Methods of increasing T cell immune response in the treatment of cancer

Abstract

Combinations of anti-cancer antibodies and inhibitory antibodies to CD223 overcome immune suppression in cancer patients. The inhibitory antibodies may be generated in an animal by injection of fragments of CD223. Antibodies may be monoclonal antibodies or single chain antibodies or humanized antibodies.

Claims

1. A method of inhibiting tumor growth and/or cellular invasion in a subject, comprising: administering to the subject an agent which inhibits CD223 activity, thereby inhibiting tumor growth and/or cellular invasion.

2. The method of claim 1 wherein the administering to the subject inhibits tumor growth.

3. The method of claim 1 wherein administration of the agent inhibits CD223 expression and/or inhibits function of CD223.sup.+ T regulatory cells.

4. The method of claim 1 wherein the agent comprises an antibody or antibody fragment.

5. The method of claim 1 wherein the agent comprises an antibody.

6. The method of claim 1 further comprising administering one or more chemotherapeutic agents.

7. A method of treating cancer in a subject, comprising: administering to the subject an agent which inhibits CD223 activity, thereby treating cancer in the subject.

8. The method of claim 7 wherein administration of the agent inhibits CD223 expression and/or inhibits functions of CD223.sup.+ T regulatory cells.

9. The method of claim 7 wherein the agent comprises an antibody or antibody fragment.

10. The method of claim 7 wherein the agent comprises an antibody.

11. The method of claim 7 further comprising administering one or more chemotherapeutic agents.

12. A method of increasing an antigen specific T cell immune response in a subject, comprising administering to the subject an agent which inhibits CD223 expression activity.

13. The method of claim 12 wherein said organism is a human suffering from cancer.

14. The method of claim 12 wherein administration of the agent inhibits CD223 expression and/or inhibits functions of CD223.sup.+ T regulatory cells.

15. The method of claim 12 wherein the agent comprises an antibody, antibody fragment, or combinations thereof.

16. The method of claim 12 further comprising administering one or more chemotherapeutic agents.

17. The method of claim 12 wherein the subject is suffering from cancer.

18. The method of claim 1 wherein inhibition of CD223 activity by the agent increases T-cell numbers in the subject.

19. The method of claim 7 wherein inhibition of CD223 activity by the agent increases T-cell numbers in the subject.

20. The method of claim 14 wherein inhibition of CD223 activity by the agent increases T-cell numbers in the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A to 1B. HA specific CD4+ T cells become tolerant and develop regulatory T cell activity upon adoptive transfer into C3-HAhigh transgenic mice. (FIG. 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 2.5106 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. Transfer of smaller numbers of 6.5 CD4+ T cells results in less severe pulmonary pathology and the C3-HAhigh recipients survive the transfer. Residual 6.5 T cells become anergic as defined by their inability to produce -interferon or proliferate to HA antigen in vitro. Mice receiving a sublethal dose of 6.5 T cells are protected from subsequent infusion of 2.5106 naive 6.5 T cells. Thus, the initial tolerized T cells develop Treg activity that suppresses lethal pneumonitis induced by the second high dose of 6.5 T cells. (FIG. 1B) Localization of effector/memory vs. suppressed T cells in C3-HAhigh mice. Naive 6.5 T cells (Thy 1.1+/1.2) were adoptively transferred into C3-HAhigh recipients (Thy 1.1/1.2+), either in the absence or in the presence of 6.5 anergic/Treg cells (Thy 1.11/.2+). Spleens and lungs were harvested 3 days after adoptive transfer and Thy 1.1+ cells were stained by immunohistochemistry. In the absence of Treg cells, T effector cells are scattered in the splenic follicles (FIG. 1B(A)) and infiltrate the pulmonary vessels (FIG. 1B(B)). In the presence of Treg cells, suppressed HA-specific 6.5 T cells become sequestered in the splenic peri-arteriolar lymphatic sheath (FIG. 1B(c)) and fail to infiltrate the pulmonary vessels (FIG. 1B (D)).

(2) 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 (FIG. 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. (FIG. 2B) Cell surface LAG-3 protein levels were assessed by antibody staining. Splenocytes were harvested from C3-HAhigh, wild type Bl0.D2 mice immunized with Vac-HA, or wt Bl0.D2 mice 5 days after i.v. injection with 6.5+/Thy1.1+/ splenocytes, and prepared into a single cell suspension. All samples were first incubated with whole rat IgG to block Fc receptors. Cells were stained with TCR specific anti-6.5-biotin+SA-APC, LAG-3-PE, or the corresponding isotype controls. Cells were double gated on the total lymphocyte population and 6.5 positive lymphocytes. Isotype control-dashed line, Nave cellslight gray line, effector/memory cellsdark gray line, anergic/Treg cellsblack line. (FIG. 2C) Analysis of multiple samples of anergic/Treg populations over many experiments confirms a direct correlation between LAG-3 level and IL-10 mRNA level.

(3) FIG. 3A-3B. LAG-3 is expressed on induced Treg cells independently of CD25 and is a marker of Treg function. (FIG. 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. (FIG. 3B) Cells were sorted into 4 populations based on their surface LAG-3 and CD25 staining: LAG-3highCD25high, LAG-3highCD25 low, LAG-3lowCD25high, and LAG-3lowCD25low. 1105 of each of the different sorted subsets of cells were added as suppressors in an in vitro suppression assay with 1104 naive 6.5 CD4+ as responders. LAG-3lowCD25 low cells were least suppressive. LAG-3highCD25high, LAG-3highCD25low, and LAG-3lowCD25high 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.

(4) FIG. 4. Anti-LAG-3 antibodies block in vitro Treg activity. Monoclonal anti-LAG-3 antibody added to the in vitro suppression assay at a concentration of 2 g/ml, totally reverses the suppression of nave 6.5 CD4+ T cell proliferation in vitro by 6.5 CD4+ suppressors at a suppressor:responder ratio of 0.04:1.

(5) FIG. 5A to 5C. Anti-LAG-3 antibody eliminates the in vivo suppression by 6.5 CD4+Treg cells by directly inhibiting Treg cells. (FIG. 5A) C3-HAhigh mice pretreated with 8,000 6.5 CD4+ T cells survived subsequent challenge with 2.5106 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. to the C3-HAhigh mice with the lethal dose of 6.5 T cells 4 days after they were pretreated with 8,000 6.5 CD4+ T cells and another dose of 200 g was given 2 days later. Anti-LAG-3 antibody treated mice could no longer tolerate the subsequent lethal challenge (Protection+aLAG-3). In contrast, treatment with isotype control antibody rat IgG1 could not eliminate the in vivo suppression (Protection+RatIgG1). (FIGS. 5B and 5C) Anti-LAG-3 mAb does not hyper-activate naive 6.5 CD4+ T cells in the absence of Treg. C3-HAhigh mice received either 2.5105 (sublethal dose; FIG. 5B) or 8105 (partial lethality between 7 and 14 days after transfer; FIG. 5C) nave 6.5 CD4+ T cells in combination with anti-LAG-3 antibody, control rat IgG1, or no antibody. No lethality was observed with the anti-LAG-3 antibody infusions at the 2.5105 dose whereas lethality at 8105 dose was not affected by anti-LAG-3 antibody.

