular in subject with Sjogren Syndrome.
  • Example 7 demonstrates that peripheral blood Tfr/Tfh ratio correlates with salivary gland infiltration by lymphocytes, providing an indicator of these pathological changes in SS patients.
  • the present inventors have surprisingly found that the ratio of Tfr/Tfh is predictive of ectopic lymphoid structure activity, the presence of autoantibodies and levels of disturbed germinal centre activity.
  • a Tfr/Tfh ratio of greater than 0.2 is indicative of pathological changes in Sjogren Syndrome including ectopic lymphoid structure activity, salivary gland infiltration by lymphocytes, focal sialoadentitis, autoantibody production and/or disturbed germinal centre activity.
  • the predictive nature of the ratio of Tfr/Tfh cells in a subject with Sjogren Syndrome can avoid the need for a salivary gland biopsy. This provides the advantage of reducing the risk to the patient and provides the clinician with a minimally invasive test for identifying patients with certain characteristics and who will respond to certain therapeutic approaches.
  • an increased Tfr/Tfh ratio is a biomarker of ectopic GC responses in target organ of Sjogren Syndrome.
  • Subjects with an ectopic GC response in a target organ are suitable subjects for treatment with therapies targeting T-B interactions, such as therapies targeting T cells, B cells, co-stimulation/immune checkpoints, antigen presentation, or cytokines affecting T or B cell function.
  • therapies targeting T-B interactions such as therapies targeting T cells, B cells, co-stimulation/immune checkpoints, antigen presentation, or cytokines affecting T or B cell function.
  • this invention may aid in clinical stratification of Sjogren's syndrome patients.
  • Specific therapeutic agents include: B- cell depleting therapies (e.g., rituximab); Epratuzumab (anti-CD22); Baminercept (LTbetaR- Ig); Rebamipide; glucocorticoids; anti-BAFF mAb (Belimumab); anti-CD40 mAb; anti- CD28; CTLA4-Fc Ig (e.g abatacept and belatacept); low-dose IL-2; anti IL-2, IL-2 immune complexes, or modified IL-2 molecules; Iguratimod; JAK inhibitors; anti-TNF mAb; anti- IL6/IL-6R; cathepsin S antagonists; cyclophosphamide; azathioprine; cyclosporine; mycophenolate mofetil; cyclophosphamide; hydroxychloroquine or methotrexate.
  • B- cell depleting therapies e.g., rituximab
  • the invention therefore provides a method of identifying subjects suitable for treatment with therapies targeting T-B interactions comprising determining the Tfr/Tfh ratio; identifying subjects with an elevated Tfr/Tfh ratio; and optionally providing said subjects with a therapy targeting T-B interactions.
  • the present inventors have also found that circulating PD-1 + ICOS + T cells are elevated in subjects with an autoimmune disease.
  • the invention therefore provides the use of PD- 1 IC0S + Tfh cells as a biomarker of autoimmune disease and/or autoimmune disease activity or severity.
  • the presence of PD-l + ICOS + Tfh cells and/or the level of PD-l + ICOS + Tfh cells in a subject may be determined as a stand-alone diagnostic test or as a stand-alone biomarker of autoimmune disease.
  • the presence of PD-1 IC0S + Tfh cells and/or detecting an elevated level of PD-1 IC0S + Tfh cells in a subject may be used in combination with other diagnostic methods and biomarkers of the invention.
  • detecting presence or absence of PD-l + ICOS + Tfh cells and/or detecting presence or absence of an elevated level of PD-1 IC0S + Tfh cells in a subject may be used in combination with determining the Tfr/Tfh ratio in said subject.
  • Example 7 demonstrates a striking increase in PD-l + ICOS + Tfh cells in SS.
  • Biomarkers may be detected in any sample from a subject.
  • the biomarker is detected in a blood sample, a plasma sample, a tissue sample such as skin, tonsil, lymph nodes, spleen, or other organs with tertiary lymphoid structures.
  • the sample is a blood sample.
  • the invention provides kits for the detection of the biomarkers disclosed herein and kits for carrying out the methods of diagnosis of autoimmune diseases as described herein.
  • kits of the invention may comprise 2 or more, for example 3 or more, 4 or more, 5 or more, 6 or more reagents for the detection of Tfr and Tfh cells.
