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Definitions

• the invention relates to therapeutic and drug screening methods.
• a variety of diseases are characterized by the development of progressive immunosuppression in a patient.
• the presence of an impaired immune response in patients with malignancies has been particularly well documented.
• Cancer patients and tumor-bearing mice have been shown to have a variety of altered immune functions such as a decrease in delayed type hypersensitivity, a decrease in lytic function and proliferative response of lymphocytes.
• Many other diseases or interventions are also characterized by the development of an impaired immune response.
• T lymphocytes are critical in the development of all cell-mediated immune reactions. Helper T-cells control and modulate the development of immune responses. Cytotoxic T-cells (killer T-cells) are effector cells which play an important role in immune reactions against intracellular parasites and viruses by means of lysing infected target cells. Cytotoxic T-cells have also been implicated in protecting the body from developing cancers through an immune surveillance mechanism. Regulatory T cells block the induction and/or activity of T helper cells. T-cells do not generally recognize free antigen, but recognize it on the surface of other cells. These other cells may be specialized antigen-presenting cells capable of stimulating T cell division or may be virally- infected cells within the body that become a target for cytotoxic T-cells.
• Cytotoxic T-cells usually recognize antigen in association with class I Major Histocompatibility Complex (MHC) products which are expressed on all nucleated cells. Helper T-cells, and most T-cells which proliferate in response to antigen in vitro, recognize antigen in association with class II MHC products. Class II products are expressed mostly on antigen-presenting cells and on some lymphocytes. T-cells can be also divided into two major subpopulations on the basis of their cell membrane glycoproteins as defined with monoclonal antibodies.
• MHC Major Histocompatibility Complex
• the CD4+ subset which expresses a 62 kD glycoprotein usually recognizes antigen in the context of class II antigens, whereas the CD8+ subset expresses a 76 Kd glycoprotein and is restricted to recognizing antigen in the context of Class I MHC.
• T-cell mediated autoimmune and inflammatory diseases are characterized by deleterious T- cell activity in which T-cells which recognize self antigens proliferate and attack cells which express such antigens.
• Other examples include the occurrence of graft rejection mediated by host T-cells and graft vs. host disease.
• immunosuppressive therapies available to treat these conditions include administration of immunosuppressive compounds such as cyclosporine A, FK506 and rapamycin.
• these therapies are not completely effective and are associated with significant adverse side effects such as nephrotoxicity, hepatotoxicity, hypertension, hirsutism, and neurotoxicity.
• additional therapies which can more effectively suppress T-cell activity with fewer side effects are needed to treat these conditions.
• Lymphocyte homeostasis is a central biological process that is tightly regulated. Tanchot, C. et al, Semin.Immunol. 9: 331-337 (1997); Marrack, P. et al., Nat.Immunol. 1: 107-111 (2000); C. D. Surh, CD. and Sprent, J., Microbes.Infect. 4: 51-56 (2002); Jameson, S.C., Nat.Rev.Immunol. 2: 547-556 (2002). While the molecular control of this process is poorly understood, molecules involved in mediating two signaling pathways are thought to be essential.
• MHC self major histocompatibility
• T cell receptor (TCR) expression is required for the continued survival of na ⁇ ve T cell. Polic, B. et al, Proc.Natl. Acad. Sci. 98: 8744-8749 (2001); Labrecque, N. et al., Immunity 15: 71-82 (2001). Second, cytokines that signal via the common gamma ( ⁇ c) chain are critical for na ⁇ ve T cell survival and homeostasis, particularly interleukin-7 (IL-7).
• IL-7 interleukin-7
• LAG-3 is particularly interesting due to its close relationship with CD4. LAG-3 has a similar genomic organization to CD4 and resides at the same chromosomal location. Bruniquel, D. et al, Immunogenetics 47: 96-98 (1997). LAG-3 is expressed on activated CD4 + and CD8 + ⁇ T lymphocytes and a subset of ⁇ T cells and NK cells. Baixeras, E. et al, J.Exp.Med. 176: 327-337 (1992); Triebel, F. et al, J.Exp.Med. 171: 1393-1405 (1990); Huard, B. et al, Immunogenetics 39: 213-217 (1994); Workman, C.J.
• a method for treating a patient suffering from an autoimmune disease is provided.
• Auto-immune T cells isolated from the patient are transfected in vitro with an expression construct comprising a coding sequence for CD223.
• the transfected auto-immune T cells are then reinfused to the patient.
• composition comprises antibodies which specifically bind to CD223 and an anti-cancer vaccine.
• kits comprising antibodies which specifically bind to CD223 and an anti-cancer vaccine.
• an improved method is provided for treating a cancer patient with an anti-cancer vaccine.
• An antibody which specifically binds to CD223 is administered to the cancer patient.
• An anti-cancer vaccine is also administered. The antibody increases magnitude of anti-cancer response ofthe cancer patient to the anti-cancer vaccine.
• a fifth embodiment of the invention provides a method to overcome suppression of an immune response to an anti-cancer vaccine.
• An antibody which specifically binds to CD223 is administered to a cancer patient with regulatory T-cells which suppress an immune response to an anti-cancer vaccine.
• An anti-cancer vaccine is also administered to the patient. The antibody increases the response of the cancer patient to the anti-cancer vaccine.
• a method for increasing number of T cells in a mammal An inhibitory agent which binds to CD223 protein or CD223 mRNA is administered to the mammal.
• the inhibitory agent inhibits activity or expression of CD223.
• a method for decreasing number of T cells in a mammal is provided.
• An expression construct which encodes CD223 is administered to the mammal.
• CD223 is expressed from the expression construct and concentration of CD223 in the mammal is increased.
• the number of T cells in the mammal is decreased.
• a method for decreasing number of T cells in a mammal A population of CD223+ T cells is administered to the mammal. The concentration of CD223 in the mammal is increased and the number of T cells in the mammal is thereby decreased.
• a polypeptide consisting of 50 or less contiguous amino acid residues of CD223 is provided.
• the polypeptide comprises an amino acid sequence KIEELE as shown in SEQ ID NO: 5.
• Another aspect of the invention is a fusion polypeptide which comprises at least two segments.
• a first segment consists of 50 or less contiguous amino acid residues of CD223.
• the first segment comprises an amino acid sequence KIELLE as shown in SEQ ID NO: 5.
• the second segment comprises an amino acid sequence which is not found in CD223 as shown in SEQ ID NO: 2 or 4.
• a method for testing substances for potential activity as a drug for treating cancer, autoimmune disease, chronic infections, AIDS, or bone marrow transplantation recipients A test substance is contacted with a CD223 protein or CD223 protein fragment comprising an amino acid sequence KIELLE as shown in SEQ ID NO: 5. Then one determines whether the test substance bound to the CD223 protein or CD223 protein fragment. The test substance is identified as a potential drug for treating cancer, autoimmune disease, chronic infections, AIDS, or bone marrow transplantation recipients if the test substance bound to the CD223 protein or CD223 protein fragment.
• Another embodiment provided by the present invention is a method for testing substances for potential activity as a drug for treating cancer, chronic infections, AIDS, or bone marrow transplantation recipients.
• a test substance is contacted with a CD223 protein.
• CD223 activity is determined in the presence and absence of the test substance.
• a test substance is identified as a potential drag for treating cancer, chronic infections, AIDS, or bone marrow transplantation recipients if the test substance inhibits the CD223 activity.
• a method for testing substances for potential activity as a drag for treating autoimmune disease A test substance is contacted with a CD223 protein. CD223 activity is determined in the presence and absence of the test substance. A test substance is identified as a potential drug for treating autoimmune disease if the test substance increases the CD223 activity.