(6) FIG. 6A to 6D. Role of LAG-3 in natural CD4+CD25+ T cells. (FIG. 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. (FIG. 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 0.04:1), with anti-CD3 antibodies (0.5 g/ml) as the T cell stimulus. Anti-LAG-3 antibodies at the concentration of 50 /m1 reverse the in vitro suppression of natural CD4+CD25+ regulatory T cells whereas isotype control antibody does not. (FIG. 6D) After the suppressor assay in C, the CD4+CD25+ cells (distinguished from the effector cells by Thy1.2 marking) were stained with anti-LAG-3 or isotype control antibody.

(7) FIG. 7. Ectopic expression of wild type but not mutant LAG-3 in CD25 depleted 6.5 CD4+ T cells confers potent in vitro regulatory activity. 6.5 CD4+ T cells were first depleted of any CD25+natural Tregs and then transduced with MSCV-based retroviral vectors encoding either GFP alone, GFP+wild type LAG-3 or GFP+a mutant LAG-3.Y73FCY that has diminished binding to MHC class II and cannot mediate downstream signaling. After a 10 day rest period, essentially no endogenous LAG-3 staining was observed on 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.Y73FCY/GFP vectors. GFP+ cells from the MSCV-LAG-3/GFP and MSCV-LAG-3.Y73FCY/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 naive 6.5 CD4+CD25 cells in a proliferation assay.

(8) FIG. 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.sup.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 K, into NOD/SCID mice. NOD/SCID mice (5/group) were monitored for diabetes.

DETAILED DESCRIPTION OF THE INVENTION

(9) LAG-3 is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. We have found that aged LAG-3 deficient mice have twice as many CD4.sup.+ and CD8.sup.+ 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.sup.+ and CD8.sup.+ T cell homeostasis, and present LAG-3 as a therapeutic target for accelerating T cell engraftment following bone marrow transplantation.

(10) CD223, also known as lymphocyte antigen gene-3 or LAG-3, is a CD4-related activation-induced cell surface protein that binds to MHC class II molecules with high affinity. Baixeras, E. et al., J. Exp. Med. 176: 327 (1992). See Triebel, F., Lag-3(CD223), Protein Reviews on the Web (PROW) 3:15-18(2002) at the URL address: http file type, www host server, domain name ncbi.nlm.gov, directory PROW, subdirectory guide, document name 16548175 1_g.htm.; Triebel, F. et al., LAG-3, a novel lymphocyte activation gene closely related to CD4, J. Exp. Med. 171: 1393-1405 (1990). 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.

(11) Regulatory T cells are a subgroup of T cells that function by inhibiting effector T cells. Regulatory T cells are CD223.sup.+ and are typically also CD4.sup.+CD25.sup.+. 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.sup.+ 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 autoantigen. CD223.sup. cells can be similarly transferred to cancer patients, bacterial or vial infection patients, or AIDS patients.

(12) 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.

(13) 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.

(14) 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. Similarly, if treating patients with infections, other anti-infection agents can be coordinated with the treatment of the present invention targeted to CD223. Such agents may be small molecule drugs, vaccines, antibodies, etc.

(15) The number of 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. Alternatively, the CD223.sup. 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.

(16) 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 an unrelated individual.

(17) To inhibit the function of CD223, CD223 antibodies or small molecule inhibitors 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 of the 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.

(18) Antibodies generated against CD223 polypeptides can be obtained by direct injection of the 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.

(19) For preparation of monoclonal antibodies, 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).

(20) Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be readily used to produce single chain antibodies to CD223 polypeptides. Also, transgenic mice may be used to express humanized antibodies to immunogenic CD223 polypeptides.

(21) To enhance or activate the function of CD223, 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.

(22) 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. Autoimmune 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. These include without limitations plasmids and viral vectors. 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.

(23) Compositions comprising a mixture of antibodies which specifically bind to CD223; and an anti-cancer vaccine can be made in vitro. Preferably 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. 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.

(24) Anti-CD223 antibodies of the invention can be used to increase the magnitude of anti-cancer response of the 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.

(25) 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.

(26) Translation of 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 411:494-498 (2001)) or small molecule inhibitors of this process which target CD223 mRNA. 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. For example, 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 helixsee 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 (AntisenseOkano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described above can also be delivered to cells by antisense expression constructs 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.

(27) Antisense constructs, antisense oligonucleotides, RNA interference constructs or siRNA duplex RNA molecules can be used to interfere with expression of CD223. Typically at least 15, 17, 19, or 21 nucleotides of the complement of CD223 mRNA sequence are sufficient for an antisense molecule. Typically at least 19, 21, 22, or 23 nucleotides of CD223 are sufficient for an RNA interference molecule. Preferably 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 Hannon, G J, 2002, RNA Interference, Nature 418: 244-251; Bernstein E et al., 2002, RNA 7:1509-1521; Hutvagner G et al., RNAi: Nature abhors a double-strand. Curr. Opin. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553; Lee N S, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K. (2002). U6-promoter-driven siRNAs with four uridine 3 overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison P J, Caudy A A, Bernstein E, Hannon G J, and Conklin D S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul C P, Good P D, Winer I, and Engelke D R. (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 W C, 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 S L, and Turner D L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052.

(28) In addition to known modulators, 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. As one example, the inventors have identified a hen egg lysozyme (HEL), 48-62-specific, H-2A.sup.k-restricted murine CD4.sup.+ 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.

(29) 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.

(30) 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.

(31) 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 through 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.

(32) 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 amino 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. Thus 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.

(33) In accordance with the teachings of the invention, 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 of the body by foreign microorganisms (bacteria or viruses) or tumor growth or cancer.

(34) 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. By inhibiting CD223 in the donor T cells or using donor T cells that have been selected or engineered for a CD223 deficiency, 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.

(35) CD223 inhibitors may also be useful when administered in combination with conventional therapeutics to treat T cell proliferation sensitive disorders. For instance a tumor, which is a T cell proliferation sensitive disorder, is conventionally treated with 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.

(36) In accordance with the teachings of the invention, 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.

(37) When methods of the invention are carried out in vivo, 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. For example, an effective amount can depend upon the degree to which an individual has abnormally depressed levels of T cells.

(38) When administered, 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. When used in medicine, 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. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

(39) CD223 modulators may be combined, optionally, with a pharmaceutically-acceptable carrier. The term pharmaceutically-acceptable carrier as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration into a human. The term 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.

(40) 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. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

(41) 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, 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.

(42) A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular drug selected, the severity of the condition being treated and the dosage required for therapeutic efficacy. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, topical, nasal, interdermal, or parenteral routes. The term 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.

(43) The pharmaceutical 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.

(44) 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.

(45) 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. Specific examples include, but are not limited to: (a) erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

(46) Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, are used herein, means that the implant is constructed and arranged 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.

(47) While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention as set forth in the appended claims.

EXAMPLES

Example 1Negative Regulation of T Cell Homeostasis by LAG-3 (CD223)

(48) The following example shows that LAG-3 (CD223) negatively regulates CD4.sup.+ and CD8.sup.+ T cell homeostasis, supporting its identification as a novel therapeutic target for accelerating T cell engraftment following bone marrow transplantation.