  • kits comprise antibodies for the detection of Tfr and Tfh cells, including at least 2 of the following: anti-CD4 (optionally, OKT4, BioLegend), anti-CD45 (optionally, HBO, BioLegend), anti-CD45RO (optionally, UCHL1, BioLegend), anti-CXCR5 (optionally, J252D4, BioLegend), anti-Foxp3 (optionally, PCH101, eBioscience), anti-PD-1 (optionally, EH12.2H7, BioLegend), and/or anti-ICOS (C398.4A, BioLegend). Kits may also optionally include instruction for use.
  • the invention provides a system for assessing whether a subject has an autoimmune disease the system comprising: detection means able and adapted to detect in a sample, optionally a blood or plasma sample, the ratio of Tfr/Tfh and/or the level of Tfr and/or Tfh cells; and a processor able and adapted to determine from the detected ratio and/or level of cells an indication of the patient having an autoimmune disease.
  • the system optionally contains a data connection to an interface, particularly a graphical user interface, capable of presenting information.
  • the autoimmune disease may include autoimmune haematological disorders (including e.g. haemolytic anaemia, aplastic anaemia, pure red cell anaemia and idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, scleroderma, Wegener granulomatosis, Churg-Strauss syndrome, dermatomyositis, myasthenia gravis, psoriasis, Steven- Johnson syndrome, celiac disease, autoimmune inflammatory bowel disease (including e.g.
  • ulcerative colitis and Crohn's disease Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, diabetes, e.g. juvenile diabetes (diabetes mellitus type I), Bechts Disease, Sjogren Syndrome, psoriasis and psoriatic arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, spondyloarthropathies, and glomerulonephritis (with and without nephrotic syndrome, e.g. including idiopathic nephrotic syndrome or minimal change nephropathy), asthma and other inflammatory airways diseases including an autoimmune component, thyroiditis (Hashimoto disease), inflammatory conditions of the central nervous system, and similar autoimmune disorders.
  • diabetes e.g. juvenile diabetes (diabetes mellitus type I), Bechts Disease, Sjogren Syndrome, psoriasis and psoriatic arthritis, rheumatoid
  • the autoimmune leads to the formation of ectopic lymphoid structures, such as those see in rheumatoid arthritis and Sjogren Syndrome.
  • the autoimmune disease is Sjogren Syndrome.
  • the subject is a human
  • the sample is a blood sample
  • the autoimmune disease is Sjogren Syndrome.
  • the invention provides screening methods for the identification of agents that can alter the Tfr/Tfh ratio.
  • the invention provides a method of screening for an agent that can alter the Tfr/Tfh ratio comprising the steps of: obtaining a first baseline sample from a subject; administering an agent to a subject; obtaining a second sample from a subject subsequent to administering the agent; and comparing the Tfr/Tfh ratio in the first and second samples.
  • the method may further optionally comprise the step of selecting an agent that increases or reduces the Tfr/Tfh ratio.
  • the invention provides methods of treatment of autoimmune diseases comprising administering an agent to normalise the Tfr/Tfh ratio.
  • the invention also provides the use of an agent that normalises the Tfr/Tfh ratio for the treatment of an autoimmune disease.
  • the agent may have been identified using the screening method of the previous embodiment.
  • the invention provides methods of treating Sjogren syndrome and methods of screening for agents that alter the Tfr/Tfh ratio in subjects with Sjogren syndrome.
  • Expression of cell surface markers may be determined, for example, by means of flow cytometry and/or FACS for a specific cell surface marker using conventional methods and apparatus (for example a Beckman Coulter Epics XL FACS system used with commercially available antibodies and standard protocols known in the art) to determine whether the signal for a specific cell surface marker is greater than a background signal.
  • the background signal is defined as the signal intensity generated by a non-specific antibody of the same isotype as the specific antibody used to detect each surface marker.
  • the specific signal observed is typically more than 20%, preferably stronger than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000%, 10000% or above, greater relative to the background signal intensity.
  • Tfr cells, Tfh cells, PD-1 TCOS CD4 + T cells and CXCR5 Foxp3 CD4 + T cells can be detected by flow cytometry.
  • Tfr cells are CXCR5 + Foxp3 + CD4 + T cells, and are identified by flow cytometry using anti-CXCR5 antibodies and anti-Foxp3 antibodies.
  • Tfh cells are CXCR5 + CD45RO cells and are identified by flow cytometry using anti-CXCR5 antibodies and anti- CD45RO antibodies.