• Another embodiment of the invention is a method of testing substances for potential activity as a drug for treating cancer, chronic infections, AIDS, or bone marrow transplantation recipients.
• a CD223+ T cell is contacted with a test substance. CD223 expression is determined in the cell in the presence and absence of the test substance.
• a test substance is identified as a potential drag for treating cancer, chronic infections, AIDS, or bone marrow transplantation recipients if the test substance inhibits the CD223 expression in the T cell.
• Yet another aspect of the invention is another method of testing substances for potential activity as a drag for treating autoimmune disease.
• a test substance is contacted with a CD223+ T cell.
• CD223 expression in the cell is determined in the presence and absence of the test substance.
• a test substance is identified as a potential drug for treating autoimmune disease if the test substance increases the CD223 expression in the T cell.
• Still another aspect of the invention is a method of isolating CD223+ T cells or CD223- T cells.
• a mixed population of T cells is contacted with an antibody which specifically binds to CD223 according to SEQ ID NO: 2 or 4.
• T cells which are bound to the antibody are separated from T cells which are not bound to the antibody.
• a population of CD223+ T cells and a population of CD223- T cells are thereby formed.
• Another embodiment of the invention is an isolated soluble murine CD223 protein comprising residues 1 to 431 and lacking residues 467 to 521.
• Still another aspect of the invention is an isolated soluble human CD223 protein comprising residues 1 to 440 and lacking residues 475 to 525.
• Yet another aspect of the invention is a method for decreasing number of T cells in a mammal.
• a soluble CD223 protein is administered to the mammal.
• MHC class II- restricted/ CD4+ T cell responses in the mammal are thereby modulated.
• HA specific CD4+ T cells become tolerant and develop regulatory T cell activity upon adoptive transfer into C3-HAhigh transgenic mice.
• Figure 1A C3-HAhigh transgenic mice express high levels of HA in various epithelial compartments, with the highest level expressed in pulmonary epithelia.
• C3-HAhigh recipients die 4-7 days after adoptive transfer of 2.5X106 HA-specific TCR transgenic (6.5) CD4+ T cells due to pneumonitis associated with a transient effector phase of activation occurring prior to development of an anergic phenotype.
• Naive 6.5 T cells (Thy 1.1+/1.2-) were adoptively transferred into C3-HAl ⁇ igh recipients (Thy 1.1-/1.2+), either in the absence or in the presence of 6.5 anergic/Treg cells (Thy 1.1-/1.2+). Spleens and lungs were harvested 3 days after adoptive transfer and Thy 1.1+ cells were stained by immunohistochemistry.
• T effector cells are scattered in the splenic follicles ( Figure IB) and infiltrate the pulmonary vessels ( Figure IC).
• Treg cells suppressed HA-specific 6.5 T cells become sequestered in the splenic peri-arteriolar lymphatic sheath ( Figure ID) and fail to infiltrate the pulmonary vessels ( Figure IE).
• FIG. 2A-2C LAG-3 is differentially expressed between anergic/Treg and effector/memory CD4+ T cells and LAG-3 expression in anergic/Treg CD4+ T cells is correlated with IL-10 expression.
• the differential expression revealed by gene chip analysis was confirmed by ( Figure 2A) quantitative real-time RT-PCR.
• the differential expression of LAG-3 in earlier days (Day 2 to Day 4) extends to 30 days after adoptive transfer.
• Figure 2B Cell surface LAG-3 protein levels were assessed by antibody staining. Splenocytes were harvested from C3-HAhigh, wild type B10.D2 mice immunized with Vac-HA, or wt B10.D2 mice 5 days after i.v.
• FIG. 3A-3B LAG-3 is expressed on induced Treg cells independently of CD25 and is a marker of Treg function.
• Figure 3A Anergic/Treg 6.5 CD4+ T cells from C3- HAhigh recipient spleens 5 days after transfer were stained for LAG-3 and CD25 expression, compared to isotype controls.
• Figure 3B Cells were sorted into 4 populations based on their surface LAG-3 and CD25 staining: LAG-3highCD25high, LAG-3highCD251ow, LAG-31owCD25high, and LAG-31owCD251ow.
• LAG-31owCD251ow cells were least suppressive.
• LAG-3highCD25high, LAG-3highCD251ow, and LAG- 31owCD25high are comparable in suppressive activity, with LAG-3highCD25high double positive cells exhibiting the most suppressive activity. This is the representative result of three reproducible experiments.
• FIG. 5A to 5C Anti-LAG-3 antibody eliminates the in vivo suppression by 6.5 CD4+ Treg cells by directly inhibiting Treg cells.
• Figure 5 A C3-HAhigh mice pretreated with 8,000 6.5 CD4+ T cells survived subsequent challenge with 2.5X106 6.5 CD4+ T cells given 4 days after the initial transfer establishment of Treg population (w/ Protection). Without the sublethal pretreatment, the C3-HAhigh recipients died 4-6 Days after lethal challenge (No Protection). Monoclonal anti- LAG-3 antibody (200 ⁇ g) was given i.v.
• Fig. 5B or 8X105 (partial lethality between 7 and 14 days after transfer; Fig. 5C) na ⁇ ve 6.5 CD4+ T cells in combination with anti-LAG-3 antibody, control rat IgGl, or no antibody. No lethality was observed with the anti-LAG-3 antibody infusions at the 2.5X105 dose whereas lethality at 8X105 dose was not affected by anti-LAG-3 antibody.
• FIG. 6A to 6D Role of LAG-3 in natural CD4+CD25+ T cells.
• Figure 6A Natural CD4+CD25+ T cells have higher levels of LAG-3 mRNA expression compared to their CD4+CD25- counterpart.
• CD4+CD25+ and CD4+CD25- T cells were purified from wild type BALB/c lymph nodes.
• CD4+CD25+ T cells the population documented to contain natural regulatory T cells, have significantly higher mRNA levels for CD25 and LAG-3, as well as for CTLA-4, GITR and Foxp3, as compared to their CD4+CD25- counterpart (Expression of each mRNA in the CD4+CD25- subset was normalized to a value of 1).
• FIG. 6B LAG-3 surface staining is negative on CD4+CD25+ natural regulatory T cells, as in their CD4+CD25- counterpart. However, intracellular staining for LAG-3 reveals a positive population in CD4+CD25+, but not in CD4+CD25- T cells.
• Figure 6C Sorted CD4+CD25+ T cells from BALB/c mouse lymph nodes were used as suppressors and CD4+CD25- T cells as responders in an in vitro suppression assay (suppressor: effector ratio of .04: 1), with anti-CD3 antibodies (0.5 ⁇ g/ml) as the T cell stimulus.
• Anti-LAG-3 antibodies at the concentration of 50 ⁇ g/ml reverse the in vitro suppression of natural CD4+CD25+ regulatory T cells whereas isotype control antibody does not.
• Figure 6D After the suppressor assay in C, the CD4+CD25+ cells (distinguished from the effector cells by Thyl.2 marking) were stained with anti- LAG-3 or isotype control antibody.
• GFP+ 6.5 CD4+ T cells transduced with the MSCV- GFP vector while high levels of LAG-3 staining were observed on GFP+ 6.5 cells transduced with the MSCV-LAG-3/GFP and MSCV-LAG-3.Y73F ⁇ CY/GFP vectors.
• GFP+ cells from the MSCV-LAG-3/GFP and MSCV-LAG-3.Y73F ⁇ CY/GFP transductions stained brightly with anti-LAG-3 antibodies while MSCV-GFP transduced cells displayed virtually no LAG-3 staining.
• GFP+ cells from each group were sorted and mixed at different ratios with APC, 5 ⁇ g/ml HA110-120 peptide and na ⁇ ve 6.5 CD4+CD25- cells in a proliferation assay.
