TREATMENT OF AUTOIMMUNE AND INFLAMMATORY DISORDERS BY

INHIBITION OF BLIMP-1

FIELD OF THE INVENTION

[0001] The present invention relates generally to methods of treating autoimmune and inflammatory disorders by inhibiting the transcription regulator Blimp- 1 (PRDM1), methods of enhancing immune response by enhancing the activity of Blimp- 1, and methods of screening for compounds that inhibit or activate Blimp- 1.

BACKGROUND OF THE INVENTION

[0002] Aberrant immune response is associated with a number of human diseases, including autoimmune disease and chronic inflammatory conditions. Interleukin-23 (IL-23) is an important pro-inflammatory cytokine that has been shown to be an essential factor in the maturation and maintenance of pathogenic Thl7 cells, which are involved in a variety of human autoimmune diseases, including psoriasis, multiple sclerosis, and rheumatoid arthritis.

[0003] CD4 ⁺ T cells that produce interleukin-17 (Thl7 cells) are recognized as a unique population of cells that have critical roles in both host defense against extracellular pathogens and the pathogenesis of human inflammatory diseases including multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, and asthma. Annunziato et al. (2007) J. Exp. Med. 204:1849; Wilson et al . (2007) Nat. Immunol. 8:950; Kebir et al. (2009) Ann. Neurol. 66:390; and Chabaud et al. (2001) Arthritis Res. 3: 168. The early development of Thl7 cells is initiated by pro-inflammatory cytokines TGF-β and IL-6 (Mangan et al.

(2006) Nature 441 :231; Veldhoen et al. (2006) Immunity 24: 179; Bettelli et al. (2006) Nature 441 :235) that induce transcription factor RORyt (Ivanov et al. (2006) Cell 126:1121), promote STAT-3 activation and upregulate IL-23 receptor (IL-23R) expression (Yang et al.

(2007) J. Biol. Chem. 282:9358). Signaling through IL-23R is essential for Thl7 cells to differentiate into a "pathogenic" cell population. McGeachy et al. (2009) Nat. Immunol. 10:314; McGeachy et al. (2007) Nat. Immunol. 8: 1390; Langrish et al. (2005) J. Exp. Med. 201 :233; Ghoreschi et al. (2010) Nature 467:967; Hirota et al. (2011) Nat. Immunol. 12:255. In the absence of IL-23 signal, Thl7 cell development is stalled at the early activation stage (McGeachy et al. (2009) Nat. Immunol. 10:314) and the cells fail to express CXCR3, T-bet and IL-18R1 (Ghoreschi et al. (2010) Nature 467:967), which promote tissue migratory and inflammatory potential. Two recent studies have also reported that IL-23 promotes GM-CSF expression by T cells and that GM-CSF is essential for the ability of Thl7 cells to drive inflammation in central nervous system (CNS). El-Behi et al. (2011) Nat. Immunol. 12:568; Codarri et al. (2011) Nat. Immunol. 12:560; Becher et al. (2011) Curr. Opin. Immunol. 23:1. Similarly in humans, recent genome-wide association studies (GWAS) have demonstrated that IL-23R gene polymorphisms are important susceptibility factors in the progression of Thl7 cell-linked inflammatory disorders. Duerr et al. (2006) Science 314:1461; Smith et al. (2008) J. Invest. Dermatol. 128: 1325.

[0004] The need exists for improved methods of treating diseases mediated by pathogenic Thl7 cells, for example in treatment of chronic inflammatory diseases and autoimmune diseases. The need also exists for inhibiting Thl7-mediated inflammatory response in subjects where this response is inhibiting an alternative, beneficial immune response. Examples include inhibition of Thl7-mediated inflammation to promote elimination of tumors (Langowski et al. (2006) Nature 442:461; WO 2004/081990) and to promote clearance of chronic infections, such as chronic fungal infections (Zelante et al. (2007) Eur. J. Immunol. 37:2695; WO 2008/153610).

[0005] A reciprocal need also exists for improved methods of enhancing Thl7 immune responses in subject for whom such response would be expected to be beneficial, for example to enhance the memory response caused by vaccination.

SUMMARY OF THE INVENTION

[0006] The methods of the present invention address these needs. The invention

Provides methods of treating Thl7-mediated inflammatory disorders, such as autoimmune diseases, by inhibition of the activity of the transcriptional regulator Blimp- 1 (PRDM1). In various embodiments, the subject has multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and anklyosing spondyitis. In various embodiments, Blimp- 1 activity is inhibited by administration of a Blimp- 1 antagonist, including but not limited to a small molecule drug, and antisense nucleic acid molecule, a small interfering nucleic acid molecule {e.g. siRNA), antibodies or fragments thereof, or other antagonists.

[0007] The invention also provides methods of treating cancers and chronic infections, such as chronic fungal infections, by inhibition of the activity of the transcriptional regulator Blimp- 1 (PRDM1). In various embodiments, Blimp- 1 activity is inhibited by administration of a Blimp- 1 antagonist, including but not limited to a small molecule drug, an antisense nucleic acid molecule, a small interfering nucleic acid molecule {e.g. siRNA), antibodies or fragments thereof, or other antagonists. [0008] In some embodiments, Blimp-1 activity is selectively inhibited in Thl7 cells.

In one embodiment, a Blimp-1 antagonist is delivered selectively to Thl7 cells by

conjugation to an antibody, or antigen binding fragment thereof, that binds to one or more surface markers on Thl7 cells selected from the group consisting of CCR6, CD161 and IL- 23R. In another embodiment, a Blimp-1 antagonist is a nucleic acid sequence, such as an antisense nucleic acid or siRNA, in an expression vector and under the control of a RORyT or IL-23R transcriptional promoters, such that its expression is enhanced in Thl7 cells.

[0009] The invention further provides methods of enhancing Thl7 response in subjects in which such Thl7 responses would be expected to be beneficial, by enhancing Blimp-1 (PRDM1) activity. In one embodiment, the subject is receiving a vaccination and the Blimp-1 agonist enhances generation of a memory response to the immunogen. In various embodiments, Blimp-1 activity is enhanced by administration of a Blimp-1 agonist, including but not limited to a small molecule drug, and nucleic acid encoding Blimp-1 or an active fragment thereof, Blimp-1 protein or an active fragment thereof, agonist antibodies or fragments thereof, or other agonists.

[0010] In some embodiments, Blimp-1 activity is selectively enhanced in Thl7 cells.

In one embodiment, a Blimp-1 agonist is delivered selectively to Thl7 cells by conjugation to an antibody, or antigen binding fragment thereof, that binds to one or more surface markers on Thl7 cells selected from the group consisting of CCR6, CD161 and IL-23R. In another embodiment, a Blimp-1 agonist is a nucleic acid sequence encoding Blimp-1 cloned into an expression vector in which it is under the control of a RORyT or IL-23R transcriptional promoters, such that its expression is enhanced in Thl7 cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 A shows Prdml and Bcl6 gene expression (in arbitrary units) in Thl7

(shaded bars, IL-17 ⁺ ) and Thl (open bars, IFNy ⁺ ) cell populations, as described in Example 2.

[0012] FIG. IB shows Prdml and Bcl6 gene expression (in arbitrary units) in wt

CD4 ⁺ / IL-17 ⁺ and CD4 ⁺ / IL-17 ^" cells, and in IL-23R ^"7" (IL-23R KO) CD4 ⁺ / IL-17 ⁺ and CD4 ⁺ / IL-17 ^" cells, as described in Example 2. IL-17 ⁺ cells as shown as shaded bars, and IL-17 ^" cells are shown as open bars. Wt cells are shown as non-hatched bars, and IL-23R ^{" "} cells are shown as hatched bars.

[0013] FIG. 1C shows Prdml and 112 gene expression (in arbitrary units) in CD4 ⁺ /

IL-17 ⁺ (shaded bars) and CD4 ⁺ / IL-17 ^" cells (open bars) after treatment with IL-23, IL-6, or no cytokine treatment, as indicated. See Example 2. [0014] FIG. 2A shows the percentage IL-17 ⁺ cells in a population of CD4 YFP ⁺ T cells from draining lymph nodes of MOG ₃₅ _55 immunized PrdmV ^1' after ex vivo stimulation with PMA and ionomycin at day seven post immunization (left plot), and the percentage of GM-CSF ⁺ and IFNy ⁺ cells within these IL-17 ⁺ / CD4 ⁺ T cells (right plots) See Example 3. For all of FIGS. 2A - 2C, percentages were determined by flow cytometry with intracellular staining for the respective cytokines, and data are representative of three independent experiments with four to five mice per group. Error bars in all graphs are SEM.

[0015] FIG. 2B shows the percentage of T-bet ⁺ cells in a population of CD4 YFP ⁺ T cells and IL-17 CD4 YFP ⁺ T cells at days seven and 12 post immunization, respectively. See Example 3.

[0016] FIG. 2C shows the percentages of IL-17 ⁺ IL-2 ⁺ , IL-17 ⁺ IL-2 ^" , IL-17TL-2 ⁺ cells in a population of CD4 YFP ⁺ T cells . See Example 3.

[0017] FIG. 3 A shows EAE clinical score for various mice (wt-Prdml ^+/+ , Prdml ^+/~ ,

Prdml ⁷ ), as a function of the number of days post immunization with MOG ₃₅ _55 in CFA. See Example 4 for details of the conditional knockout mice used in the experiments.

[0018] FIG. 3B presents summaries of the maximal clinical scores for three experiments of the type described for FIG. 3A, and at Example 4.