  • the PD-l + ICOS + CD4 + T cells can be identified by flow cytometry using anti-PD-1 antibodies and anti-ICOS antibodies.
  • Anti-CD4 OKT4, BioLegend
  • anti-CD45 HI30, BioLegend
  • anti-CD45RO UCHL1, BioLegend
  • anti- CXCR5 J252D4, BioLegend
  • anti-Foxp3 PCHlOl, eBioscience
  • anti-PD-1 EH12.2H7, BioLegend
  • anti-ICOS C398.4A, BioLegend
  • the term "positive” and “+” when used in regard to a marker means that, in a cell population, more than 20%, preferably more than, 30%, 40%, 50%, 60%, 70%, 80%, 90%) 95%), 98%), 99%) or even all of the cells express said marker.
  • negative or "-" as used with respect to markers means that in a cell population, less than 20%, 10%, preferably less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 % or none of the cells express said marker.
  • a positive signal is more than 20%>, preferably stronger than 30%>, 40%>, 50%>, 60%>, 70%>, 80%, 90%, 100%, 500%, 1000%, 5000%, 10000% or above, greater relative to the background signal intensity.
  • the term "positive” and “+” with reference to multiple markers means that the cells expresses all of the positive markers at a detectable level.
  • the positive" and “+” with reference to multiple markers means that the cell population is made up of more than 20%, preferably more than, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 98%, 99% cells that express each of said markers.
  • Figure 3 a) Variation of blood Tfr:Tfh ratio accordingly to c-reactive protein (CRP). Analysis by linear regression, b) Variation of blood Tfr:Tfh ratio accordingly to disease severity score (ESSDAI). Analysis by linear regression.
  • FIG. 4 Activated programmed death 1 -positive (PD-1 + ) inducible costimulator-positive (ICOS ) follicular helper T (Tfh) cells in peripheral blood indicate disease activity in primary Sjogren's syndrome (SS).
  • SS primary Sjogren's syndrome
  • Figure 9 Expression of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1 Bcl-6, and CD57 by Tfh cells (blue), CXCR5 Tregs (black) and Tfr cells (red), in children blood (top rows) and in tonsils (bottom rows). Na ' ive CD4 + T cells were used as control (gray). Representative plots from 6 healthy children. CXCR5 subsets in tonsils were defined as CXCR5 + ICOS + cells.
  • Figure 10 a) Immunofluorescence microscopy of formalin- fixed paraffin-embedded human tonsils stained for DAPI (blue), CXCR5 (yellow), CD4 (red) and Foxp3 (green). Top, middle and bottom outlined areas indicate top, middle and bottom enlarged areas on the right, respectively. Data are representative of tonsil sections from 5 healthy children.
  • FIG. 11 Blood Tfr cells are a distinct subset of suppressive Tregs.
  • FIG. 17 Blood Tfr cells are immature but are not committed as such in the thymus, a) Backgate of CXCR5- and CXCR5 + Tregs accordingly to CD45RO and Foxp3 expression, b) Expression of D45RO, CD45RA, CCR7, CD62L, HLA-DR and CD27 by Tfr cells (red) and CXCR5 Tregs (black) in blood.
  • Figure 22 a) Model of CXCR5 + follicular helper and regulatory cells T cells generation and recirculation in humans, upon antigen stimulation. Tfh cells in red and Tfr cells in blue.
  • Figure 26 Blood Tfr cell:Tfh cell ratio identifies pathologic lymphocytic infiltration in the target organ of SS.
  • values are the mean ⁇ SEM.
  • significance was determined by Student's unpaired t-test with Wel
  • Figure 30 Identification, by immunohistochemistry, of CXCR5 FoxP3 + Tfr cells (arrows) within focal sialadenitis containing CD20 + B cells in MSG biopsy samples from patients with primary SS.
  • Far left image shows focal sialadenitis containing CD20 + B cells (brown).
  • FIG 31 Blood Tfr celkTfh cell ratio as a marker of primary SS and focal sialadenitis (FSA).
  • FSA focal sialadenitis
  • ROC Receiver operating characteristic
  • AUC area under the curve
  • significance was determined by Student's unpaired t-test with Welch's correction for variance except when variance was not significantly different between groups (patients with non-SS sicca syndrome versus healthy donors); one-way analysis of variance was used to compare all 3 groups.