(49) Wild type C57BL/6 mice have a constant number of .sup.+ T cells from 4 to 52 weeks of age. As previously reported, young 4 week old LAG-3.sup./ mice have normal T cell numbers. Miyazaki, T. et al., Science 272: 405-408 (1996). In contrast, the number of .sup.+ T cells in LAG-3.sup./ mice steadily increases from 3 months of age to numbers 2-fold higher than wild type mice. This difference is highly significant given the tight homeostatic regulation of .sup.+ T cell number evidenced by the very low standard deviation. Both CD4+ and CD8+ cells were increased in LAG-3.sup./ mice but the CD4:CD8 ratio was unchanged. Similarly, LAG-3.sup./ mice transgenic for the OT-II TCR (ovalbumin 326-339-specific, H-2A.sup.b-restricted) had an increased number CD4.sup.+ V2.sup.+ T cells compared with wild type control OT-II transgenic mice, except that these differences were evident at 5 weeks of age. Approximately 20% .sup.+ T cells and CD49b.sup.+ NK cells constitutively express LAG-3 in wild type mice (Workman, C. J. et al., Eur. J. Immunol. 32: 2255-2263 (2002)), and their numbers were also significantly increased in LAG-3.sup./ mice. Surprisingly, several other cell types such as B220.sup.+ B cells, Gr-1.sup.+ granulocytes and Mac-1.sup.+ macrophages, none of which express LAG-3, were also increased in LAG-3.sup./ compared with control mice. The increased cell numbers observed in LAG-3.sup./ mice was consistent with a 50% increase in the number of dividing BrdU.sup.+ cells in vivo. It is important to note that the differences in cell number observed between LAG-3.sup./ and wild type mice was highly consistent and reproducible. The absence of LAG-3 did not appear to have any significant effect on the cell surface phenotype of T cells from LAG-3.sup./ mice. These data support the idea that LAG-3 regulates the number of T cells in mice, and indirectly affects leukocyte numbers in general.

(50) To determine if LAG-3 influences the homeostatic expansion of T cells in a lymphopenic environment, purified T cells were adoptively transferred into RAG.sup./ mice, which lack T and B cells, and T cell number in the spleen determined 15 days post-transfer. There was a 2.8-fold increase in the number of LAG-3.sup./ T cells compared with the wild type control. Remarkably, only a small percentage of the wild-type T cells expressed LAG-3 despite the clear effect that the absence of LAG-3 has on T cell expansion. This infers that brief, transient expression of LAG-3 may be sufficient for it to exert its effect on dividing cells. Increased expansion of CD4.sup.+ and CD8.sup.+ T cells was observed demonstrating that both cell types were equally affected by the absence of LAG-3. Interestingly, there was also a two-fold increase in the number of .sup. host-derived cells in recipients of LAG-3.sup./ versus LAG-3.sup.+/+ T cells. This was consistent with the increased number of macrophages and granulocytes observed in unmanipulated LAG-3.sup./ mice. To ensure that the increased expansion of LAG-3.sup./ T cells observed in RAG.sup./ mice is independent of antigen specificity, we used purified T cells from OVA [Ovalbumin 257-264-specific, H-2K.sup.b-restricted; Hogquist, K. A. et al., Cell 76: 17-27 (1994)] and OT-II [Ovalbumin 326-339-specific, H-2A.sup.b-restricted; Bamden, M. J. et al., Immunol. Cell Biol. 76: 34-40 (1998)] transgenic mice. Wild-type CD4+V2.sup.+ OT-II T cells expanded poorly in RAG.sup./ mice, consistent with previous reports indicating that these cells show little homeostatic expansion in lymphopenic hosts. Ernst, B. et al., Immunity 11: 173-181 (1999). In contrast, this limitation did not apply to T cells from LAG-3.sup./ OT-II transgenic mice, which expanded vigorously in lymphopenic hosts to numbers that were 3.2-fold more than the wild-type T cells by 15 days post-transfer. Similarly, the number of LAG-3.sup./ CD8+V2.sup.+ OVA transgenic T cells recovered from RAG-1.sup./ mice was 4-fold higher than wild-type control OVA T cells. Remarkably, this difference persisted for at least a month post transfer. These data again demonstrated that both CD4.sup.+ and CD8.sup.+ T cells are equally affected by the loss of LAG-3. To assess the importance of LAG-3 ligation by MHC class II molecules, LAG-3.sup./ and wild type OVA transgenic T cells were transferred into mice lacking both MHC class I and class II molecules (2m.sup./H-2A.sup.b/). The data clearly show that the enhanced expansion of LAG-3.sup./ T cells is abrogated in the absence of MHC class II molecules, demonstrating the importance of this interaction.

(51) LAG-3.sup./ mice or adoptive recipients of LAG-3.sup./ 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. To test this directly, B cells were co-transferred with either LAG-3.sup./ or wild-type T cells into RAG.sup./ mice. We also took advantage of this approach to assess the contrasting roles of 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. et al., Immunity 5: 217-228 (1996); Rooke, R., et al., Immunity 7:123-134 (1997). In contrast, it is possible that the interaction between LAG-3 and MHC class II molecules would have the opposite effect. As seen previously, there was a 3.0-fold increase in the number of LAG-3.sup./ T cells compared with the wild type control when transferred with MHC class II.sup./ B cells. However in the presence of wild-type B cells, the difference between LAG-3.sup./ and LAG-3.sup.+/+ T cell numbers increased to 4.9-fold. The increased LAG-3.sup./ T cell number is likely due to increased MHC: TCR interaction, thus potentiating expansion. In contrast, the LAG-3.sup./ T cells would be subjected to both positive (via MHC: TCR interaction) and negative (via MHC:LAG-3 interaction) homeostatic control which results in comparable expansion of wild-type T cells.

(52) In the presence of wild-type T cells, the number of B cells recovered from the spleen 7 days post-transfer was identical to mice receiving B cells alone. In contrast, there was a 2.7-fold increase in the number of B cells recovered from LAG-3.sup./ T cell recipients, providing a direct demonstration that the increased B cell number was due to the deregulation of LAG-3.sup./ T cells. Interestingly, there was an increase in the number of MHC class II.sup./ B cells in the presence of wild-type T cells compared with mice receiving B cells alone. This supports the idea that the local absence of LAG-3:MHC class II interaction can result in increased B cell expansion due to transient deregulation of wild-type T cells even though the recipient RAG.sup./ mice have MHC class II.sup.+ macrophages and dendritic cells in the spleen. An alternate possibility is that ligation of MHC class II molecules by LAG-3 delivers a negative regulatory signal to B cells thereby preventing expansion. While this is plausible for B cells, it would not explain the increased numbers of MHC class II.sup. cells, such as granulocytes, in LAG-3.sup./ mice. One possibility, which is currently being investigated, is that the deregulated expansion of LAG-3.sup./ T cells results in their production of cytokines that induce the broad expansion of many cell types.