• Figure 8 shows that ectopic expression of LAG-3 on a Phogin-specific T cell clone confers protection from diabetes following co-transfer with splenyocytes from NOD mice.
• 10 7 pre-diabetic NOD splenocytes were transferred alone (none) or in combination with Phogrin T-cell clone 4 (obtained from John Hutton) cells transduced with vector (MIG), LAG-3, or a signaling-defective mutant, LAG-3 DK, into NOD/SCID mice.
• MIG vector
• LAG-3 a signaling-defective mutant, LAG-3 DK
• LAG-3 is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity.
• LAG-3 deficient mice have twice as many CD4 + and CD8 + T cells than wild type controls.
• LAG-3 deficient T cells show enhanced homeostatic expansion in lymphopenic hosts, which is dependent on LAG-3 ligation of MHC class II molecules. This was abrogated by ectopic expression of wild type LAG-3 but not by a signaling defective mutant. This deregulation of T cell homeostasis results in the expansion of multiple cell types.
• Our data suggest that LAG-3 negatively regulates CD4 + and CD8 + T cell homeostasis, and present LAG-3 as a therapeutic target for accelerating T cell engraftment following bone marrow transplantation.
• a representative murine DNA and amino acid sequence for CD223 is set forth as SEQ ID NOS: 1 and 2, respectively. See also GenBank Accession Code X9113.
• a representative human DNA and amino acid sequence for CD223 is set forth as SEQ ID NOS: 3 and 4, respectively. See also GenBank Accession Number X51985. These sequences are derived from single individuals. It is expected that allelic variants exist in the population which differ at less than about 5% of the positions. Such allelic variants are included within the meaning of CD223 of murine or human origin.
• Regulatory T-cells are a subgroup of T-cells that function by inhibiting effector T- cells. Regulatory T-cells are CD223 + and are typically also CD4 + CD25 . Regulatory T-cells play a central role in balancing autoimmune tolerance and immune responsiveness. Such cells can be isolated from CD223- cells using antibodies and separation techniques known in the art. These include but are not limited to immunoaffinity chromatography, FACS, immunoprecipitation, etc.
• the CD223 + cells can be administered to autoimmune disease, allergy, or asthma patients. In the case of an autoimmune disease patient the cells can be pre-activated with auto-antigen. CD223 " cells can be similarly transferred to cancer patients, bacterial or vial infection patients, or AIDS patients.
• a comparative analysis of gene expression arrays from antigen specific CD4+ T cells differentiating to either an effector/memory or a regulatory phenotype revealed Treg- specific expression of LAG-3, a CD4 homologue that binds MHC class II.
• LAG- 3high CD4+ T cells display in vitro suppressor activity and antibodies to LAG-3 inhibit the suppression both in vitro and in vivo.
• These findings identify LAG-3 as a Treg specific receptor or co-receptor modulating suppressor activity.
• Manipulation of Treg cells via LAG-3 can therefore be used to enhance immunotherapy of autoimmune diseases, cancer and infectious diseases as well as enhance lymphocyte engraftment in settings of donor lymphocyte infusion, bone marrow transplantation and adoptive T cell transfer.
• CD223 is a regulatory T-cell specific cell surface molecule that regulates the function of regulatory T-cells.
• the function of a regulatory T-cell may be enhanced by enhancing or increasing CD223 activity, or by increasing the number of CD223+ cells in a T-cell population. Enhancing the function of regulatory T-cells in an organism may be used to limit the immune T-cell response in those circumstances where such a response is undesirable, such as when a subject suffers from autoimmune disease. Conversely, the function of a regulatory T-cell may be inhibited by inhibiting CD223 activity or by decreasing the number of CD223+ cells in a T-cell population. Inhibiting the function of regulatory T-cells in an organism may be used to enhance the immune T-cell response in those circumstances where such a response is desirable, such as in a patient suffering from cancer, chronic infection, or a bone marrow transplant recipient.
• an anti-tumor vaccine When treating a cancer patient with an inhibitory agent that binds to CD223 protein or mRNA, one may optionally co-administer an anti-tumor vaccine. Such vaccines may be directed to isolated antigens or to groups of antigens or to whole tumor cells. It may be desirable to administer the inhibitory agent with chemotherapeutic agents. Treatment with multiple agents need not be done using a mixture of agents but may be done using separate pharmaceutical preparations. The preparations need not be delivered at the same exact time, but may be coordinated to be delivered to a patient during the same period of treatment, i.e., within a week or a month or each other. Thus a composition comprising two active ingredients may be constituted in the body of the patient.
• any suitable anti-tumor treatment can be coordinated with the treatments of the present invention targeted to CD223.
• other anti-infection agents can be coordinated with the treatment of the present invention targeted to CD223.
• agents may be small molecule drugs, vaccines, antibodies, etc.
• CD223 + cells in a T-cell population can be modified by using an antibody or other agent that selectively binds to CD223.
• CD223 + cells represent an enriched population of regulatory T-cells that can be introduced back into the original source of the T-cells or into another compatible host to enhance regulatory T-cell function.
• the CD223 " cells represent a population of T-cells deficient in regulatory T-cell activity that can be reintroduced into the original source of the T- cells or another compatible host to inhibit or reduce regulatory T-cell function while retaining general T-cell activity.
• any desired means for either increasing or decreasing (modulating) CD223 activity can be used in the methods of the invention. This includes directly modulating the function of CD223 protein, modulating CD223 signal transduction, and modulating expression of CD223 in T-cells by modulating either transcription or translation or both. Those means which selectively modulate CD223 activity are preferred over nonselective modulators. Also, those inhibitory means which create a transient CD223 deficiency in a population of T-cells which then return to normal levels of CD223 activity may be preferred for treating a temporary T-cell deficiency.
• the transiently deficient T-cells may be used to reconstitute a diminished T-cell population with T-cells that will be genetically normal with respect to CD223.
• Such a temporary T-cell deficiency occurs, for example, in patients receiving a stem cell transfer following myoablation.
• Modulation of CD223 activity can be performed on cells in vitro or in whole animals, in vivo. Cells which are treated in vitro can be administered to a patient, either the original source of the cells or au unrelated individual.
• CD223 antibodies or small molecule inlubitors can be used.
• Antibodies or antibody fragments that are useful for this purpose will be those that can bind to CD223 and block its ability to function.
• Such antibodies may be polyclonal antibodies, monoclonal antibodies (see, e.g. Workman, C. J. et al, "Phenotypic analysis ofthe murine CD4-related glycoprotein, CD223 (LAG-3)", Eur. J. Immunol. 32:2255-2263 (2002)), chimeric antibodies, humanized antibodies, single-chain antibodies, soluble MHC class II molecules, antibody fragments, etc.
• Antibodies generated against CD223 polypeptides can be obtained by direct injection ofthe CD223 polypeptides into an animal or by administering CD223 polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the CD223 polypeptides itself. In this manner, even a sequence encoding only a fragment of the CD223 polypeptide can be used to generate antibodies binding the whole native CD223 polypeptide.
• any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
• any agent which increases the level of CD223 or the activity of existing CD223 in the T-cell may be used. Such agents may be identified using the screening assays described below.
• Expression vectors encoding CD223 can also be administered to increase the gene dosage.
• the expression vectors can be plasmid vectors or viral vectors, as are known in the art. Any vector can be chosen by the practitioner for particularly desirable properties.
• Autoimmune disease which are amenable to treatments according to the present invention include autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, mixed essential cryoglobulinemia, systemic lupus erythematosus, insulin-dependent diabetes mellitus, rheumatoid arthritis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, and multiple sclerosis.
• Auto-immune T cells can be isolated from autoimmune disease patients as is known in the art. These can be transfected with a coding sequence for CD223. Any desirable expression vector can be used for expressing CD223.