[0019] FIG. 3C shows percentage of CD4 ⁺ T cells and total CD4 ⁺ T cells in the CNS of the heterozygous and Prdml KO mice described for FIG. 3A, and at Example 4, 12 days after EAE induction.

[0020] FIGS. 3D, 3E and 3F show the percentages and absolute numbers of IL-17 ⁺ ,

GM-CSF ⁺ and IFN-y ⁺ cells, respectively, in the CNS of the heterozygous and Prdml KO mice described for FIG. 3A, and at Example 4.

[0021] FIG. 4A shows the percentage of CCR6 ⁺ and CXCR3 ⁺ cells in a population of

CD4 ⁺ YFP ⁺ T cells in Prdml heterozygotes and knockouts, as described in greater detail at Example 5.

[0022] FIGS. 4B and 4C show percentages of Ki-67 ⁺ and Bcl-2 ⁺ cells in a population of CD4 ⁺ YFP ⁺ and IL-17 ⁺ CD4 ⁺ YFP ⁺ T cells, respectively, as indicated. See Example 5.

DETAILED DESCRIPTION

[0023] As used herein, including the appended claims, the singular forms of words such as "a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise. Unless otherwise indicated, or where clear from the context {e.g. in the discussion of animal experiments or models), discussion herein refers to human proteins (e.g. Blimp-1) and methods of treatment of humans. Table 1 below provides a listing of sequence identifiers used in this application. All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and individually indicated to be incorporated by reference. Citation of the references herein is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

I. Definitions

[0024] "Blimp-1 antagonist" refers to any agent that inhibits Blimp-1 activity.

"Blimp-1 agonist" refers to any agent that enhances or mimics Blimp-1 activity.

[0025] "Administration" and "treatment," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition, or by another cell. "Treatment," as it applies to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications. "Treatment" as it applies to a human, veterinary, or research subject, or cell, tissue, or organ, encompasses contact of an agent with animal subject, a cell, tissue, physiological compartment, or physiological fluid.

[0026] The phrase "consists essentially of," or variations such as "consist essentially of or "consisting essentially of," as used throughout the specification and claims, indicate the inclusion of any recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified dosage regimen, method, or composition. As a non-limiting example, a binding compound that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues, that do not materially affect the properties of the binding compound. [0027] "Effective amount" encompasses an amount sufficient to ameliorate or prevent a symptom or sign of the medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the method route and dose of administration and the severity of side affects. See, e.g., U.S. Pat. No. 5,888,530. An effective amount can be the maximal dose or dosing protocol that avoids significant side effects or toxic effects. The effect will result in an improvement of a diagnostic measure or parameter by at least 5%, usually by at least 10%, more usually at least 20%>, most usually at least 30%>, preferably at least 40%>, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%>, and most ideally at least 90%>, where 100% is defined as the diagnostic parameter shown by a normal subject. See, e.g., Maynard et al. (1996) A Handbook of SOPs or Good Clinical Practice, Interpharm Press, Boca Raton, FL; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK.

[0028] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0029] To examine the extent of inhibition of Blimp- 1 activity, for example, samples or assays comprising a given, e.g., protein, gene, cell, or organism, are treated with a potential activating or inhibiting agent and are compared to control samples without the agent. Control samples, i.e., not treated with agent, are assigned a relative activity value of 100%. Inhibition is achieved when the activity value relative to the control is about 90%> or less, typically 85%> or less, more typically 80%> or less, most typically 75%> or less, generally 70%> or less, more generally 65%> or less, most generally 60%> or less, typically 55%> or less, usually 50%> or less, more usually 45%> or less, most usually 40%> or less, preferably 35%> or less, more preferably 30%) or less, still more preferably 25%> or less, and most preferably less than 25%>. Activation is achieved when the activity value relative to the control is about 110%, generally at least 120%, more generally at least 140%, more generally at least 160%, often at least 180%, more often at least 2-fold, most often at least 2.5-fold, usually at least 5-fold, more usually at least 10-fold, preferably at least 20-fold, more preferably at least 40-fold, and most preferably over 40-fold higher.

[0030] Endpoints in activation or inhibition can be monitored as follows. Activation, inhibition, and response to treatment, e.g., of a cell, physiological fluid, tissue, organ, and animal or human subject, can be monitored by an endpoint. The endpoint may comprise a predetermined quantity or percentage of, e.g., an indicia of inflammation, oncogenicity, or cell degranulation or secretion, such as the release of a cytokine, toxic oxygen, or a protease. The endpoint may comprise, e.g., a predetermined quantity of ion flux or transport; cell migration; cell adhesion; cell proliferation; potential for metastasis; cell differentiation; and change in phenotype, e.g., change in expression of gene relating to inflammation, apoptosis, transformation, cell cycle, or metastasis See, e.g., Knight (2000) Ann. Clin. Lab. Sci. 30: 145; Hood and Cheresh (2002) Nature Rev. Cancer 2:91; Timme et al. (2003) Curr. Drug Targets 4:251; Robbins and Itzkowitz (2002) Med. Clin. North Am. 86: 1467; Grady and Markowitz (2002) Annu. Rev. Genomics Hum. Genet. 3:101; Bauer, et al. (2001) Glia 36:235;

Stanimirovic and Satoh (2000) Brain Pathol. 10:113.

[0031] An endpoint of inhibition is generally 75% of the control or less, preferably

50% of the control or less, more preferably 25% of the control or less, and most preferably 10% of the control or less. Generally, an endpoint of activation is at least 150% the control, preferably at least two times the control, more preferably at least four times the control, and most preferably at least 10 times the control.

II. General

[0032] The present invention relates to the discovery that zinc finger domain containing molecule Blimp- 1 is a key regulator of the IL-23 -mediated Thl7 effector differentiation pathway. In light of this discovery, it will be possible to treat Thl7-mediated diseases by antagonizing the activity of Blimp- 1. Excessive Thl7 responses are believed to be responsible for the pathogenesis of a number of autoimmune and chronic inflammatory disorders, including but not limited to multiple sclerosis, psoriasis, rheumatoid arthritis, Crohn's disease, and ankylosing spondylitis.

[0033] Enhancement of Blimp- 1 expression or activity will find use in decreasing

Thl7 responses in subjects for which such an inflammatory response is detrimental, such as those with cancers or chronic infections, e.g. chronic fungal infections. [0034] Alternatively enhancement of Blimp- 1 expression or activity will find use in enhancing Thl7 responses in subjects for which such responses are beneficial, such as those receiving vaccinations.

[0035] Methods of screening for Blimp- 1 antagonists and agonists will enable discovery of compounds for use in the methods of treatment of the present invention.

[0036] Blimp- 1 (B lymphocyte-induced maturation protein- 1), encoded by PRDM1, is a zinc finger domain-containing transcription factor that is induced by cytokine signaling during early T cell activation. It directly represses il-2 transcription to ensure properly controlled immune response. Martins et al. (2008) J. Exp. Med. 205:1959. It was first discovered 16 years ago as a transcriptional repressor of human interferon-beta (IFN-β) promoter that was induced upon viral infection. Ectopic expression of Blimp- 1 is sufficient to drive B cells into mature Ig-secreting plasma cells. Blimp- 1 is also a key regulator of terminal differentiation in various cell types. Blimp- 1 has been reported to control effector cytokine production in human NK cells. Smith et al. (2010) J. Immunol. 185:6058. Blimp-1 promotes differentiation of B cells to antibody secreting plasma cells (Turner et al. (1994) Cell 77:297; Shapiro-Shelef et al. (2003) Immunity 19:607), CD8 ⁺ T cells to cytotoxic T lymphocytes (CTLs) (Rallies et al. (2009) Immunity 31 :283; Shin et al. (2009) Immunity 31 :309; Rutishauser et al. (2009) Immunity 31 :296) and regulatory T cells (Tregs) to IL-10- expressing effector Tregs (Cretney et al. (2011) Nat. Immunol. 12:304; Martins et al. (2008) Ann. Rev. Immunol. 26:133). In case of CD4 ⁺ T cells, three new studies have shown that Blimp-1 inhibits Bcl-6-driven differentiation of CD4 ⁺ T cells to T follicular helper cells (T _FH ) and accordingly, non-T _FH express higher levels of Blimp-1 compared to T _FH cells. Johnston et al. (2009) Science 325:1006; Nurieva et al. (2009) Science 325:1001; Yu et al. (2009) Immunity 31 :457.

[0037] Blimp-1 (PRDM1) is also known as PR domain zinc finger protein 1; positive regulatory domain 1 -binding factor 1; as well as BLIMP 1; PRDI-BF1; PRDIBF1;

MGC 118922; MGC 118923; MGC 118924; and MGC 118925. Human Blimp-1 (PRDM1) is described further at Gene ID No. 639 and MIM 603423. Two alternatively spliced transcript variants that encode different isoforms have been reported. Nucleic acid sequences are provided at RefSeqs NM 001198.3 (longer isoform 1, PRDMla) and NM 182907.2 (shorter isoform 2, PRDMip), and corresponding polypeptide sequences are provided at RefSeqs NP 001189.2 and NP 878911.1, the sequences of which are hereby incorporated by reference in their entireties, and also provided at SEQ ID NOs: 1 and 3 (nucleic acid sequences) and SEQ ID NOs: 2 and 4 (polypeptide sequences). See Table 1. Polymorphisms in Blimp-1 (PRDM1) have been associated with risk of a severe and/or aggressive form ulcerative colitis (WO 2011/116111) and systemic lupus erythematosus (SLE) (WO 2011/044205).