(53) The influence of LAG-3 expression on homeostatic expansion in lymphopenic mice is not limited to nave T cells. Transfer of antigen-experienced memory OT-II T cells also resulted in a substantially accelerated expansion of LAG-3.sup./ 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.sup./ 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.sup.M. Workman, C. J. et al., Eur. J. Immunol. 32: 2255-2263 (2002). The vector also contained an internal ribosomal entry site (IRES) and green fluorescent protein (GFP) cassette to facilitate analysis of transduced cells. Persons, D. A. et al., Blood 90: 1777-1786 (1997). LAG-3.sup./ and LAG-3.sup.+/+ OT-II T cells were also transduced with an empty vector/GFP alone control. Transduced cells were transferred into RAG-1.sup./ recipients and the number of OT-II T cells recovered 15-days post-transfer determined. As expected, the LAG-3.sup./ GFP alone control T cells expended more than the wild-type GFP cells [2.8-fold]. Ectopic expression of LAG-3 reduced the number of OT-II T cells to a level comparable to the wild-type control, while expression of the LAG-3 signaling defective mutant had no effect on homeostatic expansion. These data demonstrate that LAG-3 is directly responsible for the effects observed.

(54) Our data clearly show that LAG-3 negatively regulates homeostatic expansion of T cells. They also support the idea that T cells may contribute to the homeostasis of many cell types. Despite the clear effect that the absence of LAG-3 had on T cells numbers in knockout mice and the expansion of T cells in lymphopenic mice, it was remarkable that only a very small percentage of T cells expressed LAG-3. Interestingly, ectopic expression of LAG-3 on all T cells did not have a greater effect on homeostatic expansion than the low-level, transient expression of LAG-3 seen on wild-type cells. This suggests that the threshold for LAG-3 signaling may be very low, and that there may be other factors that limit the effect of LAG-3 signaling. Identifying the downstream signaling molecules(s) that interact with LAG-3 and determining the mechanism by which LAG-3 regulates homeostatic expansion will clearly be an important focus of future research.

(55) A surprising observation was the increased number of cells that do not express LAG-3, such as B cells and macrophages. Co-transfer experiments clearly demonstrated that the absence of LAG-3 on T cells was responsible for the increase in other cells types observed. This could be due to a soluble or cell surface protein that is either induced by LAG-3 signaling that limits the number and/or expansion of other cell types or produced due to the absence of negative regulation by LAG-3 that limits the number and/or expansion of other cell types. The precise nature of this bystander expansion and its physiological role remain to be determined.

(56) Patients receiving bone marrow or mega dose stem cell transplants are particularly susceptible to infections in the first 4-6 months due to the slow rate of lymphocyte reconstitution. Our studies support the idea that LAG-3 is a viable therapeutic target and that blocking LAG-3 expression or function will accelerate T cell engraftment and significantly reduce this window of susceptibility.

Example 2Materials and Methods

(57) This example provides the experimental methods and materials for example 1.

(58) Mice:

(59) The following mice were used: LAG-3.sup./ [obtained from Yueh-Hsiu Chen, Stanford University, Palo Alto, Calif., with permission from Christophe Benoist and Diane Mathis, Joslin Diabetes Center, Boston, Mass.; Miyazaki, T. et al., Science 272: 405-408 (1996)]; C57BL/6J [Jackson Labs, Bar Harbor, Me.]; B6.PL-Thy1.sup./Cy (Thy1.1 congenic) [Jackson Labs]; RAG-1.sup./ [Jackson Labs, Bar Harbor, Me.; Mombaerts, P. et al., Cell 68: 869-877 (1992)]; MHC class II.sup./[provided by Peter Doherty, St. Jude Children's Research Hospital, Memphis, Tenn.; Grusby, M. J. et al., Science 253:1417-1420 (1991)]; MHC class I.sup.//II.sup./ [Taconic, Germantown, N.Y.; Grusby, M. J. et. al., Proc. Natl. Acad. Sci. U.S.A 90: 3913-3917 (1993)]; OT-II TCR transgenic mice [provided by Stephen Schoenberger, La Jolla Institute for Allergy and Immunology, La Jolla, Calif., with permission from William Heath, Walter and Eliza Hall Institute, Parkville, Victoria Australia; Barnden, M. J. et al., Immunol. Cell Biol. 76: 34-40 (1998)] and OT-I (OVA) TCR transgenic mice [Jackson Labs; Hogquist, K. A. et al., Cell 76: 17-27 (1994)]. Genome-wide microsatellite analysis demonstrated that 97% of the 88 genetic markers tested for the LAG-3.sup./ mice were derived from B6 mice (Charles River Laboratories, Troy, N.Y.). LAG-3.sup./, MHC class II.sup./, OT-I.LAG-3.sup./ and OT-II.LAG-3.sup./ colonies were maintained in the St. Jude Animal Resource Center. All animal experiments were performed in an AAALAC-accredited, SPF facility following national, state and institutional guidelines. Animal protocols were approved by the St. Jude IACUC.

(60) LAG-3 Constructs and Retroviral Transduction:

(61) 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.sup.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 constructs 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 than 10.sup.5/ml after 24 hrs was obtained (Markowitz, D. et al., J. Virol. 62: 1120-1124 (1988)).

(62) Flow Cytometry:

(63) Single cell suspensions were made from spleens and RBC lysed with Gey's solution. Splenocytes were first stained with Fc Block, anti-CD16/CD32 (2.4G2) (BD PharMingen, San Diego, Calif.) for 10 min on ice. The cells were then stained for the following cell surface markers using various conjugated antibodies from BD PharMingen: 0+ TCR (H-57-597), V2 (B20.1), TCR (GL3), CD4 (RM4-4), CD8a (53-6.7), CD45R/B220 (RA3-6B2), CD11b/Mac1 (M1/70), 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, IgG1 ; Workman, C. J. et al., Eur. J. Immunol. 32: 2255-2263 (2002)) or the same antibody obtained as a PE conjugate (BD PharMingen). The cells were then analyzed by flow cytometry (Becton Dickinson, San Jose, Calif.).

(64) Bromodeoxyuridine Incorporation:

(65) At 5, 16, 28, and 52 weeks of age, LAG-3.sup.+/+, LAG-3.sup./, OTII.LAG-3.sup.+/+ and OTII.LAG-3.sup./ mice were given BrdU (Sigma, St. Louis, Mo.) in their drinking water for 8 days (0.8 mg/ml). The mice were then sacrificed by CO.sub.2 inhalation and the spleens removed. Staining for BrdU incorporation was performed as described (Flynn, K. J. et al., Proc. Natl. Acad. Sci. U.S.A 96: 8597-8602 (1999)). Briefly, the LAG-3.sup./ and LAG-3.sup.+/+ splenocytes were stained for TCR, CD4, CD8 and B220 expression. The OTII.LAG-3.sup.+/+ and OTII.LAG-3.sup./ splenocytes were stained for V2 and CD4 expression (PharMingen). The cells were then fixed with 1.2 ml 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 50 KU of DNase (Sigma) in 0.15M NaCl+4.2 mM MgCl.sub.2 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.