• the expression regulatory signals can be derived from CD223 itself or from other genes. After transfection with CD223 expression construct the T cells can be reintroduced to the patient. Methods for infusing blood cells to a patient are well known in the art.
• compositions comprising a mixture of antibodies which specifically bind to CD223; and an anti-cancer vaccine can be made in vitro.
• the composition is made under conditions which render it suitable for use as a pharmaceutical composition.
• Pharmaceutical compositions may be sterile and pyrogen-free.
• the components of the composition can also be administered separately to a patient within a period of time such that they are both within the patient's body at the same time. Such a time- separated administration leads to formation of the mixture of antibodies and vaccine within the patient's body. If the antibody and vaccine are to be administered in a time-separated fashion, they may be supplied together in a kit. Within the kit the components may be separately packaged or contained.
• kits Other components such as excipients, carriers, other immune modulators or adjuvants, instructions for administration of the antibody and the vaccine, and injection devices can be supplied in the kit as well. Instructions can be in a written, video, or audio form, can be contained on paper, an electronic medium, or even as a reference to another source, such as a website or reference manual.
• Anti-CD223 antibodies ofthe invention can be used to increase the magnitude of anti- cancer response ofthe cancer patient to the anti-cancer vaccine. It can also be used to increase the number of responders in a population of cancer patients. Thus the antibodies can be used to overcome immune suppression found in patients refractory to anti-cancer vaccines.
• the anti-cancer vaccines can be any that are known in the art, including, but not limited to whole tumor cell vaccines, isolated tumor antigens or polypeptides comprising one or more epitopes of tumor antigens.
• Expression of CD223 in T-cells can be modulated at the transcriptional or translational level. Agents which are capable of such modulation can be identified using the screening assays described below.
• CD223 mRNA can be inhibited by using ribozymes, antisense molecules, small interference RNA (siRNA; See Elbashir, S. M. et al, "Duplexes of 21 -nucleotide RNAs mediate RNA interference in cultured mammalian cells", Nature 411:494-49% (2001)) or small molecule inhibitors of this process which target CD223 mRNA.
• siRNA small interference RNA
• Antisense technology can be used to control gene expression through triple- helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
• the 5' coding portion of the polynucleotide sequence which codes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of CD223.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the CD223 polypeptide (Antisense-- Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
• the ohgonucleotides described above can also be delivered to cells by antisense expression constracts such that the antisense RNA or DNA may be expressed in vivo to inhibit production of CD223.
• Such constructs are well known in the art.
• Antisense constructs, antisense ohgonucleotides, RNA interference constracts or siRNA duplex RNA molecules can be used to interfere with expression of CD223.
• at least 15, 17, 19, or 21 nucleotides of the complement of CD223 mRNA sequence are sufficient for an antisense molecule.
• at least 19, 21, 22, or 23 nucleotides of CD223 are sufficient for an RNA interference molecule.
• an RNA interference molecule will have a 2 nucleotide 3' overhang. If the RNA interference molecule is expressed in a cell from a construct, for example from a hairpin molecule or from an inverted repeat of the desired CD223 sequence, then the endogenous cellular machinery will create the overhangs.
• siRNA molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Harmon, GJ, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al, RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232; Brammelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells.
• Short hairpin RNAs induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol 20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester WC, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y, DeRuiter SL, and Turner DL. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99(9): 6047-6052.
• additional modulators of CD223 activity that are useful in the methods of the invention can be identified using two-hybrid screens, conventional biochemical approaches, and cell-based screening techniques, such as screening candidate molecules for an ability to bind to CD223 or screening for compounds which inhibit CD223 activity in cell culture.
• the inventors have identified a hen egg lysozyme (HEL), 48-62-specif ⁇ c, H-2A -restricted murine CD4 + T cell hybridoma 3A9 that does not express CD223, even after activation. Ectopic expression of wild type, but not signaling defective, CD223 significantly reduced the IL-2 response of this T cell hybridoma to its specific peptide.
• This provides a simple in vitro assay system to screen for CD223 activity modulators.
• This latter method may identify agents that directly interact with and modulate CD223, as well as agents that indirectly modulate CD223 activity by affecting a step in the CD223 signal transduction pathway.
• Cell-based assays employing cells which express CD223 can employ cells which are isolated from mammals and which naturally express CD223. Alternatively, cells which have been genetically engineered to express CD223 can be used. Preferably the genetically engineered cells are T-cells.
• Agents which modulate CD223 activity by modulating CD223 gene expression can be identified in cell based screening assays by measuring amounts of CD223 protein in the cells in the presence and absence of candidate agents.
• CD223 protein can be detected and measured, for example, by flow cytometry using anti-CD223 specific monoclonal antibodies.
• CD223 mRNA can also be detected and measured using techniques known in the art, including but not limited to Northern blot, RT-PCR, and array hybridization.
• One particularly useful target sequence for identifying CD223 modulators is the amino acid motif KIEELE (SEQ ID NO: 5) in the CD223 cytoplasmic domain which is essential for CD223 function in vitro and in vivo. Screening assays for agents which bind this motif will identify candidate CD223 modulators whose activity as an inhibitor or activator of CD223 can be further characterized tlirough further testing, such as in cell based assays.
• This motif can be contained with in a polypeptide which consists of 50 or fewer contiguous amino acid residues of CD223. Alternatively, the motif can be contained within a fusion protein which comprises a portion of CD223 and all or a portion of a second (non-CD223) protein.
• the second protein may be a natural protein or can be a synthetic polypeptide, for example containing a Histidine tag, or other useful polypeptide feature.
• Protein-protein binding assays are well known in the art and any of a variety of techniques and formats can be used.
• CD223 can be post-translationally processed to yield a soluble form of the protein.
• the soluble form comprises at least amino acid residues 1 to 431 of murine CD223, and at least amion acid residues 1 to 440 of human CD223.
• the cytoplasmic tail is missing in each case. All or part of the transmembrane domain is missing as well.
• This soluble form modulates responses of MHC class II-restricted/CD4+ T cells.
• the soluble form may be useful for administration to autoimmune disease patients, allergy patients, asthma patients, or cancer patients, for example. Administration of the soluble form may be by any of convenient means, including infusion, topical, or intravenous administration.
• CD223 inhibitors may be administered to an organism to increase the number of T-cells in the organism.
• This method may be useful for treating organisms suffering from conditions resulting in a low T-cell population.
• Such conditions include diseases resulting from immunodeficiency such as AIDS, as well as disorders involving unwanted cellular invasion or growth, such as invasion ofthe body by foreign microorganisms (bacteria or viruses) or tumor growth or cancer.
• Such a T-cell deficiency is also an expected hazard for patients receiving a stem cell transfer following myoablation.
• the T-cells of such patients are compromised and deliberately targeted for destruction so that they can be replaced with healthy donor T-cells.
• the process of reconstituting a healthy T-cell population from a stem cell transfer can take several months, during which time the patient is very susceptible to opportunistic infections which can be life threatening.
• T-cell division is enhanced and the process of T-cell reconstitution can be accelerated and the period of T-cell deficiency can be reduced.
• CD223 inlubitors may also be useful when administered in combination with conventional therapeutics to treat T-cell proliferation sensitive disorders.
• a tumor which is a T-cell proliferation sensitive disorder
• a chemotherapeutic agent which functions by killing rapidly dividing cells.
• the CD223 inhibitors of the invention when administered in conjunction with a chemotherapeutic agent enhance the tumoricidal effect of the chemotherapeutic agent by stimulating T-cell proliferation to enhance the immunological rejection of the tumor cells.
• CD223 activators or expression enhancers may be administered to an organism to decrease the number of T-cells in the organism and thereby decrease deleterious T-cell activity.