[0038] Mouse Blimp- 1 may find use in screening for antagonists and agonists of

Blimp- 1 for use as drugs in humans. Mouse Blimp- 1 (PRDM1) is described further at Gene ID No. 12142. Nucleic acid and polypeptide sequences are provided at RefSeqs

NM_007548.3 and NP_031574.2, the sequences of which are hereby incorporated by reference in their entireties, and also provided at SEQ ID NOs: 5 and 6. See Table 1.

[0039] Here we show that Blimp-1 is a key transcription factor that regulates IL-23- mediated differentiation of pathogenic Thl7 cells. Blimp-1 represses IL-2 expression, allowing Thl7 cells to transition to a mature phenotype. Blimp-1 facilitates co-expression of effector cytokines including GM-CSF and IFNy in activated IL-17-producing T cells and endows the Thl7 cells with inflammatory functions, and enhances the expression of survival and homing markers associated with pathogenic function. T cell-specific Blimp-1 deficiency renders mice resistant to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, consistent with its essential role in the disease. The results reported herein indicate that Blimp-1 is one of the key elements that promote Thl7 maturation, in that downregulates IL-2, promotes GM-CSF and CXCR3 expression and allows maturation of inflammatory Thl7 cells.

Blimp-1 Involvement in IL-23 Signaling and Thl 7 cell Development

[0040] The literature suggests that Blimp-1 promotes priming of CD4 ⁺ T cell subsets into fully differentiated effector cells (including Thl, Th2 and Thl 7 cells) unless otherwise repressed by Bcl-6 to promote differentiation to T _FH cells. To understand whether Blimp-1 differentially regulates Thl 7 versus Thl cells, we performed gene expression analysis of sorted IL-17 and IFNy-producing cells from the draining lymph nodes of MOG (myelin oligodendrocyte glycoprotein (MOG)35-55 peptide in CFA) immunized mice. See

Example 2. Results are provided at FIG. 1A. Prdml expression was specific to Thl 7 cells as compared to Thl cells, which had reduced Prdml but increased Bcl6 expression. Bcl6 is an antagonistic regulator of Blimp-1. These results indicate that Blimp-1 expression is specifically elevated in Thl 7 cells.

[0041] To determine whether IL-23 signaling induces Blimp-1 expression, gene expression was measured in sorted wt and IL-23R-deficient Thl 7 cells, as described in Example 2. IL-23 signaling induces Prdml expression in Thl 7 cells, and reduced Bcl6 expression, as show in FIG. IB. [0042] Based on these results, and without intending to be limited by theory, it is possible that IL-2 is involved in Blimp- 1 -mediated effects described herein. It has been previously observed that IL-23R ^{" "} fail to downregulate IL-2 expression, and the resulting continued IL-2 expression inhibits maturation of Thl7 to their fully differentiated

(pathogenic) state. McGeachy et al. (2009) Nat. Immunol. 10:314. It has also been observed that Blimp- 1 directly represses IL-2 gene transcription. Martins et al. (2008) J. Exp. Med. 205: 1959; Martins et al. (2008) Ann. Rev. Immunol. 26: 133. The data presented herein (FIGS. 1A and IB) are consistent with the hypothesis that IL-23 promotes effector Thl7 differentiation by inducing Blimp- 1, which inhibits the IL-2 pathway, thus promoting terminal differentiation of Thl7 cells. Regardless of whether IL-2 is involved in the mechanism of action of Blimp- 1, the results described herein conclusively demonstrate a role for Blimp- 1 in promoting Thl7 differentiation, and hence pathology.

[0043] Further experiments were performed to determine whether IL-23 can directly induce Prdml expression in Thl7 cells, as described in Example 2. The results, shown in FIG. 1C, show that that Prdml gene transcription is a proximal event following IL-23 stimulation. The results further show that 112 expression is highest in cells with lowest Prdml expression (IL-17 ^" cells), and lowest in cells with the highest Prdml expression (IL- 23-treated IL-17 ⁺ cells), consistent with the proposed role of Prdml as a downregulator of IL-2 expression during Thl7 development.

Blimp- 1 Involvement in Thl 7 Pathogenicity

[0044] Additional experiments confirmed that the T cell population promoted by

Blimp-1 exhibit the hallmark properties of pathogenic Thl7 cells, including elevated IL-17, INF-γ, GM-CSF, and T-bet expression. Details are provided at Example 3. To test whether Blimp-1 deletion affects Thl 7 differentiation we assessed draining lymph nodes at day seven post immunization with MOG ₃ 5_55 peptide in CFA. As shown in FIG. 2A (left plot), the proportion of YFP ⁺ CD4 ⁺ T cells producing only IL-17 was reduced by Prdml deficiency. However, there was a significant defect in the ability of Prdml ^"7" IL-17 producers to co- express GM-CSF and IFNy. Two recent studies have reported that IL-23 promotes GM-CSF expression by T cells and that GM-CSF is essential for the ability of Thl 7 cells to drive inflammation in the central nervous system (CNS). El-Behi et al. (2011) Nat. Immunol. 12:568; Codarri et al. (2011) Nat. Immunol. 12:560.

[0045] To further understand the mechanism of Blimp-1 mediated differentiation we assessed T-bet expression in Prdml ^1' YFP ⁺ CD4 T cells. The absence of Prdml expression prevented T-bet upregulation in CD4 ⁺ T cells (T-bet was upregulated in only 3.3% of cells, rather than 28%), and in IL-17 ⁺ CD4 ⁺ T cells (see FIG. 2B), suggesting that T-bet may be influenced by Blimp-1 expression.

[0046] Together these results support our hypothesis that IL-23 mediates functional maturation of Thl7 cells through induction of Blimp-1, which promotes co-expression of GM-CSF and IFNy by IL-17-producing cells. These findings are also in agreement with a previous fate mapping study using III 7a ^Cre R26R ^eYFF mice, which revealed that IL-23R signaling is essential for Thl7 cells to co-express IFNy and for their eventual deviation towards solely IFNy-producing "ex -Thl 7 cells." Hirota et a/. (2011) Nat. Immunol. 12:255.

[0047] Published reports have also indicated that in the absence of IL-23 signal, Thl7 cells are unable to downregulate their IL-2 expression and are stalled at the early activation stage. McGeachy et al. (2009) Nat. Immunol. 10:314. Since Blimp-1 is a known repressor of IL-2 transcription, we proposed that IL-23 could promote Blimp-1 -dependent inhibition of IL-2 in Thl7 cells, allowing transition to a mature phenotype. At day seven post

immunization we found that almost half of the Thl7 cells had transitioned towards a mature IL-17 single producer phenotype, and were no longer IL-2 co-producers. See FIG. 2C. But in the absence of IL-23 signal (due to Prdml ablation), nearly all the Thl7 cells were trapped at the early activation (IL-2 co-producer) stage and were unable to downregulate their IL-2 expression. All together these results confirm that IL-23 drives full effector differentiation of Thl7 cells in lymph nodes by inducing Blimp-1.

Blimp-1 Ablation Prevents Thl 7 -Mediated Disease in a Mouse Model

[0048] In light of the results suggesting that Prdml -I- Thl 7 cells are unable to undergo functional maturation, we next tested whether this affects their ability to induce EAE, a Thl 7 cell-driven model of human multiple sclerosis. See Example 4. As shown in FIGS. 3A and 3B, upon immunization with MOG ₃₅ _55 in CFA, Prdml-/- mice were completely protected from EAE. As shown in FIG. 3C, these mice also had significantly fewer CD4 ⁺ T cells accumulated in the central nervous system (CNS) as compared with Prdml ^~/+ and Prdml ^+I+ mice. The -90% reduction in absolute numbers of CD4 T cells further emphasizes the defect in the ability of Prdml ^1' CD4 ⁺ T cells to infiltrate the CNS. Furthermore, fewer of the Prdml ^1' YFP ⁺ CD4 ⁺ T cells that infiltrated the CNS were IL-17 producers (FIG. 3D). More importantly, there was a significant defect in the ability of Prdml ^'1' Thl 7 cells to co-express GM-CSF (FIG. 3E) and IFNy (FIG. 3F). These results suggest that IL-23 signaling could be stabilizing the maturation of pathogenic Thl 7 cells by inducing Blimp- 1, which promotes expression of GM-CSF and ΙΚΝγ by Thl7 cells. IFNy producing Thl7 cells are known to be present in the lesional tissue in EAE and preferentially accumulate in the CNS of patients with multiple sclerosis (Kebir et al. (2009) Ann. Neurol. 66:390), highlighting their importance in promoting inflammation.

Blimp- 1 Effects on Thl 7 Cell Survival and Homing

[0049] Various other experiments were performed to determine the mechanism by which Blimp- 1 ablation prevents EAE. The experiments described in Examples 2 and 3 already establish that Blimp- 1 is important for development of a fully mature, pathogenic Thl7 cells. The results of the EAE experiments described above, however, also showed a reduced number (accumulation) of inflammatory CD4 ⁺ T cells in the CNS in the absence of Blimp- 1. Such a result could be explained if Blimp- 1 enhanced homing of Thl7 cells to the target tissue (in the case the CNS), or enhanced proliferation (or prevented apoptosis) in these Thl7 cells, or exhibited some combination of these effects.

[0050] Experiments were performed to address whether Blimp- 1 controls the ability of Thl7 cells to migrate, expand or survive and regulate cellular infiltration into the CNS. Towards this end, we assessed expression of homing receptors on YFP ⁺ CD4 ⁺ T cells. The proportion of cells expressing CCR6 (Thl7-specific chemokine receptor) was reduced slightly but there was a significant reduction in CXCR3 expressing cells in the absence of Blimp- 1. See FIG. 4A. This suggests that Blimp- 1 promotes expression of chemokine receptors on Thl7 cells required for trafficking to sites of inflammation.