  • significance was determined by Mann- Whitney U test due to skewed distribution of values and nondifferent variance according to the Brown- Forsythe test.
  • Figure 32 a) Odds ratio (OR), P value, and AUC of logistic regression models predicting SS diagnosis (versus healthy donors and versus patients with non-SS sicca syndrome) and focal sialadenitis (versus normal histology or nonspecific lymphocytic infiltration [LI]) based on the Tfr cell:Tfh cell ratio as a continuous variable or as specific cutoffs.
  • the Tfr cell:Tfh cell ratio is transformed by 1 decimal place (x 10 1 ) for better interpretation of the OR. Shown are percentages of sensitivity, specificity, and correct classification of patients based on given cutoffs.
  • NA not applicable
  • b) ROC curve for prediction of focal sialadenitis diagnosis (versus normal histology/nonspecific lymphocytic infiltration) based on the Tfr cell:Tfh cell ratio.
  • Figure 33 a) Microscopy of formalin- fixed paraffin-embedded salivary glands stained for CD4 (top) and CD20 (bottom) by immunohistochemistry. Data are representative of salivary gland sections from 25 SS patients, with unspecific lymphocytic infiltration (LI) (left) and focal sialoadenitis (FSA) (right), b) ROC curve analysis of peripheral blood Tfr/ Tfh ratio for SS diagnosis.
  • LI lymphocytic infiltration
  • FSA focal sialoadenitis
  • Figure 37 Expression of regulatory and follicular markers by adult blood Tfh cells, CXCR5- T regs and Tfr cells. Expression of Foxp3, CD25, CD69, CTLA-4, CXCR5, ICOS, PD-1 Bcl- 6, and CD57 by Tfh cells (blue), CXCR5 Tregs (black) and CXCR5 + Tfr cells (red) in adult blood. Naive CD4+ T cells were used as control (gray). Representative plots from 42 healthy volunteers.
  • CD38 hl IgD- germinal center B cells sorted from human tonsils were used as positive control, c) Negative control for immunofluorescence microscopy, merge (left) and composite of the four immunofluorescence channels (right).
  • Negative control for immunofluorescence microscopy merge (left) and composite of the four immunofluorescence channels (right).
  • DAPI Alexa-Fluor 488 (anti-mouse), Alexa-Fluor 546 (anti-rabbit) and AlexaFluor (anti-Rat) secondary antibodies, without primary antibodies.
  • DAPI was used as nuclei counter staining. Unspecific binding of secondary antibodies and cross-reactivity between secondary antibodies were excluded. Error bars represent SEM.
  • Figure 40 Sorting strategy for human blood na ' ive B cells, Tfh cells, CXCR5- T regs and Tfr cells. Sorting strategy for FACS-sort of CXCR5 CD25 CD127 CD4 + Tfh cell, CXCR5 CD25 CD127 + CD4 + Tconv cell, CXCR5 CD25 CD127 CD4 + Tfr cells, CXCR5 CD25 CD127 CD4 Tregs, and CD27-IgD CD 19 naive B cell populations from peripheral blood (buffy-coats).
  • Figure 45 Effector memory, central memory and na ' ive subsets of human blood CXCR5- T regs and Tfr cells in SS and BTK patients.
  • Figure 46 Optimization of enzymatic digestion of salivary gland tissue from SS patients.
  • Figure 47 Validation of ICOS + PD-l + CD4 + T cells for identification of Tfh cells, a) CXCR5 and ICOS expression by salivary gland CD19 + B cells and CD4 + T cells after enzymatic digestion by Liberase TM (O. lmg/mL) plus DNAse I (O.lmg/mL). Representative plots of 25 salivary gland biopsies, b) Microscopy of formalin- fixed paraffin-embedded salivary glands stained for CXCR5 (right), with correspondent negative control (left) by immunohistochemistry. Data are representative of salivary gland sections from 4 SS patients, c) CXCR5 and Bcl-6 expression by ICOS + PD-l + CD4 + T cells from human tonsils. Representative plots from 6 healthy children.
  • Figure 48 Characteristics of minor salivary gland biopsies of primary Sjogren's syndrome patients. Impact of different enzymes in membrane markers used for identification of cellular components of minor salivary gland tissue infiltrates, including CXCR5 -expressing T cells. Human tonsil fragments from healthy children were used for optimization. Liberase TM (0.1 mg/mL), DNAse I (0.1 mg/mL), Collagenase P (0.1 mg/mL). All enzymes led to significant duction of CXCR5 on PD-1 + CD4 + T cells. Representative plots of 2 independent experiments.