• This method may be useful for treating organisms suffering from conditions resulting in an abnormally high T-cell population or deleterious T-cell activity, for example graft rejection mediated by host T-cells, graft vs. host disease and T-cell mediated autoimmune and inflammatory diseases such as rheumatoid arthritis, type 1 diabetes, muscular sclerosis, etc.
• the methods of the invention may be applied to any organism which contains T-cells that express CD223. This includes, but is not limited to, any mammal and particularly includes humans and mice.
• the effective amount of CD223 modulator used will vary with the particular modulator being used, the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration and similar factors within the knowledge and expertise of the health practitioner.
• an effective amount can depend upon the degree to which an individual has abnormally depressed levels of T cells.
• the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptably compositions.
• Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
• the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention.
• Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
• pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
• CD223 modulators may be combined, optionally, with a pha ⁇ uaceutically-acceptable carrier.
• pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human.
• carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
• The- components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
• the pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
• suitable buffering agents including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
• suitable preservatives such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
• compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the anti-inflammatory agent, which is preferably isotonic with the blood of the recipient.
• This aqueous preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono-or di-glycerides.
• fatty acids such as oleic acid may be used in the preparation of injectables.
• Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
• a variety of administration routes are available. The particular mode selected will depend, of course, upon the particular drag selected, the severity of the condition being treated and the dosage required for therapeutic efficacy.
• the methods of the invention may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels ofthe active compounds without causing clinically unacceptable adverse effects.
• modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes.
• parenteral includes subcutaneous, intravenous, intramuscular, or infusion. Intravenous or intramuscular routes are not particularly suitable for long-term therapy and prophylaxis. They could, however, be preferred in emergency situations. Oral administration will be preferred because of the convenience to the patient as well as the dosing schedule.
• compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
• compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active agent.
• Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
• Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the active agent, increasing convenience to the subject and the physician.
• Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Pat. No. 5,075,109.
• Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like.
• lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
• hydrogel release systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
• sylastic systems such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides
• peptide based systems such as mono-di-and tri-glycerides
• wax coatings such as those described in U.S. Pat. Nos.
• Long-term release are used herein, means that the implant is constructed and a ⁇ anged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days.
• Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
• LAG-3 (CD223) negatively regulates CD4 and CD8 + T cell homeostasis, supporting its identification as a novel therapeutic target for accelerating T cell engraftment following bone marrow transplantation.
• LAG-3 '7" mice transgenic for the OT-II TCR had an increased number CD4 + V ⁇ 2 + T cells compared with wild type control OT-II transgenic mice, except that these differences were evident at 5 weeks of age.
• Approximately 20% ⁇ + T cells and CD49b NK cells constitutively express LAG-3 in wild type mice (Workman, C.J. et al, Eur . Immunol. 32: 2255-2263 (2002)), and their numbers were also significantly increased in LAG-3 " " mice.
• LAG-3 "7” and wild type OVA transgenic T cells were transfe ⁇ ed into mice lacking both MHC class I and class II molecules ( ⁇ 2m " ⁇ x H-2A ⁇ “7” ).
• the data clearly show that the enlianced expansion of LAG-3 "7” T cells is abrogated in the absence of MHC class II molecules, demonstrating the importance of this interaction.
• LAG-3 "7" mice or adoptive recipients of LAG-3 "7” T cells have increased numbers of cells that are normally negative for LAG-3, such as B cells and macrophages. This supports the idea that an alteration in the homeostatic control of T cells, due to the absence of LAG-3, directly alters the control of other leukocyte cell types.
• B cells were co-transferred with either LAG-3 ' " or wild-type T cells into RAG " " mice.
• MHC class II molecules in regulating T cells homeostasis. Previous studies have clearly demonstrated that the homeostatic expansion and long-term survival of CD4 + T cells requires periodic interaction with MHC class II molecules. Takeda, S.
• LAG-3 expression on homeostatic expansion in lymphopenic mice is not limited to na ⁇ ve T cells. Transfer of antigen-experienced 'memory' OT-II T cells also resulted in a substantially accelerated expansion of LAG-3 " " T cells compared with the wild-type control cells [7.2-fold]. It was important to verify that LAG-3 was directly responsible for this 'deregulated' T cells expansion and not a closely linked gene that was disrupted by the original targeting strategy. Thus, LAG-3 "7" OT-II T cells were transduced with murine stem cell virus (MSCV)-based retrovirus that contained either wild-type LAG-3 or a signaling defective mutant, LAG-3. ⁇ K M . Workman, C.J.
• MSCV murine stem cell virus
• the vector also contained an internal ribosomal entry site (IRES) and green fluorescent protein (GFP) cassette to facilitate analysis of transduced cells.
• IRS internal ribosomal entry site
• GFP green fluorescent protein
• mice The following mice were used: LAG-3 "7" [obtained from Yueh-Hsiu Chen, Stanford University, Palo Alto, CA, with permission from Christophe Benoist and Diane Mathis, Joslin Diabetes Center, Boston, MA; Miyazaki, T. et al, Science 272: 405-408 (1996)]; C57BL/6J [Jackson Labs, Bar Harbor, ME]; B6.PL-I7?yi Cy (Thyl.l congenic) [Jackson Labs]; RAG-1 " " [Jackson Labs, Bar Harbor, ME; Mombaerts, P.
• MHC class II "7” [provided by Peter Doherty, St. Jude Children's Research Hospital, Memphis, TN; Grusby, M.J. et al, Science 253:1417-1420 (1991)]; MHC class r 7 7H "7” [Taconic, Germantown, NY; Grusby, M.J.
• LAG-3 constructs and retroviral transduction LAG-3 constructs were produced using recombinant PCR as described (Vignali, D.A.A. and K. M. Vignali, J.Immunol. 162: 1431-1439 (1999)).
• the LAG-3.WT and LAG-3. ⁇ K M LAG-3 with a deletion of the conserved KIEELE motif in the cytoplasmic tail have been described (Workman, C.J. et al, J.Immunol. 169: 5392-5395 (2002)).
• LAG-3 constracts were cloned into a murine stem cell virus (MSCV)-based retroviral vector, which contained an internal ribosomal entry site (IRES) and green fluorescent protein (GFP), and retrovirus produced as described (Persons, D.A. et al, Blood 90: 1777-1786 (1997); Persons, D.A. et al, Blood Cells Mol Dis. 24: 167-182 (1998)).
• Retroviral producer cell lines were generated by repeatedly transducing GPE+86 cells (7-10) times until a viral titer of greater then 10 5 /ml after 24 hr was obtained (Markowitz, D. et al, J Virol. 62: 1120-1124 (1988)).
• the cells were then stained for the following cell surface markers using various conjugated antibodies from BD PharMingen: ⁇ + TCR (H-57-597), V ⁇ 2 (B20.1), ⁇ TCR (GL3), CD4 (RM4-4), CD8a (53-6.7), CD45R/B220 (RA3-6B2), CDllb/Macl (Ml/70), Gr-1 (RB6-8C5), CD44 (IM7), CD25/IL2R (7D4), CD69 (H1.2F3) and CD244.2/NK cells (2B4).
• LAG-3 expression was assessed with a biotinylated rat anti-LAG-3 mAb (C9B7W, IgGl K; Workman, C.J.
• the LAG-3 "7" and LAG-3 +7+ splenocytes were stained for TCR ⁇ , CD4, CD8 and B220 expression.
• the 0TII.LAG-3 +7+ and OTII.LAG-3 "7" splenocytes were stained for V ⁇ 2 and CD4 expression (PharMingen).
• the cells were then fixed with 1.2ml ice-cold 95% ethanol for 30 min on ice.