(66) Adoptive Transfer Experiments:

(67) T cells and/or B cells from splenocytes were either positively sorted by FACS or negatively sorted by magnetic bead cell sorting (MACS). For FACS purifications, splenocytes were stained for TCR, CD4 and CD8 expression and sorted by positive selection on a MoFlow (Cytomation, Ft. Collins, Colo.). For negative MACS purification, splenocytes were stained with PE-coupled anti-B220, anti-Grl, anti-Macl, anti-TER119 (erythrocytes), anti-CD244.2 (NK cells) and anti-CD8 (for negative purification of OTII transgenic T cells). The cells were then incubated with magnetic beads coupled with anti-PE antibody (Miltenyi Biotech, Auburn, Calif.) and then negatively sorted on an autoMACS (Miltenyi Biotech, Auburn, Calif.) to 90-95% purity. In some experiments, T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells were washed twice with PBS, resuspended in PBS plus 0.1% BSA at 110.sup.7 cells/ml and incubated with 5 M CFSE for 10 min at 37 C. The cells were washed twice with PBS plus 0.1% BSA. The purified CFSE labeled or unlabeled T cells (510.sup.6 or 110.sup.7) and in some cases B cells (510.sup.6) were injected i.v. into RAG-1.sup./ or Thy1.1.sup.+ (B6.PL) mice.

(68) Retroviral Transduction of Normal T Cells:

(69) Spleens from OTII.LAG-3.sup.+/+ and OTII.LAG3.sup./ mice were removed and single cell suspensions made at 2.510.sup.6 cell/ml. The splenocytes were activated with OVA 326-339 peptide [10 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.sup.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 V2.sup.+/CD4.sup.+/GFP.sup.+ expression by FACS. The cells were allowed to rest for two additional days and then 510.sup.6 cells were injected into RAG.sup./ mice via the tail vein. Fifteen days post-transfer, the mice were sacrificed by CO.sub.2 inhalation and the spleens removed. The splenocytes were stained and analyzed by flow cytometry.

Example 3Induced Treg Cells with Potent Regulatory Activity

(70) In order to identify Treg specific molecules, we performed a differential gene expression analysis of antigen-specific T cells differentiating to either effector/memory cells in response to viral infection or Treg cells upon encounter of cognate antigen as a self-antigen. This analysis revealed that the LAG-3 gene was selectively upregulated in Treg cells. The physiologic role of LAG-3, an MHC class II binding CD4 homologue, has not been clearly elucidated. Several in vitro studies have suggested that LAG-3 may have a negative regulatory function (Hannier et al., 1998; Huard et al., 1994; Workman et al., 2002a; Workman et al., 2002b). Here we show that membrane expression of LAG-3 selectively marks Treg cells independently of CD25 and that LAG-3 modulates both the in vitro and in vivo suppressive activity of Treg cells.

(71) In order to study differences between T cell effector/memory and tolerance induction, we have utilized adoptive transfer of T cell receptor (TCR) transgenic CD4+ T cells (clone 6.5) specific for a model antigen-hemagglutinin (HA). In wild-type mice infected with recombinant HA-expressing vaccinia virus (Vac-HA), 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. When removed from the adoptively transferred 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. However, after the activation phase, 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.

(72) 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. Thus, C3-HA.sup.low mice tolerate the transfer of 2.510.sup.6 6.5 CD4+ T cells quite well, but C3-HA.sup.high mice, which have 1000 fold higher HA expression than C3-HA.sup.low mice, die within 4-7 days after transfer of 2.510.sup.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. Adoptive transfer of less than 2.510.sup.5 6.5 CD4+ T cells into C3-HA.sup.high mice causes pulmonary vasculitis of less severity and the recipients survive (FIG. 1A) (Huang et al., 2003). Interestingly, 6.5 CD4+ T cells transferred at a sublethal dose acquire a regulatory phenotype as they are capable of protecting mice from death upon subsequent infusion of what would be a lethal dose of 6.5 CD4+ T cells in unprotected C3-HA.sup.high mice. This in vivo regulatory function is extremely potent, since transfer of as few as 8,000 cells (0.3% of the lethal dose) will completely protect animals from death upon subsequent infusion of 2.510.sup.6 naive 6.5 CD4+ T cells. Protection is observed as early as 4 days after the initial transfer and remains active up to 6 months (FIG. 1A). Depletion of CD4+ T cells, but not CD8+ T cells, before adoptive transfer totally eliminates the protective effect, thereby defining the Treg phenotype of anergized clonotypic 6.5 CD4+ T cells.

(73) Suppression of lethal pneumonitis is accompanied by an accumulation of the initial input (Treg) 6.5 T cells in the lungs and a drastic reduction in the number of infiltrating T effector cells from the second infusion. Instead of accumulating in the lungs, as occurs in the absence of Treg cells, the T effector cells accumulate in the splenic peri-arteriolar lymphatic sheath (FIG. 1B). Further evidence that the anergic cells demonstrate Treg function comes from the finding that they inhibit the activation of cytotoxic HA-specific CD8+ T cells in vivo (data not shown). Elimination of CD25+ T cells prior to the first (protective) adoptive transfer did not affect the development of Treg cells capable of protecting animals from a subsequent lethal challenge of 6.5 T cells. Therefore, it is likely that the Treg phenotype of the initial input T cells was acquired after adoptive transfer as opposed to being a consequence of naturally occurring Treg cells among the adoptively transferred population. These findings are highly compatible with the published findings of Von Boehmer and colleagues, who demonstrated that 6.5 CD4+ T cells rendered tolerant after transfer into transgenic mice expressing HA in the B cell compartment in fact exhibit Treg function (Jooss et al., 2001).

Example 4LAG-3 is Differentially Expressed on Induced Treg Cells

(74) In order to identify genes associated with the anergic/Treg phenotype in our in vivo system, we performed Affymetrix chip analysis on purified 6.5 CD4+ T cells either after adoptive transfer into non-transgenic recipients followed by Vac-HA immunization to generate effector/memory T cells or after transfer into C3-HA.sup.high mice to generate anergic/Treg cells. Thy1.1(+)Thy1.2() congenic 6.5 T cells were purified from Thy1.1()Thy1.2(+) Vac-HA infected wild-type (effector/memory) or C3HA.sup.high (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.

(75) RNA was isolated from naive 6.5 CD4+ T cells as the day 0 sample and isolated from 6.5 CD4+ T cells at days 2, 3 and 4 post-adoptive transfer for chip analysis. 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. Among the genes that had been previously identified, LAG-3 was among the most differentially expressed in anergic/Treg populations relative to effector/memory populations. This result was subsequently validated by quantitative RT-PCR analysis with a LAG-3 primer-probe pair for various time points extended to 1 month post adoptive transfer. After a minimal initial increase in the effector/memory cells, LAG-3 expression returns to baseline by 20 days post adoptive transfer. In striking contrast, 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).

(76) Cell surface expression of LAG-3 on populations of anergic/Treg 6.5 CD4+ T cells relative to effector/memory 6.5 CD4+ T cells was then analyzed using an anti-LAG-3 monoclonal antibody (Workman et al., 2002b) (FIG. 2B). While there are very low levels of LAG-3 staining on effector/memory cells, a significant proportion of anergic/Treg cells from C3-HA.sup.high transgenic mice display moderate to high levels of LAG-3 staining, correlating with the gene expression results. As 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.sup.high 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 correlation coefficient (R.sup.2) of 0.87 (FIG. 2C).