[0051] Next we assessed whether Blimp- 1 regulates proliferation of Thl7 cells during differentiation by measuring Ki67, a cellular proliferation marker. As shown in FIG. 4B, in the absence of Blimp- 1 fewer Thl7 cells were Ki67 positive, suggesting that Blimp- 1 promotes optimal expansion of Thl7 cells.

[0052] Reduced accumulation of PrdmV ^1' CD4 ⁺ T cells in the CNS could also be due to enhanced susceptibility to apoptosis. For this we measured the expression of Bcl-2, a factor associated with cell survival. The proportion of CD4 ⁺ T cells, and specifically Thl7 cells, expressing Bcl-2 did not differ between PrdmV ^1' and Prdml ^~/+ mice. See FIG. 4C.

[0053] Together these results suggest that Blimp-1 is required for optimal Thl7 cell differentiation and expansion, thereby allowing Thl7 cells to migrate to the site of inflammation and mediate pathogenic functions. III. Uses

[0054] The invention provides a method of treating autoimmune diseases or chronic inflammatory diseases, comprising administering an antagonist of Blimp- 1. In various embodiments, the inflammatory or autoimmune disease is selected from the group consisting of diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, chronic obstructive pulmonary disease, atherosclerosis, and inflammatory bowel diseases. See, e.g., Duvallet et al. (2011) Annals Med. 43:503. In some embodiments, the inflammatory bowel disease is selected from the group consisting of Crohn's disease, ulcerative colitis, sprue and food allergies.

Inflammatory Bowel Disease

[0055] The methods of the present invention may be used in the treatment of inflammatory bowel disease (IBD). Ulcerative colitis (UC) and Crohn's disease are the two major forms of idiopathic Inflammatory Bowel Disease (IBD) in humans. Kirsner et al. eds. (1988) Inflammatory Bowel Disease: 3rd ed. Goldner et al. (1990) Idiopathic Inflammatory Bowel Disease, in Stein,, ed., Internal Medicine, Little Brown & Co., Boston, pp. 369-380; Cello et al. (1989) Ulcerative Colitis, in Sleisenger et al. eds. (1989) Gastrointestinal Disease: Pathophysiology Diagnosis Management, W. B. Saunders Co., Philadelphia, p. 1435.

[0056] Several animal models are used to study the pathogenesis of IBD. The criteria for an animal model of IBD have been reviewed. Strober et al. (1988) Dig. Dis. Sci. 33 Suppl.:3S-IOS; Beekan (1988) Experimental Inflammatory Bowel Disease, in: Kirsner et al. (1988) Inflammatory Bowel Disease, Lea and Febiger, Philadelphia, pp. 37-49. The most widely used models are the experimental colonic lesions produced by dinitrobenzene sulfonic acid (DNBS), 2, 4, 6-trinitro-benzensulfonic acid (TNBS) and carrageenan. These models involve tissue destruction in the colon, intrarectal administration of 5-30 mg of TNBS in 0.25 ml of 50% ethanol in the rat produces dose-dependent colonic ulcers and inflammation which are observed by gross and light microscopic examination, and by biochemical measurement of myeloperoxidase activity in the colon at 3-4 weeks. Morris et al. (1989) Gastroenterology 96:795.

Multiple Sclerosis

[0057] The methods of the present invention may be used in the treatment of multiple sclerosis (MS). MS is a multi-factorial inflammatory disease of the human central nervous system resulting in the slowing of electrical conduction along the nerve. The disease is characterized by an increase in the infiltration of inflammatory cells, loss of oligodendrocytes, and increased gliosis (astrocyte hypertrophy and proliferation). Myelin is the target of this cellular autoimmune inflammatory process, leading to impaired nerve conduction. See Thompson (1996) Clin. Immunother. 5:1. Clinical manifestations are variable, but are usually characterized by an initial relapsing-remitting course, with acute exacerbation followed by periods of clinical stability. Over time, a steady deterioration in neurological functions takes place as the disease evolves into a chronic progressive phase. This deterioration is responsible for disabling complications and side-effects, which greatly affect quality of life and increases mortality risk of affected patients. It is estimated that close to a third of a million people in the United States have MS.

[0058] There are several models that are widely used for testing therapies that may be effective in treating MS. One model is Experimental Autoimmune Encephalomyelitis (EAE), as described in greater detail at Example 4. Briefly, EAE is a T cell mediated autoimmune disease of the central nervous system (CNS). It can be induced in susceptible strains of mice (SJL mice) by immunization with CNS myelin antigens or alternatively, it can be passively transferred to susceptible mice using antigen stimulated CD4 ⁺ T cells. Pettinelli (1981) J. Immnnol. 127:1420. EAE is widely recognized as an acceptable animal model for multiple sclerosis in primates. Alvord et al. eds. (1984) Experimental allergic encephalomyelitis-! useful model for multiple sclerosis. Alan R. Liss, New York. Another common experimental MS model is a viral model, whereby an MS-like disease is induced by Theiler's murine encephalomyelitis virus (TMEV). Dal Canto et a/.(1977) Am. J. Path. 88:497. The lysolecithin model is also widely accepted as a model for demyelinating conditions such as MS.

Rheumatoid Arthritis

[0059] The methods of the present invention may be used in the treatment of rheumatoid arthritis (RA). RA is a chronic, systemic and articular inflammatory disorder characterized by an imbalance in the immune system that causes overproduction of proinflammatory cytokines, e.g. tumor necrosis factor alpha (TNFa) and interleukin 1 (IL-I), and a lack of anti-inflammatory cytokines, e.g. IL-10. RA is characterized by synovial inflammation, which progresses to cartilage destruction, bone erosion and subsequent joint deformity. The primary symptoms of RA are joint inflammation, stiffness, swelling, fatigue, difficulty moving, and pain. During the inflammatory process, polymorphonuclear cells, macrophages, and lymphocytes are released. Activated T-lymphocytes produce cytotoxins and pro-inflammatory cytokines, while macrophages stimulate the release of prostaglandins and cytotoxins. Vasoactive substances (histamine, kinins, and prostaglandins) are released at the site of inflammation and cause edema, warmth, erythema, and pain associated with inflamed joints.

[0060] One of the models used to test for new therapies for arthritis includes the collagen-induced arthritis (CIA) model. Myers et al.{\ 997) Life Sci. 61 : 1861. In this model, immunization of genetically susceptible rodents or primates with Type II collagen leads to the development of a severe polyarticular arthritis that is mediated by an autoimmune response. It mimics the synovitis and erosions of cartilage and bone that are characteristic of RA.

Diabetes

[0061] The methods of the present invention may be used in the treatment of diabetes

(IDDM). The main clinical feature of IDDM is elevated blood glucose levels

(hyperglycemia). The elevated blood glucose level is caused by autoimmune destruction of insulin producing cells in the islets of Langerhans of the pancreas. This is accompanied by a massive cellular infiltration surrounding and penetrating the islets (insulitis) composed of a heterogeneous mixture of CD4 ⁺ and CD8 ⁺ T-lymphocytes, B-lymphocytes, macrophages and dendritic cells.

[0062] One animal model that is particularly useful in testing agents for treating

IDDM is the NOD mouse. The NOD mouse represents a model in which autoimmunity against beta-cells is the primary event in the development of IDDM. Diabetogenesis is mediated through a multi-factorial interaction between a unique MHC class II gene and multiple, unlinked, genetic loci, as in the human disease. Moreover, the NOD mouse demonstrates the critical interaction between heredity and environment, and between primary and secondary auto-immunity. Its clinical manifestation, for example, depends on various external conditions, most importantly on the micro-organism load of the environment in which the NOD mouse is housed.

[0063] Another animal model for studying the effects of therapeutic agents in IDDM is the streptozotocin (STZ) model. Hartner et al. (2005) BMC Nephrol. 6:6. This model has been used extensively as an animal model to study the mechanisms involved in the destruction of pancreatic beta cells in IDDM. In this model, diabetes is induced in rodents by the beta-cell toxin streptozotocin (STZ). STZ is taken up by the pancreatic beta cell through the glucose transporter GLUT-2, decomposes intracellularly, and causes damage to DNA either by alkylation or by the generation of NO. The appearance of DNA strand breaks leads to the activation of the abundant nuclear enzyme poly(ADP-ribose) polymerase (PARP), which synthesizes large amounts of (ADP-ribose) polymer, using NAD ⁺ as a substrate. As a consequence of PARP activation, the cellular concentration of NAD ⁺ decreases to very low levels, impairing the ability to generate sufficient energy, and ultimately leading to cell death.

[0064] The methods of the present invention may also be useful in treatment of other disorders with which a Thl7 inflammatory response is undesirable. Experiments have demonstrated that Thl7-mediated inflammation may promote, rather than inhibit, the growth of tumors. Langowski et al. (2006) Nature 442:461; WO 2004/081990. Accordingly, the invention provides a method of promoting immunosurveillance and treatment of cancer comprising administering an antagonist of Blimp- 1. Exemplary tumors that may be treated using the methods of treatment of the present invention include tumors of the gastrointestinal tract, such as, but not limited to, tumors of the stomach, bowel and intestine, and also tumors of the lung, liver, pancreas, breast, bone and any other solid tumor or blood borne tumor. The treatment may result in inhibition of tumor cell growth or proliferation, or may result in preventing the further spread (metastasis) of tumor cells to other tissues or organs.