• the cells were washed and permeabilized with PBS + 1% paraformaldehyde + 0.01% Tween 20 for 1 h at room temperature.
• the cells were then washed and incubated with 50KU of DNase (Sigma) in 0.15M NaCl + 4.2mM MgCl? pH 5.0 for 10 min at 37°C.
• BrdU was detected by the addition of anti-BrdU- FITC (Becton Dickinson) for 30 min at RT and then analyzed by flow cytometry.
• T cells and/or B cells from splenocytes were either positively sorted by FACS or negatively sorted by magnetic bead cell sorting (MACS).
• FACS purifications splenocytes were stained for TCRDD, CD4 and CD8 expression and sorted by positive selection on a MoFlow (Cytomation, Ft. Collins, CO).
• MACS purification splenocytes were stained with PE- coupled anti-B220, anti-Grl, anti-Mac 1, anti-TER119 (erythrocytes), anti-CD244.2 (NK cells) and anti-CD8 (for negative purification of OTII transgenic T cells).
• T cells were then incubated with magnetic beads coupled with anti-PE antibody (Miltenyi Biotech, Auburn, CA) and then negatively sorted on an autoMACS (Miltenyi Biotech, Auburn, CA) to 90-95% purity.
• T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE).
• CFSE carboxyfluorescein diacetate succinimidyl ester
• the purified CFSE labeled or unlabeled T cells (5x10 6 or lxlO 7 ) and in some cases B cells (5xl0 6 ) were injected i.v. into RAG-1 "7" or Thyl.l + (B6.PL) mice.
• Retroviral transduction of normal T cells Spleens from OTII.LAG-3 +7+ and OTII.LAG3 " " mice were removed and single cell suspensions made at 2.5x10 6 cell ml. The splenocytes were activated with OVA 326-339 peptide [lO ⁇ M] in culture for two days. The activated splenocytes were then incubated on a monolayer of GFP alone, LAG-3.WT/GFP or LAG-3. ⁇ K M /GFP retroviral producer cells for 2 days in the presence of polybrene. The cells were allowed to rest for 10 days and then sorted for V ⁇ 2 + /CD4 + /GFP + expression by FACS.
• mice were allowed to rest for two additional days and then 5xl0 6 cells were injected into RAG " " mice via the tail vein. Fifteen days post-transfer, the mice were sacrificed by C0 2 inhalation and the spleens removed. The splenocytes were stained and analyzed by flow cytometry.
• TCR T cell receptor
• clone 6.5 a model antigen - hemagglutinin
• Vac-HA recombinant HA-expressing vaccinia virus
• adoptively transferred HA specific 6.5 CD4+ T cells differentiate into effector/memory cells upon encounter with HA.
• the effector/memory response is characterized by a typical expansion/contraction phase and the development of memory markers.
• these effector/memory cells When removed from the adoptively transfe ⁇ ed animal, these effector/memory cells are hyper-responsive to HA in vitro relative to naive 6.5 CD4+ T cells as assayed by antigen-specific proliferative response and ⁇ -interferon production. This memory response persists for months after adoptive transfer. In contrast, adoptive transfer of 6.5 CD4+ T cells into C3-HA transgenic mice, that express HA in multiple epithelial tissues, results in tolerance (Adler et al, 2000; Adler et al., 1998). Similar to the effector/memory response, there is a rapid expansion/activation phase characterized by proliferation and expression of effector cytokines, such as ⁇ -interferon.
• the total HA-specific T cell pool contracts and residual 6.5 cells fail to produce ⁇ -interferon or proliferate in vitro upon antigen stimulation 4-7 days after adoptive transfer (Adler et al., 2000; Huang et al., 2003).
• the extinction of the capacity to produce lymphokines such as IL-2 and ⁇ -interferon and proliferate in response to antigen represents the standard operational definition of the anergic phenotype.
• the intensity of the initial in vivo effector phase in C3-HA mice that precedes tolerance induction, is proportional to the number of 6.5 CD4+ T cells adoptively transferred as well as the expression level of HA antigen in the recipient mice.
• C3-HA l0W mice tolerate the transfer of 2.5 X 10 6 6.5 CD4+ T cells quite well, but C3- HA hlgh mice, which have 1000 fold higher HA expression than C3-HA low mice, die within 4-7 days after transfer of 2.5 X 10 6 6.5 CD4+ T cells (Fig. 1A).
• the cause of death is lethal pulmonary vasculitis due to infiltration of transgenic 6.5 CD4+ T cells in the lung, where HA expression is the highest.
• Example 4--LAG-3 is differentially expressed on induced Treg cells
• Thyl.l(+)Thyl.2(-) congenic 6.5 T cells were purified from Thyl.l(-)Thyl.2(+) Vac-HA infected wild-type (effector/memory) or C3HA hlgh (anergic/Treg) recipients using a sequential isolation procedure involving MACS Column depletion of CD8+ T cells, B cells and Thy 1.2(+) T cells followed by flow cytometric sorting to >95% purity. This protocol avoids the use of TCR-specific or CD4 coreceptor specific antibodies that could potentially alter TCR or CD4 dependent gene expression patterns.
• Genes that were differentially expressed between anergic/Treg populations and effector/memory populations were rank ordered according to an algorithm that summed their differential expression from days 0-4.
• a surprisingly large number of genes were selectively activated in anergic/Treg populations even at these early time points post adoptive transfer. Many of these genes represented ESTs with no known function.
• LAG-3 was among the most differentially expressed in anergic/Treg populations relative to effector/memory populations.
• LAG-3 expression After a minimal initial increase in the effector/memory cells, LAG-3 expression returns to baseline by 20 days post adoptive transfer. In striking contest, LAG-3 expression increases 20-50 fold over the first 5 days among anergic/Treg cell populations and remains high over the subsequent 4- week analysis (Fig. 2A). In contrast, levels of FoxP3, GITR and CTLA-4 showed modest increases (1.5-4 fold) that were similar in both effector/memory cells and the induced anergic/Treg cells over the first 4-5 days (data not shown).
• IL-10 is commonly associated with differentiation and function of Treg (Moore et al, 2001), we analyzed the endogenous levels of IL-10 mRNA and their correlation to the levels of LAG-3 mRNA in CD4+ T cell subsets from C3-HA hlgh transgenic mice (anergic/Treg 6.5 CD4+ T cells). Analysis of multiple samples of anergic/Treg populations over many experiments revealed correlation between LAG-3 mRNA level and IL-10 mRNA level with a co ⁇ elation coefficient (R 2 ) of 0.87 (Fig. 2C).
• Example 6 ⁇ LAG-3 is required for induced Treg activity in vivo
• Example 7 LAG-3 is expressed by natural Treg cells and is required for suppressor activity
• GFP+ 6.5 CD4+ T cells transduced with the MSCV-GFP vector while high levels of LAG-3 staining were observed on GFP+ 6.5 cells transduced with the MSCV-LAG-3/GFP and MSCV-LAG-3.Y73F ⁇ CY/GFP vectors.
• GFP+ cells from each group were sorted and mixed at different ratios with APC, HA 110"120 peptide and na ⁇ ve 6.5 CD4+CD25- cells in a proliferation assay.
• 6.5 cells expressing wild type LAG-3 potently suppressed proliferation of na ⁇ ve 6.5 cells while no suppression was observed with control GFP transduced 6.5 cells or 6.5 cells expressing the non-functional LAG- 3.Y73F ⁇ CY mutant.
• Total proliferation was in fact somewhat increased in these two latter groups, since GFP and LAG-3.Y73F ⁇ CY transduced 6.5 cells themselves proliferate in addition to the na ⁇ ve 6.5 cells in the assay.
• wild type LAG-3 transduced T cells themselves demonstrated a significant reduction in prohferative responses apart from inhibiting proliferation of the non-transduced 6.5 cells.