  • Figure 50 Identification of ectopic lymphoid structures in minor salivary glands of Sjogren's syndrome patients, a) Correlation between SS disease activity as measured by ESSDAI and histological diagnosis (normal, no infiltration by lymphocytes; LI, unspecific lymphocytic infiltration; FSA, focal sialadenitis), b) Microscopy of formalin- fixed paraffin-embedded MSG stained for CD20, CD21 and Bcl-6 in a SS patient with FSA harboring features of ectopic lymphoid structures (ELS) with ectopic germinal centre formation (top) and without ELS (bottom), c) Number of primary SS patients with normal, LI and FSA patients further characterized for the presence of ELS and ectopic germinal centre accordingly to CD21 and Bcl-6 immunohistochemistry. Bars on scatterplots represent SEM.
  • ELS ectopic lymphoid structures in minor salivary glands of Sjogren's syndrome patients
  • Figure 51 Flowchart of patient recruitment and selection.
  • DMARDs disease modifying antirheumatic drugs
  • TNF tumor necrosis factor
  • AECG American-European Consensus Group criteria for Sjogren's syndrome.
  • Figure 52 Demographic and clinical characteristics of primary Sjogren's syndrome (SS) and non-Sjogren sicca syndrome (non-SSS) patients.
  • Figure 54 An exemplary stratification scheme for Sjogren Syndrome, where ESSDAI represents disease activity (high or low) and FSA represents focalsialedenitis (ie. infiltration of salivary glands with ectopic lymphoid structure, or, in other words, biopsy with pathological changes).
  • ESSDAI disease activity
  • FSA focalsialedenitis
  • Germinal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high affinity antibodies.
  • Tfh T follicular helper
  • Tfr T follicular regulatory
  • Blood Tfr cells have a na ' ive-like phenotype, although they are absent from human thymus or cord blood. Here, we found, these cells were generated in peripheral lymphoid tissues prior to T-B interaction, as they are maintained in B-cell deficient patients. Therefore, blood CXCR5 Foxp3 + T cells in human pathology can be used as a biomarker for ongoing humoral activity, and can predict the results of diagnostic biopsies in Sjogren Syndrome. Given our results it is anticipated that level of circulating Tfr cells or blood Tfr/Tfh ratio can be useful for diagnosis in other immune-mediated and autoimmune diseases.
  • DMARDs Disease Modifying Anti-Rheumatic Drugs
  • PBMCs were isolated from blood samples by Ficoll-gradient medium (Histopaque-1077, Sigma- Aldrich) using SepMate tubes (StemCell Technologies). Lymphocytes from tonsils and thymocytes were also isolated by Ficoll-gradient medium after mechanical disruption. Before cell sorting, PBMCs from Buffy-coats were enriched for CD4 + T cells using Mojo Sort Human CD4 T Cell Isolation Kit (BioLegend). The CD4 + fraction was used for cell sorting of CD4 + T cell subsets. The CD4- fraction was used for cell sorting of na ' ive B cells ( Figure S3A for sorting strategy). A cell suspension was prepared from salivary gland tissue for flow cytometry analysis.
  • salivary gland was cut into small fragments and incubated at 37.°C with Liberase TM O. lmg/mL plus DNAse I O. lmg/mL in RPMI medium for 20 minutes. After washing, fragments were incubated again for 10 minutes incubation with the same enzyme solution. After washing, fragments were vigorously pipetted and filtered to obtain a cell suspension.
  • Cell Trace Violet Cell Proliferation Kit (Life Technologies) was used for cell proliferation assessment.
  • Cell sorting was performed in Aria IIu and Aria III instruments (BD Biosciences).
  • Flow cytometry analysis was performed in a LSR Fortessa instrument (BD Biosciences) and further analyzed with FlowJo vlO software (TreeStar).
  • TCR stimulation assays 25 x 10 3 CXCR5 CD25 CD127- CD4 + Tregs cells and CXCR5 CD25 CD127 CD4 + Tregs were plated with 1 ⁇ L/well of anti- CD3/anti-CD28 MACSiBead particles (T Cell Activation Kit, Miltenyi Biotec).