[0065] Further experiments have demonstrated that Thl7-mediated inflammation may prevent the mounting of a productive immune response to chronic infections, such a chronic fungal infections. Zelante et al. (2007) Eur. J. Immunol. 37:2695; WO 2008/153610.

Accordingly, the invention also provides a method of promoting clearance of chronic infections, such as chronic fungal infections, comprising administering an antagonist of Blimp- 1.

IV. Blimp- 1 Antagonists

[0066] The present invention involves, in one aspect, antagonism of Blimp- 1 activity as a means of treating autoimmune and inflammatory disorders, or in treating cancers or chronic infections, such as chronic fungal infections. Many of the experiments herein make use of Prdml knockout mutants, and demonstrate that cells lacking Blimp- 1 have different properties than wt cells. These different properties can be used to screen compounds for Blimp- 1 antagonist activities, since compounds that induce effects mimicking the properties Prdml knockout mutants in wt cells will be Blimp- 1 antagonists. Compounds identified on the basis of their ability to inhibit mouse Blimp- 1 in mouse models would be expected to also inhibit human Blimp- 1 in most cases, and thus be potential human therapeutics. Small Molecules

[0067] Small molecule drugs can be selected from natural or synthetic libraries of compounds, isolated from plant or animal extracts, or synthesized based on rational drug design considerations. Blimp- 1 antagonists can be obtained using any of the numerous suitable approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. Lam (1997) Anticancer Drug Des . 12: 145; U.S. Pat. Nos. 5,738,996 and 5,807,683. Exemplary methods for the synthesis of molecular libraries can be found, e.g., at DeWitt et al. (1993) Proc. Nat'l Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Nat'l.Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.

33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233, the disclosures of which are hereby incorporated by reference in their entireties. Libraries of compounds may be presented in solution (e.g. Houghten (1992) Bio/Techniques 13:412), on beads (Lam (1991) Nature 354:82), chips (Fodor (1993) Nature 364:555), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Nat'l Acad. Sci. USA 89:1865) or phage (Scott and Smith (1990) Science 249:386; Devlin (1990) Science 249:404; Cwirla et al. (1990) Proc. Nat'l Acad. Sci. USA 87:6378; and Felici (1991) J. Mol. Biol. 222:301).

[0068] Another approach uses recombinant bacteriophage to produce large peptide libraries. Using the "phage method" (Scott and Smith (1990) Science 249:386; Cwirla et al. (1990) Proc. Nat'l Acad. Sci. 87:6378; Devlin et a/.(1990) Science 249:404), very large libraries can be constructed (10 ⁶ -10 ⁸ peptides). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al. (1986) Molecular Immunology 23:709; Geysen et al. (1987) J. Immunologic Method 102:259) and the method of Fodor et al.

(Science 251 :767 (1991)) are examples. Furka et al. (14th International Congress of

Biochemistry, Volume 5, Abstract FR:013 (1988); Furka (1991) Int. J. Peptide Protein Res. 37:487), Houghton (U.S. Pat. No. 4,631,211) and Rutter et al. (U.S. Pat. No. 5,010,175) describe methods to produce a mixture of peptides that can be tested as activators or inhibitors.

[0069] Screening phage-displayed random peptide libraries offers a rich source of molecular diversity and represents a powerful means of identifying peptide ligands that bind a molecule of interest. Cwirla et al. (1990) Proc. Nat'l Acad. Sci, 87:6378; Devlin et al. (1990) Science 249:404. Phage expressing binding peptides are selected by affinity purification with the target of interest. This system allows a large number of phage to be screened at one time. Since each infectious phage encodes the random sequence expressed on its surface, a particular phage, when recovered from an affinity matrix, can be amplified by another round of infection. Thus, selector molecules immobilized on a solid support can be used to select peptides that bind to them. This procedure reveals a number of peptides that bind to the selector and that often display a common consensus amino acid sequence. Biological amplification of selected library members and sequencing allows the determination of the primary structure of the peptide(s). Peptides are expressed on the tip of the filamentous phage Ml 3, as a fusion protein with the phage surface protein pilus (at the N-terminus). Typically, a filamentous phage carries on its surface three to five copies of pili and therefore of the peptide. In such a system, no structural constraints are imposed on the N-terminus; the peptide is therefore free to adopt many different conformations, allowing for a large diversity. However, biases in the distribution of peptides in the library may be caused by biological selection against certain of the peptides, which could reduce the diversity of peptides contained in the library. In practice, this does not appear to be a significant problem. When randomly selected peptides expressed at the N-terminus of pili were analyzed (Cwirla et al. (1990) Proc. Nat'! Acad. Sci., 87:6378), most amino acids appeared at each position of the variable peptide, indicating that no severe discrimination against particular amino acids had occurred. Selection against particular combinations of amino acids would however not have been detected in this analysis.

[0070] Synthetic libraries can also be used to screen for novel peptides, or mimetics or fragments thereof, according to the present invention. Needels et al . (1993) Proc. Nat'l Acad. Sci. USA 90:10700; Ohlmeyer et al . (1993) Proc. Nat'l Acad. Sci. USA 90:10922; Lam et al . Infl Pat. Appl. Pub. WO 92/00252; Kocis et al . Infl Pat. Appl. Pub. WO 94/28028, each of which is incorporated herein by reference in its entirety. siRNA

[0071] Additional compounds that can be used to selectively inhibit Blimp- 1 include siRNA (see, e.g., Stevenson (2004) New. England. J. Med. 351 : 1772), antisense nucleic acids, and ribozymes, as disclosed at U.S. Pat. Nos. 6,211,164, 6,677,445 and 6,846,921; U.S. Pat. App. Publication Nos. 2004/0097446 and 2005/01533925; and PCT publications

WO 2003/070888 and WO 2001/057206. Such nucleic acid-based antagonists can be designed in light of the known nucleic acid sequence for human Blimp- 1, as provided at SEQ ID NOs: 1 and 3 herein.

[0072] Methods of producing and using siRNA are disclosed, e.g., at U.S. Pat. Nos.

6,506,559 (WO 99/32619); 6,673,611 (WO 99/054459); 7,078,196 (WO 01/75164);

7,071,311 and PCT publications WO 03/70914; WO 03/70918; WO 03/70966; WO

03/74654; WO 04/14312; WO 04/13280; WO 04/13355; WO 04/58940; WO 04/93788; WO 05/19453; WO 05/44981; WO 03/78097 (U.S. patents are listed with related PCT

publications). Exemplary methods of using siRNA in gene silencing and therapeutic treatment are disclosed at PCT publications WO 02/096927 (VEGF and VEGF receptor); WO 03/70742 (telomerase); WO 03/70886 (protein tyrosine phosphatase type IVA (Prl3)); WO 03/70888 (Chkl); WO 03/70895 and WO 05/03350 (Alzheimer's disease);

WO 03/70983 (protein kinase C alpha); WO 03/72590 (Map kinases); WO 03/72705 (cyclin D); WO 05/45034 (Parkinson's disease). Exemplary experiments relating to therapeutic uses of siRNA have also been disclosed at Zender et al. (2003) Proc. Nat'l. Acad. Sci. (USA) 100:7797; Paddison et al. (2002) Proc. Nat'l Acad. Sci. (USA) 99: 1443; and Sah (2006) Life Sci. 79: 1773. siRNA molecules have also been used in clinical trials, e.g., of chronic myeloid leukemia (CML) (ClinicalTrials.gov Identifier: NCT00257647) and age-related macular degeneration (AMD) (ClinicalTrials.gov Identifier: NCT00363714).

[0073] Although the term "siRNA" is used herein to refer to molecules used to induce gene silencing via the RNA interference pathway (Fire et al. (1998) Nature 391 :806), such siRNA molecules need not be strictly polyribonucleotides, and may instead contain one or more modifications to the nucleic acid to improve its properties as a therapeutic agent. Such agents are occasionally referred to as "siNA" for short interfering nucleic acids. Although such changes may formally move the molecule outside the definition of a "ribo"nucleotide, such molecules are nonetheless referred to as "siRNA" molecules herein. For example, some siRNA duplexes comprise two 19 - 25 nt (e.g. 21 nt) strands that pair to form a 17 - 23 basepair (e.g. 19 base pair) polyribonucleotide duplex with TT (deoxyribonucleotide) 3' overhangs on each strand. Other variants of nucleic acids used to induce gene silencing via the RNA interference pathway include short hairpin RNAs ("shRNA"), for example as disclosed in U.S. Pat. App. Publication No. 2006/0115453. Such shRNA molecules may be delivered, e.g., using a retroviral vector, as disclosed at U.S. Pat. App. Pub. 2007/0154487 (see Figures 42 and 43, and Example 9, therein), the disclosure of which is hereby

incorporated by reference in its entirety.

[0074] Although the sense strand is provided for the Blimp- 1 sequence herein (SEQ

ID NOs: 1, 3 and 5), other sequences may be used to generate siRNA molecules for use in silencing these genes. The sequence of the opposite strand of the siRNA duplexes is simply the reverse complement of the sense strand, with the caveat that both strands have

2-nucleotide 3' overhangs. That is, for a sense strand "n" nucleotides long, the opposite strand is the reverse complement of residues 1 to (n-2), with 2 additional nucleotides added at the 3' end to provide an overhang. Where an siRNA sense strand includes two U residues at the 3 ' end, the opposite strand also includes two U residues at the 3 ' end. Where an siRNA sense strand includes two dT residues at the 3 ' end, the opposite strand also includes two dT residues at the 3 ' end.