• LAG-3 transduction did not induce other genes associated with Tregs, including Foxp3, CD25, CD103 and GITR (data not shown). This result, together with the lack of significant differential expression of these genes between 6.5 T cells differentiating to effector/memory vs anergic/Treg phenotypes, suggests that LAG-3 may mediate a distinct pathway of regulatory T cell function independent of the Foxp3 pathway.
• LAG-3 is a cell surface molecule selectively upregulated on Treg cells that may be directly involved in mediating Treg function. Given the many systems in which both natural and induced Treg activity has been defined, it remains to be determined whether LAG-3 is a "universal" Treg marker or selectively marks only certain Treg subsets. Our results suggest that in addition to induced CD4+ Treg cells, LAG-3 plays at least some role in mediating suppression by natural CD4+CD25+ Treg cells. Furthe ⁇ uore, other experimental data demonstrate a role for LAG-3 in the regulation of homeostatic lymphocyte expansion by natural Treg (Workman and Vignali, accompanying paper).
• LAG-3 expression is significantly greater among CD4+CD25+ T cells from wildtype mice suggests that it may play a role in the function of natural, as well as induced, Tregs.
• the combination of LAG-3 and CD25 may define Treg subsets with the most potent suppressive activity.
• LAG-3 is a "lineage marker" for Treg as it is expressed at variable levels that correlate with the magnitude of regulatory activity in in vitro assays.
• Treg represent a stable lineage or differentiation state capable of promoting tolerance in a non-cell autonomous fashion (von Boehmer, 2003). Different mechanisms have been identified for Treg function in different systems (reviewed in Shevach, 2002).
• LAGS' 11611 cells produce increased amounts of IL-10 and display enhanced in vitro suppressor activity but the role of IL-10 in mediating suppressive function in our system remains to be determined.
• Antibodies to LAG-3 inhibit the suppressor activity of Treg cells both in vitro and in vivo.
• LAG-3 is a Treg specific receptor or coreceptor that modulates the suppressor activity of this T cell subset.
• LAG-3 knockout mice would display multi-system autoimmunity (i.e., similar to Fox ⁇ 3 knockout or scurfy mice), which has not been reported in these mice.
• multi-system autoimmunity i.e., similar to Fox ⁇ 3 knockout or scurfy mice
• LAG-3 knockout mice We are in the process of reexamining older LAG-3 knockout mice for more subtle evidence of late-onset autoimmunity, as was observed in PD-1 knockout mice (Nishimura et al., 1999; Nishimura et al., 2001). It is also conceivable that other regulatory mechanisms might have been enhanced in these mice to compensate for the loss of LAG-3 expression. [110] Because it is expressed at higher levels on Treg cells, LAG-3 provides an excellent potential target for selective manipulation of Treg activity to treat both cancer and autoimmune disease. CD25, the "gold standard" Treg marker, is induced at high levels in activated cells, as it is a critical component of the IL-2 receptor complex.
• CD4+CD25+ cells are enriched in Treg activity is not because CD25 is specific to Treg function, but rather because Treg cells are chronically stimulated by continuous encounter with self-antigen in the periphery.
• the TNF receptor super-family member 18 molecule also called GITR
• GITR TNF receptor super-family member 18 molecule
• antibodies to GITR have been reported to inhibit Treg activity both in vivo and in vitro.
• GITR is equivalently up-regulated on activated T cells and therefore is apparently no more selective as a marker for Treg cells than is CD25 (McHugh et al., 2002; Shimizu et al., 2002).
• CD4+CD25- cell populations can suppress certain immune functions (Annacker et al., 2001; tendou et al., 2002; Curotto de Lafaille et al, 2001; Graca et al., 2002; Shimizu and Moriizumi, 2003; Stephens and Mason, 2000). Nonetheless, the finding that CD25 high LAG-3 high cells exhibit the greatest suppressive activity suggests that antibodies against both of these cell surface molecules may be used coordinately to manipulate Treg activity.
• Treg cells suppress the reactivity of CD4+ and CD8+ effector cells through direct T-T interactions or through DC intermediaries.
• LAG-3 a MHC class II binding molecule
• the C3-HA transgenic mice have been previously described (Adler et al., 2000; Adler et al, 1998).
• the hemagglutinin (HA) gene derived from the influenza virus A/PR/8/34 (Mount Sinai strain) has been placed under the control of the rat C3(l) promoter.
• Two founder lines were established in the B10.D2 genetic background. These two founder lines, C3-HA high and C3-HA low , which contain 30-50 and 3 transgene copies respectively, express the C3-HA hybrid mRNA in the same set of non-lymphoid tissues including the lung and prostate.
• the TCR transgenic mouse line 6.5 that expresses a TCR recognizing an I-E - restricted HA epitope ( 110 SFERFEIFPKE 120 ; SEQ ID NO: 7) (generously provided by Dr. Harald von Boehmer, Harvard University, Boston, MA), was back-crossed 9 generations onto the B10.D2 genetic background.
• the other TCR transgenic mouse line Clone-4 that expresses a TCR recognizing a K d -restricted HA epitope ( 518 IYSTVASSL 526 ;SEQ ID NO: 8) (generously provided by Dr.
• Thy 1.1 was used as a surrogate marker.
• Thy 1.1 + CD8 + T cells express the HA-specific clonotypic TCR as nearly all of the mature CD8 + T cells in the Clone-4 mice directly recognize the K d -restricted HA epitope (Morgan et al, 1996).
• Transgenic mice used for experiments were between the age of 8 to 24 weeks. All experiments involving the use of mice were performed in accordance with protocols approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine.
• Clonotypic CD4 + or CD8 + T cells were prepared from pooled spleens and lymph nodes of 6.5 or Clone-4 transgenic mice. Clonotypic percentage was determined by flow cytometry analysis. The activation marker CD44 was analyzed to ensure that these clonotypic cells were not activated in donor mice and were naive in phenotype. After washing 3 times with HBSS, an appropriate number of cells were resuspended in 0.2 ml of HBSS for i.v. injection through the tail vein.
• Tissues were harvested from mice three days after adoptive transfer. Tissue was fixed in ImmunoHistoFix (A Phase sprl, Belgium) for 3 days at 4°C and then embedded in ImmunoHistoWax (A Phase sprl, Belgium). Serial sections were stained using biotin- labeled anti-Thyl.l mAb (PharMingen, San Diego, CA). The Vectastain ABC kit (Vector, Burlingame, CA) and NovaRed (Vector) were used for development. Sections were counterstained with hematoxylin QS (Vector). Sections were analyzed using a Nikon Eclipse E800. Final image processing was performed using Adobe PhotoShop (Mountain View, CA).
• CD4+ T cells were first enriched by using biotinylated anti-CD8 (Ly-2, 53-6.7), anti-B220 (RA3-6B2), and anti-Thyl.2 (30-H12) antibodies (all purchased from BD Biosciences PharMingen, San Diego, CA) and MACS streptavidin microbeads and MACS LS separation column (Miltenyi Biotech, Auburn, CA) to deplete CD8+ T cells, B cells and the recipient T cells (Thy 1.2+).
• CD4+ T cells and CD8+ T cells are the only populations bearing Thy 1.1, and because CD8+ T cells had been depleted during enrichment, sorting for Thyl.l(+) cells using FACSVantage SE cell sorter (BD Biosciences) resulted in highly purified 6.5 CD4+ T cells (95%). This technique avoids the use of TCR-specific or CD4 coreceptor specific antibodies that could potentially alter TCR or CD4 dependent gene expression patterns.
• Sorted cells were sheared with Qiashredder columns (Qiagen, Valencia CA), followed by total RNA isolation using the RNeasy kit (Qiagen).