  • 25 x 10 3 CXCR5 + CD25-CD127 + CD4 + Tfh cells were plated with CXCR5 CD25 CD127 CD4 + Tregs cells or CXCR5 CD25 CD127 CD4 + Tregs cells in 1 : 1 ratio, in the presence of 30 x 10 3 CD27-IgD + CD19 + naive B cells.
  • Tfh cells were cultured with ⁇ g/mL SEB (Sigma- Aldrich). After 5 days responder Tfh cells were analyzed for CTV dilution, B cells for CD38 upregulation and Tregs for follicular and activation markers.
  • Tfh cells were plated with CXCR5 CD25 CD127 CD4 + Tregs cells or CXCR5 CD25 CD127 CD4 + Tregs cells in 1 : 1 ratio, in the presence of 30 x 10 3 CD27-IgD CD19 + naive B cells.
  • SS Sjogren syndrome
  • AECG American European Consensus Group
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • PBC primary biliary cirrhosis
  • ESSDAI EULAR Sjogren's syndrome disease activity index
  • PDN prednisolone
  • HCQ hydroxychloroquine.
  • Example 2 Blood and tissue Tfr cells present different follicular and regulatory markers
  • Realtime PCR was set up with Power SYBR Green PCR Master Mix (Applied Biosystems) and performed on ViiA 7 Real-Time PCR System (Applied Biosystems), according to manufacturer instructions.
  • each gene was normalized to housekeeping genes (B2M, ACTB or G6PD) and calculated by change-in-threshold method (ACT), using QuantStudio Real-Time PCR software vl . l (Applied Biosystems).
  • DAPI was used as nuclei counterstaining. Images were acquired with ZEN 2012 software on a Zeiss LSM 710 confocal point-scanning microscope (Carl Zeiss, Oberkochen, Germany) using a dry plan-apochromat 20x objective (200x magnification) and with a numerical aperture of 0.80. Images were further analyzed using Image-J Fiji software. For immunohistochemistry, salivary gland tissues were stained with anti-human CXCR5-Alexa-Fluor 488, anti-human CD4 and CD20, using DAKO EnVision+ System-HRP labeled polymer. Hematoxylin was used as counterstaining. Images were acquired with NanoZoomer-SQ (Hamamatsu, Japan) using a 20x objective with a numerical aperture of 0.75. Images were further analyzed using NDP.view software.
  • CXCR5 is used to identify human circulating Tfh cells
  • Peripheral blood CXCR5 Foxp3 + T cells share characteristics with both circulating Tfh cells and CXCR5- Tregs ( Figure 9).
  • Taking advantage of routine tonsillectomies performed due to tonsil hypertrophy in otherwise healthy children we compared the cell phenotype of paired blood and tissue samples from the same child ( Figure 9, Figure 36b).
  • CXCR5 expression can transiently occur upon human T cell activation (34, 39, 40).
  • human T cells can also transiently express Foxp3 upon in vitro TCR stimulation in a TGF- ⁇ dependent manner (33, 41).
  • Tfr cells in tonsils have follicular and regulatory markers and were found within germinal centers, whereas blood Tfr cells do not express ICOS, PD-1, or Bcl-6, apparently diverging these cells from follicular imprinting.
  • Previous studies have described low ICOS and PD-1 expression, and no Bcl-6 expression in human blood Tfh cells (18).
  • blood Tfr cells have also lower expression of ICOS (13).
  • murine circulating Tfr cells can bypass the B cell zone and do not gain full activation as part of a memory programmed state (13). In line with these studies, the absence of ICOS, PD-1 and Bcl-6 from human blood CXCR5 + Tfr cells does not exclude their follicular ontogeny.
  • ELISA IgA, IgM and total IgG concentration were determined in supernatants from T-B co-culture (as described above) by ELISA using Human ELISA Ready Set Go Kit, according to manufacturer instructions (eBioscience).
  • 96-well permeable supports (5 ⁇ pore size) (Corning). Plain RPMI medium (RPMI 1640, Life Technologies) or that supplemented with O ⁇ g/mL CXCL13 (Peprotech) was added to the bottom wells of the plate. After 4 hours of incubation (37. °C, 5% C0 2 ), filters were removed and cells that migrated to the lower chamber were counted in a LSR Fortessa instrument (BD Biosciences) and further analyzed with Flow Jo vlO software (TreeStar). Chemotaxis index was calculated as the ratio of cells migrating toward CXCL13 and cells randomly migrating.