[0075] Double stranded RNA or a small interfering RNA, including shRNA, can be delivered to the target cell in a patient in a number of different ways. For example, it may be conveniently administered by microinjection; by bombardment by particles covered by the dsRNA; by soaking the cell or organism in a solution of the dsRNA; by electroporation of cell membranes in the presence of the dsRNA; by liposome-mediated delivery of dsRNA and transfection mediated by chemicals such as calcium phosphate; by viral infection; by transformation; and the like. The dsRNA may be introduced along with components that enhance RNA uptake by the cell, stabilize the annealed strands, or otherwise increase inhibition of Prdml gene expression. Exemplary methods of administration include oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, intranasal, ophthalmic, or intraperitoneal administration. In addition, the dsRNA or shRNA can be administered via an implantable extended release device. In some embodiments, the siRNA or shRNA is conjugated to an antibody, or antigen-binding fragment thereof, that specifically binds to the surface of Thl7 cells, such as antibodies specific for CCR6, CD161 and/or IL-23R, including bispecific constructs binding to two or more of these surface markers. See, e.g., WO 08/106131. Antisense

[0076] Antisense nucleic acids can also be used as Blimp- 1 antagonists in the methods of the present invention. A Blimp- 1 /Prdml antisense nucleic acid sequence can comprise the complement of any contiguous segment within the sequence of the Prdml genes of the invention (SEQ ID NOs: 1 and 3). "Antisense" nucleic acids are DNA or R A molecules that are complementary to at least a portion of a specific mRNA molecule. See Weintraub (1990) Sci. Amer. 262:4046; Marcus-Sekura (1987) Nucl. Acid Res. 15:5749; Marcus-Sekura (1988) Anal. Biochem. 172:289; Brysch et al. (1994) Cell. Mol. Neurobiol. 14:557. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. Because the cell does not translate an mRNA in this double stranded form, antisense nucleic acids interfere with the expression of mRNA into protein. Oligomers of greater than about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient. Antisense methods have been used to inhibit the expression of many genes in vitro (Marcus-Sekura (1988) Anal. Biochem. 172:289;

Hambor et al. (1988) Proc. Nat'l Acad. Sci. U.S.A. 85:4010) and in situ (Arima et al. (1998) Antisense Nucl. Acid Drug Dev. 8:319; Hou et al. (1998) Antisense Nucl. Acid Drug Dev. 8:295).

[0077] Oligonucleotides that are complementary to the 5' end of the message, e.g., the

5' untranslated sequence up to and including the AUG initiation codon, are considered preferred for antisense applications because, in general, they efficiently inhibit translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have also been shown to be effective at inhibiting translation of mRNAs. See generally Wagner (1994) Nature 372:333. Thus, oligonucleotides complementary to the 5 '-non-translated region, the 3 '-non-translated region, or any other suitable region of the transcript {e.g., part of a coding region) could be used in an antisense approach to inhibit translation of endogenous Prdml gene mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'-, 3'-, or coding region of a gene mRNA, antisense nucleic acids should be at least six nucleotides long, and are preferably oligonucleotides ranging from six to about 50 nucleotides long. In other embodiments the oligonucleotide is at least ten nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. [0078] The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (Letsinger et al. (1989) Proc. Nat'l Acad. Sci. USA 86:6553; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648; PCT Publication No. WO 88/09810) or the blood-brain barrier (PCT Publication No. WO 89/10134), hybridization- triggered cleavage agents (Krol et al. (1988) BioTechniques 6:958) or intercalating agents (Zon (1988) Pharm. Res. 5:539). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

[0079] The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl -2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methyIguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6- isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, 5-methyl- 2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2- fluoroarabinose, xylulose, and hexose. In yet another embodiment, the antisense

oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

[0080] Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209, 1988), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Nat'l Acad. Sci. U.S.A. 85:7448), etc.

[0081] A number of methods have been developed for delivering antisense DNA or

R A to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. To enhance the efficiency of delivery, a preferred approach employs a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong transcriptional promoter. The use of such a construct to transfect target cells in the patient results in the transcription of sufficient amounts of single stranded RNAs that form complementary base pairs with the endogenous Pdrml transcripts and thereby prevent translation. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be effected by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include but are not limited to: the SV40 early promoter region (Bemoist and Chambon (1981) Nature 290:304), the promoter contained in the 3D long terminal repeat of Rous sarcoma virus (Yamamoto et al. (1980) Cell 22:787), the herpes thymidine kinase promoter (Wagner et al. (1981) Proc. Nat'l Acad. Sci. USA 78:1441), the regulatory sequences of the

metallothionein gene (Brinster et al. (1982) Nature 296:39), etc. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct.

Alternatively, viral vectors can be used that selectively infect the desired tissue or cell type (e.g. Thl7 cells).

[0082] An effective dose of an antisense oligonucleotide ranges from about 0.01 to

0.1, 0.1 to 1, or 1 to 10 mg/kg/day. In high dose embodiments, 10 to 50 mg/kg/day, is administered. The antisense oligonucleotide may be delivered subcutaneously, intravenously, or by any other suitable delivery method.

Delivery of Nucleic Acid-based Blimp- 1 Antagonists and Agonists

[0083] Nucleic acid-based antagonists and agonists of Blimp- 1, such as antisense nucleic acids, siRNAs, and Blimp- 1 encoding constructs, may be delivered to target cells as follows. A gene encoding a Blimp- 1 antagonist or agonist can be introduced either in vivo, ex vivo, or in vitro in a viral vector. Such vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. For example, in the treatment of neurological disorders or injuries, the striatal subventricular zone (SVZ) can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al. (1991) Molec. Cell. Neurosci. 2:320), an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 90:626 (1992)), and a defective adeno-associated virus vector (Samulski et al.

(1987) J. Virol. 61 :3096; Samulski et al. (1989) J. Virol. 63:3822) including a defective adeno-associated virus (AAV) vector with a tissue specific promoter {see. e.g., U.S. Pat. No. 6,040,172).

[0084] In another embodiment the inhibitor of Prdml can be introduced in a retroviral vector, e.g., as described in U.S. Pat. No. 5,399,346; Mann et al. (1983) Cell 33:153; U.S. Pat. No. 4,650,764; U.S. Pat. No. 4,980,289; Markowitz et al. (1988) J. Virol, 62: 1120; U.S. Pat. No. 5,124,263; Infl Pat. App. Pub. WO 95/07358; and Kuo et al. (1993) Blood 82:845. Targeted gene delivery is described in Int'l Pat. App. Pub. WO 95/28494.

[0085] Alternatively, the vector can be introduced by lipofection. Liposomes may be used for encapsulation and trans fection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated

transfection can be used to prepare liposomes for in vivo transfection of an expression vector encoding Blimp-1 or an antagonist thereof. Feigner et. al. (1987) Proc. Nat'l Acad. Sci.

U.S.A. 84:7413. See Mackey et al. (1988) Proc. Nat'l Acad. Sci. U.S.A. 85:8027. The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes. Feigner and Ringold (1989) Nature 337:387. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. See Mackey et al.

(1988) Proc. Nat'l Acad. Sci. U.S.A 85:8027.

[0086] It is also possible to introduce the vector as a naked DNA plasmid. Naked

DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter. See, e.g., Wu et al. (1992) J. Bioi. Chem., 267:963-967; Wu and Wu (1988) J. Biol. Chem. 263: 14621; Can. Pat. App. Pub.No. 2,012,311.

Antibodies

[0087] Antibodies, including intrabodies {e.g. Alvarez et al. (2000) Clinical Cancer

Research 6:3081), may also be used as Blimp-1 antagonists. Blimp-1 protein, or antigenic peptide fragments thereof, may be used to generate these antibodies, based on the known amino acid sequence of Blimp-1, as provided at SEQ ID NOs: 2 and 4 herein. Such antibodies or fragments may be chimeric, humanized, human or single chain antibodies or fragments. To express an antibody within a cell, a nucleic acid molecule encoding the antibody, such as a recombinant expression vector encoding the antibody, is introduced into the cell. Preferably, the antibody used to modulate protein expression or function is a single chain Fv (scFv) fragment, although whole antibodies, or antigen binding fragments thereof (e.g., Fab fragments) may also be useful. In one embodiment of the invention, an antibody is expressed intracellularly in a Thl7 cell, or precursor thereof, to inhibit the activity of Blimp-1. A nucleic acid molecule encoding the antibody may optionally be introduced using a recombinant viral vector, such a retroviral vectors {e.g. lentiviral vectors), adenoviral vectors and adeno-associated viral (AAV) vectors, or any other vector system suitable for delivery of genes to cells in vivo.

Thl 7 -specific Delivery and Expression

[0088] Blimp-1 has been reported to play a role in promoting differentiation of regulatory T cells (Tregs) to IL-10-expressing effector Tregs (Cretney et al. (2011) Nat. Immunol. 12:304) and in inhibiting Bcl-6-driven differentiation of CD4 ⁺ T cells to T follicular helper cells (T _FH ) (Johnston et al. (2009) Science 325:1006; Nurieva et al. (2009) Science 325 : 1001 ; Yu et al. (2009) Immunity 31 :457). Given these disparate effects on different T cell subsets, the methods of the present invention are best performed by antagonizing Blimp-1 activity specifically in Thl 7 cells. Such targeted inhibition would avoid effects of Blimp-1 antagonists on non-Thl7 cells, which might otherwise lead to undesired side effects caused by reduction of Treg function and enhanced T _FH function.