Example 5LAG-3 is Required for Maximal Treg Function

(77) Cell surface expression of LAG-3 and CD25 on populations of anergic/Treg 6.5 CD4+ T cells was analyzed coordinately using anti-LAG-3 and anti-CD25 monoclonal antibodies. Although similar proportions of effector/memory and anergic/Treg cells express CD25 (data not shown), LAG-3 and CD25 expression on anergic/Treg cells was not completely concordant (FIG. 3A). We therefore sorted the cells into LAG-3.sup.highCD25.sup.high, LAG-3.sup.highCD25.sup.low, LAG-3.sup.lowCD25.sup.high, and LAG-3.sup.lowD25.sup.low populations and analyzed their regulatory activity in a standard in vitro suppression assay. In vitro suppression of proliferative responses among naive 6.5 CD4+ cells showed that the LAG-3.sup.highCD25.sup.high population displayed the highest suppressive activity and the LAG-3.sup.lowCD25.sup.low population had the lowest while the suppressive activity of the LAG-3.sup.highCD25.sup.low and the LAG-3.sup.lowCD25.sup.low cells were comparable (FIG. 3B). These results suggest that, among induced Treg cells, the combination of LAG-3 and CD25 may mark Treg cells with the most suppressive activity.

(78) To further evaluate the direct role of LAG-3 in regulating suppression by induced Treg cells, we first determined whether anti-LAG-3 antibodies could block the ability of LAG-3 expressing cells to suppress the in vitro proliferative responses of naive HA-specific T cells. Anti-LAG-3 antibodies at the concentration of 2 g/ml inhibit suppression by Treg 6.5 CD4+ T cells in the in vitro assay system (FIG. 4). Over the 2-day assay period, anti-LAG-3 antibodies did not affect proliferative responses of 6.5 T cells stimulated in the absence of Treg, confirming that the effect of anti-LAG-3 antibodies was indeed on the Treg cells and not the effector cells (data not shown). The ability of anti-LAG-3 antibodies to block in vitro suppression by Treg cells demonstrates that LAG-3 is not simply a Treg selective marker, but is a molecule that modulates Treg activity.

Example 6LAG-3 is Required for Induced Treg Activity In Vivo

(79) We next evaluated the role of LAG-3 in modulating in vivo Treg function by determining whether administration of anti-LAG-3 antibodies could block suppression of lethal pneumonitis by Tregs in C3-HA.sup.high mice. C3-HA.sup.high mice were pretreated with 8,000 (sublethal dose) of 6.5 CD4+ T cells followed by a subsequent dose of 2.510.sup.6 naive 6.5 CD4+ T cells 4 days after the first transfer. As described above, Tregs have already developed at this point. Anti-LAG-3 antibody (200 g) was administered i.v. together with the subsequent challenge of 2.510.sup.6 6.5 cells and another 200 g was given 2 days later. This antibody treatment totally eliminated the in vivo suppressive activity of the Treg cells and the mice died in a time frame comparable to the C3-HA.sup.high mice lethally challenged without protective sublethal 6.5 pretreatment. On the contrary, mice with established Treg treated with isotype control antibody (Rat IgG1) or no antibody survived subsequent challenge with 2.510.sup.6 naive 6.5 T cells (FIG. 5A). While these results suggest that the anti-LAG-3 antibodies were blocking Treg activity in vivo, an alternate formal possibility was that, rather than directly inhibiting Treg cells, the anti-LAG-3 antibodies hyper-activated the T cells in the challenge population such that they overcame the inhibitory effects of the Tregs. To rule out this possibility, we asked whether in vivo administration of anti-LAG-3 antibodies together with a dose of 6.5 T cells just below the lethality threshold would cause lethality in the absence of a pre-established Treg population. We therefore administered 2.510.sup.5 6.5 T cells (the maximal dose that will not cause lethality) or 8.010.sup.5 6.5 T cells (roughly 50% lethality between 7 and 14 days after transfer) into C3-HA.sup.high mice together with anti-LAG-3 antibodies or isotype control. FIG. 5B demonstrates that the anti-LAG-3 treatment did not render the 2.510.sup.5 6.5 T cell dose lethal nor enhance the partial lethality of the 8.010.sup.5 6.5 T cell dose. Therefore, the effect of anti-LAG-3 antibodies in the experiment in FIG. 5A was to directly inhibit Treg cells.

Example 7LAG-3 is Expressed by Natural Treg Cells and is Required for Suppressor Activity

(80) Taken together, these data confirm an important role for LAG-3 in mediating suppressor function of induced Treg. Given that the relationship between induced Treg and natural Treg remains unclear, it was of interest to see whether LAG-3 was expressed selectively on CD4+CD25+ T cells from wild type mice. LAG-3 mRNA (along with CTLA-4, FoxP3 and GITR mRNA) is indeed selectively expressed on CD4+CD25+ cells compared with CD4+CD25 cells (FIG. 6A). Despite this reproducible finding, we were unable to detect surface LAG-3 on either CD4+CD25+ or CD4+CD25 cells by antibody staining. However, antibody staining of permeabilized cells clearly indicated that 10-20% of CD4+CD25+ cells expressed intracellular stores of LAG-3. In contrast staining of permeabilized CD4+CD25 cells demonstrated absolutely no LAG-3+ population (FIG. 6B). These findings suggested that at least some natural Tregs possessed intracellular stores of LAG-3 that could be rapidly recruited to the cell surface upon encounter with cognate antigen and subsequently mediate suppression. While natural Treg are contained within the T cell population defined by CD4 and CD25, it is indeed possible that the actual Treg cells are those expressing intracellular LAG-3. To directly evaluate the role of LAG-3 in the regulatory function of natural Tregs, we asked whether anti-LAG-3 antibodies could inhibit in vitro suppression mediated by purified CD4+CD25+ cells. FIG. 6C demonstrates that anti-LAG-3 antibodies indeed block suppression mediated by purified CD4+CD25+ cells, suggesting that LAG-3 plays a role in suppression mediated by natural as well as induced Treg. Staining of the CD4+CD25+ cells at the end of the in vitro suppression assay revealed that roughly 20% now express high levels of LAG-3 on their surface, supporting the notion that the intracellular LAG-3 in mobilized to the surface under circumstances of TCR engagement and mediates regulatory activity (FIG. 6D).

Example 8Ectopic Expression of LAG-3 Confers Regulatory Activity

(81) The blocking experiments in FIGS. 5 and 6 suggest that LAG-3 is required for maximal Treg function. To further validate this conclusion, we performed a series of transduction experiments to determine if ectopic expression of LAG-3 on T cells confers regulatory activity. For these experiments, 6.5 CD4+ T cells were first depleted of any CD25+ natural Tregs and then transduced with MSCV-based retroviral vectors encoding either GFP alone, GFP+wild type LAG-3 or GFP+a mutant LAG-3.Y73FCY that has substantially reduced affinity for MHC class II and cannot mediate downstream signaling (Workman et al., 2002a). After a 10 day rest period, essentially no endogenous LAG-3 staining was observed on 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.Y73FCY/GFP vectors. GFP+ cells from each group were sorted and mixed at different ratios with APC, HA.sup.110-120 peptide and nave 6.5 CD4+CD25 cells in a proliferation assay. As shown in FIG. 7, 6.5 cells expressing wild type LAG-3 potently suppressed proliferation of nave 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.Y73FCY mutant. Total proliferation was in fact somewhat increased in these two latter groups, since GFP and LAG-3.Y73FCY transduced 6.5 cells themselves proliferate in addition to the nave 6.5 cells in the assay. Indeed, wild type LAG-3 transduced T cells themselves demonstrated a significant reduction in proliferative responses apart from inhibiting proliferation of the non-transduced 6.5 cells. These results provide direct evidence confirming the functional role of LAG-3 in suppression. Interestingly, 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.