  • mice and humans show key differences between mice and humans regarding the function of blood CXCR5 Tfr cells: while murine blood Tfr cells appear to be specialized in suppressing antibody production (despite their lower suppressive capacity when compared to tissue Tfr cells) (13, 14, 16, 54), human blood Tfr cells do not have the ability to fully suppress humoral responses.
  • CXCR5- conventional Tregs did not upregulate CXCR5 upon in vitro activation, further confirming CXCR5 -expressing Tfr cells as a distinctive subset.
  • Example 5 Blood Tfr cells have distinctive naive-like phenotype
  • circulating Tfr cells were virtually devoid of CD45RO CCR7- effector-memory cells in striking contrast to CXCR5- Tregs, a phenotype more similar to circulating Tfh cells ( Figure 18b). While the vast majority of blood Tfh cells were CD45RO CCR7 central-memory cells, consistently with previous reports (18-21), a significant proportion of Tfr cells were CD45RO-CCR7 + naive cells ( Figure 18b). Furthermore, the few CD45RO- Tfh cells did not express high levels of CD45RA indicating that those cells were not really naive, in contrast to Tfr cells ( Figure 43a). Therefore, blood Tfr cells constitute a pool of na ' ive resting cells.
  • Tfr cells have a prominent na ' ive phenotype. However, they are absent from the thymus and cord blood (where activated Tregs can already be found).
  • Tfr cells recirculate from secondary lymphoid tissues before being fully committed to tissue Tfr cells.
  • Example 7 Blood Tfr/ Tfh ratio is a biomarker of ectopic GC responses in Sjogren Syndrome
  • Tfr / Tfh ratio is a biomarker of ectopic GC formation in the target tissue of SS.
  • salivary gland biopsies of our SS cohort compared to salivary gland tissue of patients referred to salivary gland biopsy not fulfilling any of SS diagnostic criteria ( Figure 46).
  • SS patients showed a significant increase in CD45 -expressing hematopoietic cells in salivary gland tissue, as well as B cells ( Figure 23).
  • peripheral blood Tfr / Tfh ratio correlated with salivary gland infiltration by lymphocytes ( Figure 24b), being a good predictor of these pathological changes in SS patients ( Figure 25 a). Therefore, our data suggest that peripheral blood Tfr / Tfh ratio is a biomarker of ectopic lymphoid structure activity in SS. Indeed, Tfr / Tfh ratio discriminated SS patients with no histological changes in their salivary gland tissue, patients with unspecific lymphocytic infiltration and patients with diagnostic focal sialoadenitis (Figure 25b, Figure 33a). Additionally, Tfr / Tfh ratio was found to be a potential new diagnostic tool to identify the subgroup of SS patients with the highest level of disturbed GC activity ( Figure 33b).
  • Tfr / Tfh ratio is a biomarker of ectopic GC responses in target organ of SS. Therefore, Tfr / Tfh ratio is a putative novel diagnostic tool to identify more suitable SS patients to therapies targeting T-B interactions.
  • Tfr cells have an immature phenotype
  • blood Tfr cells are not fully endowed with suppressive function, because the suppressive capacity of conventional Treg cells have been ascribed predominantly to those cells with a more mature phenotype.
  • the TCR repertoire of Tfr cells is different from Tfh and probably skewed towards auto-antigens (55), it is possible that circulating Tfr cells represent a pool of cells ready to be recruited into subsequent GC responses as they retain the ability to migrate towards CXCL13.
  • the coinhibitory receptor CTLA-4 controls B cell responses by modulating T follicular helper, T follicular regulatory, and T regulatory cells, Immunity 41, 1026-1039 (2014).
  • CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype, Blood 110, 2983-2990 (2007).
  • TFR follicular regulatory T

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Abstract

L'invention concerne le diagnostic de troubles auto-immuns, et concerne des méthodes, des utilisations, des produits, des biomarqueurs et des kits permettant de diagnostiquer, d'identifier ou de stratifier des patients atteints d'un trouble auto-immun. En particulier, l'invention concerne un kit, un produit, un biomarqueur, une utilisation ou une méthode permettant de diagnostiquer ou de stratifier le syndrome de Sjögren.
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