• cDNA was synthesized using the Superscript Choice kit (Gibco/BRL) and an HPLC purified T7-DT primer (Proligo, Boulder, CO).
• Biotinylated cRNA probe was prepared using the ENZO BioArray RNA transcript kit (Affymetrix, Santa Clara, CA).
• Murine gene chips U174A, B and C were hybridized and analyzed according to standard Affymetrix protocols.
• mRNA prepared from purified na ⁇ ve 6.5 clonotypic CD4 + T cells and anergic/Treg and effector/memory 6.5 clonotypic CD4 T cells on various days after adoptive transfer was analyzed by Affymetrix gene chips.
• the differential expression of genes between anergy/Treg induction and effector/memory induction was ranked by "distance". The distance was defined as the sum of the absolute differences of expression between anergic T cells and effector/memory T cells on day 2 ( ⁇ dl ⁇ ), day 3 ( ⁇ d2 ⁇ ), and day 4 ( ⁇ d3 ⁇ ) after adoptive transfer, divided by the value of na ⁇ ve CD4 + T cells ( «) for normalization.
• Anti-LAG-3 C9B7W, from PharMingen
• Anti-CD25 7D4, from PharMingen
• anti-GITR polyclonal antibody purchased from R&D Systems.
• splenocytes from 6.5+/-Thyl.l+/- transgenic mice were isolated and enriched for CD4+ using a CD4+ negative selection isolation kit (Miltenyi Biotec).
• Splenocytes and inguinal and axillary lymph nodes were harvested five days later and prepared into a single cell suspension.
• RBCs were lysed with ACK lysis buffer.
• Wild type BALB/c mice were used for out natural Treg assays. 5 X 10 4 flow cytometry sorted CD4+CD25- T cells (Responders) and 5 x 10 4 3000-rad irradiated BALB/c splenocytes (Antigen Presenting Cells) were mixed with different numbers of flow cytometry sorted CD4+CD25+ suppressor T cells and incubated in round bottom 96-well tissue culture plates with 0.5 ⁇ g/ml of anti-CD3 antibody in 200 ⁇ l of CTL media. Forty-eight to 72 hours later, cultures were pulsed with 1 ⁇ Ci 3 H- thymidine and incubated an additional 16 hours before harvest with a Packard Micromate cell harvester. Determination of the amount of incorporated radioactive counts was performed with a Packard Matrix 96 direct beta counter (Packard Biosciences, Meriden, CT).
• Primer pair and probe sets were designed using Primer Express software and then synthesized by Applied Biosystems for LAG-3, CD25, GITR and IFN- ⁇ . Primer and probe set used for Foxp3 was quoted from literature (S4).
• the relative mRNA frequencies were determined by normalization to the internal control 18S RNA. Briefly, we normalized each set of samples using the difference in the threshold cycles (Ct) between the target gene and the 18S RNA: The calibration sample was assigned as the sample with the highest ⁇ Ct in each set of assay ( ⁇ Ct ca iibration) • Relative mRNA frequencies were calculated as 2 AACt where ⁇ Ct samp i e ).
• Primers and probe sets used are: LAG-3 Primer 5'-ACA TCA ACC AGA CAG TGG CCA-3'(SEQ ID NO: 9)/Primer 5'-GCA TCC CCT GGT GAA GGT C- 3'(SEQ ID NO: 10)/Probe 5'-6FAM-CCC ACT CCC ATC CCG GCC C-TAMRA- 3 '(SEQ ID NO: 11); CD25 Primer 5'-TGT ATG ACC CAC CCG AGG TC-3'(SEQ ID NO: 12)/Primer 5'-TTA GGA TGG TGC CGT TCT TGT-3' (SEQ ID NO: 13)/Probe 5'-6FAM-CCA ATG CCA CAT TCA AAG CCC TCT CC-TAMRA- 3 '(SEQ ID NO: 14); GITR Primer 5'-TCC GGT GTG TTG CCT GTG-3'(SEQ ID NO: 15)/Primer 5'-CAA AGT CTG CA
• LAG-3 constructs and retroviral producer cell lines.
• LAG-3 constracts were produced using recombinant PCR as described (Vignali and Vignali, 1999).
• the LAG-3.WT and the functionally null mutant LAG-3.Y73F. ⁇ CY cytoplasmic tailless LAG-3 with a point mutation that greatly reduces the ability of LAG-3 to bind MHC class II
• LAG-3 constracts were cloned into a murine stem cell virus (MSCV)-based retroviral vector, which contained an internal ribosomal entry site (IRES) and green fluorescent protein (GFP), and retrovirus was produced as described (Persons et al., 1997; Persons et al., 1998).
• Retroviral producer cell lines were generated by repeatedly transducing GPE+86 cells ( ⁇ 7-10 times) until a viral titer of greater than 10 5 /ml after 24 h was obtained (Markowitz et al., 1988).
• Splenocytes from 6.5 mice were stained with biotin labeled anti-B220, anti-Grl, anti- Macl, anti-TER119, anti-CD49b, anti-CD8 and anti-CD25 antibody (PharMingen, San Diego, CA). The cells were then incubated with magnetic beads coupled with streptavidin and negatively sorted on an autoMACS (Miltenyi Biotech, Auburn CA) to 90-95% purity of CD4 + /CD25 " T cells. The purified 6.5 CD4 + /CD25 " T cells were activated by plate bound anti-CD3 (2C11) and anti-CD28 (35.71).
• the activated T cells (4xl0 5 cells/ml) were spin transduced (90min, 3000 rpm) with viral supernatant from vector alone, LAG-3. WT/GFP or LAG- 3.Y73F. ⁇ CY/GFP retroviral GPE+86 producer cell lines described above plus IL-2 and polybrene (6 ⁇ g/ml). The cells were allowed to rest for 10 days and then sorted on the top -30-35% GFP + /Thyl.2 + T cells.
• the purified GFP T cells were cultured (2 fold dilutions starting at 2.5xl0 4 ) with 2.5xl0 4 CD4 + /CD25 " 6.5 T cells (purified by negative AutoMACS), 5xl0 4 i ⁇ adiated (3000rads) splenocytes, and 5 ⁇ g/ml HA110- 120 in a 96-well round bottom plate.
• the cells were cultured for 72 h and pulsed with [ 3 H]thymidine I ⁇ Ci/well (Du Pont, Wilmington, DE) the last 7-8 h of culture.
• CD223 is cleaved from the cell surface and released in a soluble form (sLAG-3). It is generated in significant amounts by activated T cells in vitro (5 ⁇ g/ml) and is also found in the serum of mice (80 ng/ml). It is likely generated by a cell surface protease.
• sLAG-3 by Western blot. The cleavage occurs in the transmembrane region (e.g., amino acids 442-466 in SEQ ID NO: 2) or in the connector region (e.g., amino acids 432-441in SEQ ID NO: 2) immediately preceding it amino-terminally.
• LAG-3 is not only required for maximal regulatory T cells (Treg) function but is also sufficient. In other words, expression of LAG-3 alone is sufficient to convert cells from activated effector T cells into regulatory T cells.
• CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the production of EL- 10. J Immunol 166, 3008-3018.
• Lymphocyte- activation gene 3/major histocompatibility complex class II interaction modulates the antigenic response of CD4+ T lymphocytes. Eur J Immunol 24, 3216-3221.
• CD4(+)CD25(+) immunoregulatory T cells gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor. Immunity i , 311-323.
• Retroviral-mediated transfer of the green fluorescent protein gene into murine hematopoietic cells facilitates scoring and selection of transduced progenitors in vitro and identification of genetically modified cells in vivo. Blood 90, 1777-1786.
• Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation. J Exp Med 192, 295-302.

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