Example 9Discussion

(82) These findings identify LAG-3 as 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. Furthermore, other experimental data demonstrate a role for LAG-3 in the regulation of homeostatic lymphocyte expansion by natural Treg (Workman and Vignali, accompanying paper). The finding that 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. As suggested by the experiments in FIG. 3, the combination of LAG-3 and CD25 may define Treg subsets with the most potent suppressive activity. We do not propose that 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. In fact, it is not clear that 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). LAG-3.sup.high 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. We therefore propose that LAG-3 is a Treg specific receptor or coreceptor that modulates the suppressor activity of this T cell subset.

(83) A number of studies have suggested a cell autonomous inhibitory role for LAG-3 (Huard et al., 1994; Workman et al., 2002b), although initial studies with LAG-3 KO mice failed to uncover any evidence for overt autoimmunity or hyperimmunity (Miyazaki et al., 1996). Given our proposed role for LAG-3 in Treg function, it might be expected that LAG-3 knockout mice would display multi-system autoimmunity (i.e., similar to Foxp3 knockout or scurfy mice), which has not been reported in these mice. However, there are clearly regulatory T cell defects displayed by LAG-3 knockout mice, such as a defect in regulating cellular homeostasis (Workman and Vignali, accompanying paper). 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.

(84) 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. The apparent reason that 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. More recently, the TNF receptor super-family member 18 molecule (also called GITR) was demonstrated to be up-regulated on Treg cells. Furthermore, antibodies to GITR have been reported to inhibit Treg activity both in vivo and in vitro. However, 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). Moreover, there are numerous reports that CD4+CD25-cell populations can suppress certain immune functions (Annacker et al., 2001; Apostolou 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.sup.highLAG-3.sup.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.

(85) Our data show that LAG-3 is required for the maximal suppressive activity of both natural and induced Treg cells. However, is it sufficient? Thus far, the only molecule shown to confer regulatory activity on activated T cells is Foxp3 (Fontenot et al., 2003; Hori et al., 2003). Importantly we have shown here that ectopic expression of LAG-3, but not a functionally defective mutant, on CD4+ T cells can also confer regulatory activity.

(86) Another key question is whether Treg cells suppress the reactivity of CD4+ and CD8+ effector cells through direct T-T interactions or through DC intermediaries. The identification of Treg selective and functional expression of LAG-3, a MHC class II binding molecule, should provide a new handle on dissecting mechanisms and manipulating Treg function for diseases in which these cells play an important role.

Example 10Experimental Procedures

(87) Transgenic Mice

(88) The C3-HA transgenic mice have been previously described (Adler et al., 2000; Adler et al., 1998). In short, 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(1) promoter. Two founder lines were established in the B 10.D2 genetic background. These two founder lines, C3-HA.sup.high and C3-HA.sup.low, which contain 30-50 and 3 transgene copies respectively, express the C3-HA hybrid rnRNA in the same set of non-lymphoid tissues including the lung and prostate. While the difference in total HA protein expression between C3-HA.sup.high and C3-HA.sup.low was not directly measured, in the lung and prostate, where the expression levels are highest, the difference is roughly 1000-fold as shown by bioassay of tissue extract induced hybridoma cytokine release.

(89) The TCR transgenic mouse line 6.5, that expresses a TCR recognizing an I-E.sup.d-restricted HA epitope (.sup.110SFERFEIFPKE.sup.120; SEQ ID NO: 7) (generously provided by Dr. Harald von Boehrner, Harvard University, Boston, Mass.), was back-crossed 9 generations onto the B 10.D2 genetic background. The other TCR transgenic mouse line Clone-4, that expresses a TCR recognizing a K.sup.d-restricted HA epitope (.sup.5181YSTVASSL.sup.526; SEQ ID NO: 8) (generously provided by Dr. Linda Sherman, Scripps Research Institute, La Jolla, Calif.), was also back-crossed more than 9 generations onto the Thy 1.1/1.1 B10.D2 genetic background. Because no clonotypic antibody is available for Clone-4 TCR, Thy 1.1 was used as a surrogate marker. Following adoptive transfer into Thy1.2/1.2 recipients, we can assume that all the Thy 1.1.sup.+ CD8.sup.+ T cells express the HA-specific clonotypic TCR as nearly all of the mature CD8.sup.+ T cells in the Clone-4 mice directly recognize the K.sup.d-restricted HA epitope (Morgan et al., 1996).

(90) 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.

(91) Adoptive Transfer

(92) Clonotypic CD4+ or CD8.sup.+ 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.

(93) Immunohistochemistry

(94) 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-Thy1.1 mAb (PharMingen, San Diego, Calif.). The Vectastain ABC kit (Vector, Burlingame, Calif.) 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, Calif.).

(95) Enrichment and Purification of In Vivo Primed 6.5 CD4+ T Cells

(96) With either effector/memory or tolerance induction in vivo after adoptive transfer, the clonotypic percentage of 6.5 CD4+ T cells in the spleens of recipient mice is only 0.2%-5%. Deliberate enrichment and purification is mandatory to obtain enough clonotypic CD4+ T cells for further studies, such as for Affymetrix gene chip analysis. Donor 6.5 T cells were crossed onto a Thy1.1(+)Thy1.2() background which allowed for a two step enrichment and purification procedure after adoptive transfer into Thy1.1()/Thy1.2(+) recipients. 6.5 CD4+ T cells were first enriched by using biotinylated anti-CD8 (Ly-2, 53-6.7), anti-B220 (RA3-6B2), and anti-Thy1.2 (30-H12) antibodies (all purchased from BD Biosciences PharMingen, San Diego, Calif.) and MACS streptavidin microbeads and MACS LS separation column (Miltenyi Biotech, Auburn, Calif.) to deplete CD8+ T cells, B cells and the recipient T cells (Thy 1.2+). Since CD4+ T cells and CD8+ T cells are the only populations bearing Thy1.1, and because CD8+ T cells had been depleted during enrichment, sorting for Thy1.1(+) 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.

(97) Gene Chip Analysis

(98) Sorted cells were sheared with Qiashredder columns (Qiagen, Valencia Calif.), 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, Colo.). Biotinylated cRNA probe was prepared using the ENZO BioArray RNA transcript kit (Affymetrix, Santa Clara, Calif.). Murine gene chips U174A, B and C were hybridized and analyzed according to standard Affymetrix protocols.