[0089] Thl 7 cells are characterized, inter alia, by expression of the transcription factor RORyt, IL-23 receptor (IL-23R), and CCR6. See Ivanov et al. (2006) Cell 126: 1121; U.S. Pat. App. Pub. No. 2007/0154487. Accordingly, RORyt and IL-23R promoters may be used to drive expression of Blimp- 1 antagonistic nucleic acid constructs in gene therapy embodiments of the present invention. Such vectors would limit expression of Blimp- 1 antagonistic nucleic acid constructs to those cells in which RORyt or IL-23R genes are being expressed, and thus target their effects to the desired Thl7 cell population. Blimp- 1 antagonistic nucleic acid constructs may comprise, for example, antisense nucleic acids or siRNA constructs designed to block the activity of Blimp- 1. IL-23R and RORyt promoters are discussed at Gocke et al. (2007) J. Immunol. 178:1341 and Eberl et al. (2003) Immuno. Rev. 195:81, respectively, the disclosures of which are hereby incorporated by reference in their entireties.

[0090] Antibodies exhibiting specific for Thl7 cells, such as anti-CCR6 antibodies, may be used to target Blimp- 1 antagonist compounds to Thl7 cells. Anti-CCR6 antibodies have been proposed for therapeutic use. See, e.g., U.S. Pat. No. 7,465,448, and U.S. Pat. App. Pub. Nos. 2002/0138860 and 2009/0041787. Anti-CCR6 monoclonal antibodies include, e.g., clone 11 A9 (Pharmingen, San Diego, Calif, USA), clone 4C6 (Acris

Antibodies, San Diego, Calif, USA), clone R6H1 (eBioscience, San Diego, Calif, USA), and clone 53103.111 (Sigma- Aldrich, St. Louis, Missouri, USA). Blimp- 1 antagonists may be bound to the anti-CCR6 antibodies to form antibody-drug conjugates. Antibodies that bind to the cell surface receptor CCR6 would be expected to be internalized, along with any conjugated drug {e.g. an siRNA molecule), based on the internalization of CCR6 upon binding of its natural ligands (CCL20/MIP3a) and β-defensin. Biragyn et al. (2007) J.

Immunol. 179:1381. See also Liu et al. (2007) Briefings Func. Genom. Proteom. 6: 112.

[0091] Antibodies that bind to other surface markers on Thl7 cells, such as IL-23R and/or CD161, may also be used to deliver Blimp- 1 antagonists to Thl7 cells.

WO 2008/106131. Bispecific and multispecific binding compounds, such as antibodies, antigen binding fragments, or antibody-like constructs that bind to two or more of CCR6, CD161 and IL-23R may also be used in the methods of treatment of the present invention to deliver Blimp- 1 antagonists to Thl7 cells. Exemplary humanized antibodies to human IL-23R are disclosed at WO 2008/106134. Exemplary anti-human CD161 are available as clone B199.2 (Pierce Antibodies, Rockford, III, USA) and clone DX12 (BD Pharmingen, San Diego, Calif, USA). Evaluation of Antagonist Activity

[0092] Compounds that mimic the effects of the Pdrml ^"7" mutants used in the experiments herein will be considered Blimp- 1 antagonists. Potential Blimp- 1 antagonists can be tested for their ability to produce the same biological effects as the knockout mice used in many of the experiments disclosed herein. Agents that alter the phenotype of wild-type mice to mimic that of homozygous Prdml knockout mice are likely to be Blimp- 1 antagonists. For example, agents that promote IL-2 expression, or inhibit GM-CSF, IFN- and/or IL-17 expression, in CD4 ⁺ T cells will be likely Blimp-1 antagonists. Preferred antagonists include those that act specifically in Thl7 (and precursor) cells, e.g. agents that are preferentially targeted to Thl7 cells or agents that are active preferentially in Thl7 cells (such as nucleic acid constructs that are selectively expressed in those cells). Methods of determining the expression of various relevant genes in CD4 ⁺ T cells are provided in the examples.

V. Blimp-1 Agonists

[0093] The present invention involves enhancement of Blimp-1 activity as a means of enhancing Thl7 response where such enhancement may be beneficial, such as during vaccinations.

[0094] Any agent capable of enhancing Blimp-1 activity, or enhancing its expression or protein levels, can act as a Blimp-1 agonist in the methods of the present invention. Small molecule drugs can be selected from natural or synthetic libraries of compounds, or synthesized based on rational drug design considerations. Compounds that exhibit effects opposite to the effects of the Pdrml ^"7" mutants used in the experiments herein will be considered potential Blimp-1 agonists.

[0095] Blimp-1 protein itself, or a biologically active fragment or sequence variant thereof, would be a Blimp-1 agonist. Nucleic acids encoding one of Blimp-1, or one of these fragments or variants, could also find use as a Blimp-1 agonist, e.g. when delivered in a vector in gene therapy.

[0096] As mentioned supra, Blimp-1 has been reported to be involved in Treg and

T _FH biology, in addition to the role in Thl7 biology disclosed herein. In light of these effects, the methods of the present invention are best performed by enhancing Blimp-1 activity specifically in Thl7 cells. Such targeted enhancement would avoid effects of Blimp-1 agonists on non-Thl7 cells, which might otherwise lead to undesired side effects caused by enhancement of Treg function and inhibition of T _FH function. [0097] Because Thl7 cells are characterized, inter alia, by expression of the transcription factor RORyt and IL-23R, their respective promoters may be used to drive Blimp- 1 expression in gene therapy embodiments of the present invention. Such vectors would limit Blimp- 1 expression to those cells in which RORyt or IL-23R genes are being expressed, and thus target their effects to the desired Thl7 cell population. IL-23R and RORyt promoters are discussed at Gocke et al. (2007) J. Immunol. 178: 1341 and Eberl et al. (2003) Immuno. Rev. 195:81, respectively, the disclosures of which are hereby incorporated by reference in their entireties.

VI. Pharmaceutical Compositions and Medicaments

[0098] To prepare pharmaceutical or sterile compositions (or medicaments) for use in the methods of the present invention, the agent or agents are admixed with a pharmaceutically acceptable carrier or excipient, see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

Formulations of therapeutic agents or combinations thereof may be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions. See, e.g., Hardman et al. (2001) Goodman and Gilman 's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications , Marcel Dekker, NY; Lieberman et al. (eds.) (1990)

Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and

Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY.

[0099] For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of

pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington 's Pharmaceutical Sciences, 18th Edition (1990) Mack Publishing Co., Easton, Pennsylvania. [00100] Liquid form preparations include solutions, suspensions and emulsions.

Examples include water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.

[00101] Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g. nitrogen.

[00102] Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

[00103] The compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

[00104] Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.

[00105] The quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1 mg to about 100 mg, preferably from about 1 mg to about 50 mg, more preferably from about 1 mg to about 25 mg, according to the particular application and the properties of the specific active compound in question {e.g. the affinity, toxicity or pharmacokinetic profile).

[00106] The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.

[00107] The amount and frequency of administration of the compounds of the invention and/or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical recommended daily dosage regimen for oral administration can range from about 1 mg/day to about 500 mg/day, preferably 1 mg/day to 200 mg/day, in two to four divided doses.

[00108] Toxicity and therapeutic efficacy of the therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD ₅₀ (the dose lethal to 50% of the population) and the ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD ₅ o and ED ₅ o. Therapeutic combinations exhibiting high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

[00109] The mode of administration of the therapeutic agents of the present invention is not particularly important. Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.

[00110] Selecting an administration regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the agent, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. Preferably, an administration regimen maximizes the amount of therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Accordingly, the amount of agent delivered depends in part on the particular agent and the severity of the condition being treated. Guidance in selecting appropriate doses of antibodies, cytokines, and small molecules are available. See, e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al.

(2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341 : 1966- 1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343: 1594-1602.

[00111] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment or predicted to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., reduction in the rate of growth of tumor tissue, or alteration of biomarkers associated with therapeutic efficacy.

[00112] The broad scope of this invention is best understood with reference to the following examples, which are not intended to limit the inventions to the specific

embodiments. The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

EXAMPLES

Example 1

General Methods

[00113] Standard methods in molecular biology are described. Maniatis et al. (1982)

Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA. Standard methods also appear in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[00114] Methods for protein purification including immunoprecipitation,

chromatography, electrophoresis, centrifugation, and crystallization are described. Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York. Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described. See, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391. Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described. Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra. Standard techniques for characterizing ligand/receptor interactions are available. See, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York.

[00115] Methods for flow cytometry, including fluorescence activated cell sorting detection systems (FACS ^® ), are available. See, e.g., Owens et al. (1994) Flow Cytometry Principles for Clinical Laboratory Practice, John Wiley and Sons, Hoboken, NJ; Givan (2001) Flow Cytometry, 2 ^nd ed.; Wiley-Liss, Hoboken, NJ; Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken, NJ. Fluorescent reagents suitable for modifying nucleic acids, including nucleic acid primers and probes, polypeptides, and antibodies, for use, e.g., as diagnostic reagents, are available. Molecular Probes (2003) Catalogue,

Molecular Probes, Inc., Eugene, OR; Sigma-Aldrich (2003) Catalogue, St. Louis, MO.

[00116] Standard methods of histology of the immune system are described. See, e.g.,

Muller-Harmelink (ed.) (1986) Human Thymus: Histopathology and Pathology, Springer Verlag, New York, NY; Hiatt, et al. (2000) Color Atlas of Histology, Lippincott, Williams, and Wilkins, Phila, PA; Louis, et al. (2002) Basic Histology: Text and Atlas, McGraw-Hill, New York, NY.