(99) Ranking the Differential Expression of Genes in CD4.sup.+ T Cells Between Anergy/Treg Induction and Effector/Memory Induction.

(100) mRNA prepared from purified nave 6.5 clonotypic CD4.sup.+ T cells and anergic/Treg and effector/memory 6.5 clonotypic CD4.sup.+ 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 (|d/1|), day 3 (|d2|), and day 4 (|d3|) after adoptive transfer, divided by the value of naive CD4.sup.+ T cells (n) for normalization.

(101) Antibodies and Staining

(102) The following antibodies were used. Anti-LAG-3 (C9B7W, from PharMingen) (Workman et al., 2002b) either purified or PE conjugated; anti-CD25 (7D4, from PharMingen) either purified or FITC conjugated; and anti-GITR (polyclonal antibody purchased from R&D Systems). For cell surface staining for LAG-3 and CD25, splenocytes from 6.5+/Thy1.1+/ transgenic mice were isolated and enriched for CD4+ using a CD4+ negative selection isolation kit (Miltenyi Biotec). Approximately 2.510.sup.6 clonotypic 6.5 cells, as determined by flow cytometry (16% of total CD4+ cells) were resuspended in HBSS and injected via tail vein into 137 (C3-HA high) or wild type B10.D2. One group of B10.D2 mice was treated with 510.sup.6 Vac-HA, while the other group was left untreated for nave control. 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. Cells were immediately blocked with 5 g whole rat IgG (Sigma) for 15 minutes before staining with anti-6.5 TCR-biotin+SA-APC, LAG-3-PE, and CD25-FITC, or the corresponding isotype controls. All staining reagents except anti-6.5-biotin were purchased from Pharmingen (San Diego, Calif.). After short incubation, samples were washed once in PBS+1% FBS solution and read on a FACScalibur machine(BD, San Jose, Calif.).

(103) In Vitro Suppression Assay for Induced 6.5 Regulatory T Cells

(104) 110.sup.4 purified naive 6.5 CD4+ T cells (Responders) and 110.sup.5 3000-rad irradiated syngeneic B 10.D2 splenocytes (Antigen Presenting Cells) were mixed with different numbers of suppressor 6.5 CD4+ T cells and incubated in round bottom 96-well tissue culture plates with 10 g/ml of HA class II (.sup.110SFERFEIFPKE.sup.120; SEQ NO: 7) peptide in 200 l of CTL media. Forty-eight to 72 hours later, cultures were pulsed with 1 Ci .sup.3H-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, Conn.).

(105) In Vitro Suppression Assay for Natural Regulatory T Cells

(106) Wild type BALB/c mice were used for out natural Treg assays. 510.sup.4 flow cytometry sorted CD4+CD25 T cells (Responders) and 510.sup.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 .sup.3H-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, Conn.).

(107) Quantitative Real-Time PCR Analysis

(108) The sorted 6.5 CD4+ T cells were immediately used for RNA extraction using Trizol reagent (Invitrogen, Carlsbad, Calif.). Reverse transcription was performed with the Superscript First Strand Synthesis System (Invitrogen, Carlsbad, Calif.). cDNA levels were analyzed by real-time quantitative PCR with the Taqman system (Applied Biosystems, Foster City, Calif.). Each sample was assayed in duplicates or triplicates for the target gene together with 18S rRNA as the internal reference in 25 l final reaction volume, using the Taqman Universal PCR Master Mix and the ABI Prism 7700 Sequence Detection system. Pre-made reaction reagents (PDARs) were purchased from Applied Biosystems for detection of IL-10 and IL-2. 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: Ct.sub.sample=(Ct.sub.sampleCt.sub.18S). The calibration sample was assigned as the sample with the highest Ct in each set of assay (Ct.sub.calibration). Relative mRNA frequencies were calculated as 2.sup.Ct where Ct=(Ct.sub.calibrationCt.sub.sample). 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 CAG TGA CCG TCA-3(SEQ ID NO: 16)/Probe 5-6FAM-CAT GGG CAC CTT CTC CGC AGG T-TAMRA-3(SEQ ID NO: 17); IFN- Primer 5-CAT TGA AAG CCT AGA AAG TCT GAA TAA C-3(SEQ ID NO: 18)/Primer 5-TGG CTC TGC AGG ATT TTC ATG-3(SEQ ID NO: 19)/Probe 5-6FAM-TCA CCA TCC TTT TGC CAG TTC CTC CAG-TAMRA-3(SEQ ID NO: 20); Foxp3 Prime 5-CCC AGG AAA GAC AGC AAC CTT-3(SEQ ID NO: 21)/Primer 5-TTC TCA CAA CCA GGC CAC TTG-3(SEQ ID NO: 22)/Probe: 5-6FAM-ATC CTA CCC ACT GCT GGC AAA TGG AGT C-3 (SEQ ID NO: 23).

(109) LAG-3 Constructs and Retroviral Producer Cell Lines.

(110) LAG-3 constructs 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) have been described (Workman et al., 2002a). LAG-3 constructs 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.sup.5/ml after 24 h was obtained (Markowitz et al., 1988).

(111) Retroviral Transduction of CD4+/CD25 T Cells and In Vitro Suppression Assay.

(112) 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, Calif.). The cells were then incubated with magnetic beads coupled with streptavidin and negatively sorted on an autoMACS (Miltenyi Biotech, Auburn Calif.) to 90-95% purity of CD4.sup.+/CD25.sup. T cells. The purified 6.5 CD4.sup.+/CD25.sup. T cells were activated by plate bound anti-CD3 (2C11) and anti-CD28 (35.71). On days 2 and 3 post-stimulation, the activated T cells (410.sup.5 cells/ml) were spin transduced (90 min, 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.sup.+/Thy1.2.sup.+ T cells.

(113) For the in vitro suppression assay, the purified GFP.sup.+ T cells were cultured (2 fold dilutions starting at 2.510.sup.4) with 2.510.sup.4 CD4.sup.+/CD25.sup.6.5 T cells (purified by negative AutoMACS), 510.sup.4 irradiated (3000 rads) splenocytes, and 5 g/ml HA110-120 in a 96-well round bottom plate. The cells were cultured for 72 h and pulsed with [.sup.3H]thymidine 1 Ci/well (Du Pont, Wilmington, Del.) the last 7-8 h of culture.

Example 11

(114) 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. We detected 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 432-441 in SEQ ID NO: 2) immediately preceding it amino-terminally.

Example 12

(115) As shown above, 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.

(116) We next wanted to determine if cells ectopically expressing LAG-3 would also exhibit regulatory function in vivo and be protected in a disease setting. We chose to ask whether ectopic expression of LAG-3 on an autoantigen-specific T cell could protect mice from type 1 diabetes. In this experimental system, splenocytes from diabetes-prone NOD mice were adoptively transferred to NOD-Scid mice, which lack lymphocytes. All mice develop diabetes within 3 months. Our preliminary analysis suggests that diabetes onset induced by NOD splenocytes is prevented by phogrin-specific T cells transduced with LAG-3, but not a signaling defective mutant or the GFP control. These data support the idea of using ectopic expression of autoantigen specific T cells with LAG-3 as a novel therapeutic for the treatment of many autoimmune diseases, asthma, and allergy.

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