[00117] Software packages and databases for determining, e.g., antigenic fragments, leader sequences, protein folding, functional domains, glycosylation sites, and sequence alignments, are available. See, e.g., GenBank, Vector NTI ^® Suite (Informax, Inc, Bethesda, MD); GCG Wisconsin Package (Accelrys, Inc., San Diego, CA); DeCypher ^® (TimeLogic Corp., Crystal Bay, Nevada); Menne et al. (2000) Bioinformatics 16: 741-742; Menne et al. (2000) Bioinformatics Applications Note 16:741-742; Wren et al. (2002) Comput. Methods Programs Biomed. 68: 177-181; von Heijne (1983) Eur. J. Biochem. 133: 17-21; von Heijne (1986) Nucleic Acids Res. 14:4683-4690.

[00118] C57BL/6J mice were obtained from Jackson Research Labs. OT-II and

CD45.1 ⁺ mice were obtained from Jackson Research Labs and interbred at Merck. The generation of U23ra ^~ ' ^~ mice has been described. Chan et al. (2006) J. Exp. Med. 203:2577. Il23ra ^~ ' ^~ OT-II CD45.1 ⁺ mice were generated at Merck. IL-17-GFP Knockin reporter mice were generated and provided by Biocytogen. Blimp- 1 conditional knockout mice {Prdmlfl/fl) were purchased from Jackson Research Labs and Rosa-YFP^DlcJf mice were provided by Pamela J. Fink (originally from Nigel Killeen) and interbred at Merck. Animals were maintained in a barrier SPF facility and all animal procedures were approved by the Merck IACUC committee, in accordance with AAALAC guidelines. [00119] For EAE studies, C57BL/6 mice (including IL-17-GFP Knockin reporter mice and Prdml^Rosa-YFF^Dlck ^cre mice) were immunized in four sites on the back with 100 μg MOG35-55 in 200 ml CFA containing 100 μg tuberculosis strain H37Ra. All mice also received 100 ng pertussis toxin (List Biological Laboratories) intraperitoneally on days 0 and 2. Mice were assigned clinical scores for EAE according to the following scale: 1, flaccid tail; 2, impaired righting reflex and hindlimb weakness; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, hindlimb paralysis with partial forelimb paralysis; 6, moribund.

[00120] The following flow cytometry antibodies were purchased from BD

Biosciences: CD4 (RM4-5), CD45.1 (A20), CD44 (IM7), IFN-γ (XMG1.2), IL-17 (TC11- 18H10), IL-2 (JES6-5H4), CCR6 (140706), Ki-67 (B56), Bcl-2 (Bcl-2/100) and pSTAT3 (pS727). GM-CSF (MP1-22E9), T-bet (4B10), and CXCR3 (173) antibodies were purchased from eBioscience. Intracellular cytokine staining was performed with Cytofix-cytoperm kit from BD according to the manufacturer's instructions. pSTAT3 staining was performed with BD Phosflow kit according to the manufacturer's instructions. OVA(323-339) was obtained from Biosynthesis Inc (Lewisville, TX).

[00121] For OT-II tracking studies, recipient mice (CD45.2 ⁺ ) received 1 10 ⁵ CD45.1 ⁺ 23ra ' ^~ or Il23ra ^+I+ OT-II CD4 ⁺ T cells intravenously 1 day before being immunized subcutaneously in the flank with 100 mg OVA (323-339) in CFA. The phenotype of OT-II ⁺ cells was assessed by flow cytometry with gating on live CD4 ⁺ CD45.1 ⁺ cells.

[00122] For stimulation of CD4 T cells ex vivo for cytokine analysis, draining lymph nodes were collected at various time points after immunization and single-cell suspensions were obtained. Cells were stimulated for 4 hours with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) and ionomycin (500 ng/ml; both from Sigma- Aldrich) in the presence of GolgiPlug (BD Biosciences) in complete medium (RPMI medium containing 10% (vol/vol) FCS, supplemented with penicillin and streptomycin, L-glutamine, HEPES, pH 7.2, sodium pyruvate and 2-mercaptoethanol), followed by flow cytometry staining and analysis. For stimulation of CD4 ⁺ T cells ex vivo for analysis of STAT3 phosphorylation, suspensions of cells from draining lymph nodes were incubated for 15 minutes with complete medium alone, IL-23 (20 ng/ml) or IL-6 (100 ng/ml). CNS mononuclear cells were isolated as described (Langrish et al. (2005) J. Exp. Med. 201 :233) from mice with EAE and cells were cultured for 24 h in complete medium with MOG (35-55) (100 ug/ml) in the presence or absence of IL-23. GolgiPlug (BD Biosciences) was added for the final four hours of culture before flow cytometry. [00123] Cell sorting was performed as follows. To sort cell subsets based on intracellular cytokine expression, cells from draining lymph nodes of OVA ₃₂ 3_33 or MOG ₃₅ _ ₅₅ immunized mice were stimulated ex vivo for four hours with PMA and ionomycin, surface stained, fixed, permeabilized and stained intracellularly for IL-17 and IFNy. RVC

(ribonuclease vanadyl complex) RNase inhibitor (Sigma-Aldrich) was added during the fixation, intracellular staining and cell sorting steps to keep the RNA intact. Fixed cells were sorted based on IL-17 and IFNy protein expression and gene expression analysis was performed. For sorting ex vivo cytokine stimulated IL-17 ⁺ cells, draining lymph node cells were isolated from MOG immunized IL-17.GFP Knockin mice and rested for 1.5 hours, followed by stimulation with IL-23 or IL-6 for 2 hours. Stimulated cells were sorted based on IL-17.GFP and CD4 expression.

[00124] TAQMAN ^® real-time quantitative PCR gene expression analysis was performed as follows. Total RNA was isolated from cells using Arcturus PicoPure RNA Isolation method, according to manufacturer's protocol (Applied Biosystems, Life

Technologies, Foster City, Calif, USA). DNase-treated total RNA was amplified using NuGen WT-Ovation Pico RNA Amplification System per manufacturer's instructions (NuGen Technologies Inc., San Carlos, Calif, USA). Primers were designed using Primer Express (Applied Biosystems, Life Technologies, Foster City, Calif, USA), or obtained commercially from Applied Biosystems (Foster City, Calif, USA). Real-time quantitative PCR on 10 ng of amplified cDNA from each sample was performed using either two gene- specific unlabelled primers utilized at 400 nM in a Applied Biosystems SYBR green real-time quantitative PCR assay or two unlabelled primers at 900 nM each were used with 250 nM of FAM-labeled probe (Applied Biosystems, Life Technologies, Foster City, Calif, USA) in a TAQMAN ^® real-time quantitative PCR reaction. Either of the following technologies was used to collect data; an ABI 7300 or 7900 sequence detection system or the Fluidigm

Biomark (Fluidigm, South San Francisco, Calif, USA). The absence of genomic DNA contamination was confirmed using primers that recognize genomic region of the CD4 promoter. Ubiquitin levels were measured in a separate reaction and used to normalize the data by the Δ-Δ Ct method. (Using the mean cycle threshold value for ubiquitin and the gene of interests for each sample, the equation 1.8 e (Ct ubiquitin minus Ct gene of interest) x 10 ⁴ was used to obtain the normalized values.)

[00125] Statistical analysis was done by Student's t test (two groups) or one-way

ANOVA (more than two groups) with Graphpad Prism software. Where shown, error bars indicate standard deviation. Example 2

IL-23 Signaling Induces Blimp- 1 in Thl7 cells

[00126] Experiments were performed to determine the role of IL-23 signaling in

Blimp-1 expression in Thl7 cells, as follows. A relatively low number of congenically marked (CD45.1 ⁺ ) OT-II (ovalbumin peptide323-339-TCR-tg) cells from wildtype (wt) and IL-23 receptor knockout (IL-23R ^{~ ~} ) were transferred to wild-type C57B1/6 recipient mice. At day seven post-immunization with ovalbumin (OVA) peptide 323-339 in complete Freund's adjuvant (CFA), IL-17 ⁺ and IL-17 ^" OT-II cells were sorted and gene expression analysis was performed. Cells were enriched for CD4 ⁺ T cells, stimulated ex vivo with PMA and ionomycin, surface stained and fixed along with an RNase inhibitor to keep the RNA intact. Fixed cells were stained intracellularly and sorted based on IL-17 protein expression and gene expression analysis was performed. Results are presented at FIG. 1 A for Prdml (left plot) and Bcl-6 (right plot).

[00127] These experiments demonstrate that wt IL-17 ⁺ OT-II cells express high levels of Prdml, and that this expression is reduced in the absence of IL-23 signaling (i.e. in IL- 23R ^{~ ~} cells). In contrast expression of Bcl6, a known direct repressor of Prdml, is significantly inhibited in IL-17 ⁺ cells and is lost in the absence of IL-23 signaling. These results suggest that IL-23 signaling promotes Prdml expression specifically in Thl7 cells.

[00128] The relative expression of Prdml and Bcl-6 expression in Thl7 and Thl cells was determined as follows. Cells from draining lymph nodes of C57BL/6J mice immunized with MOG ₃₅ _55 in CFA were obtained at day seven post immunization. These cells were stimulated ex vivo with phorbol 12-myristate 13 -acetate (PMA) and ionomycin, surface stained, and fixed along with an RNase inhibitor to keep the RNA intact. Fixed cells were stained intracellularly and sorted based on IL-17 and IFNy protein expression. TAQMAN ^® gene expression analysis was performed on the resulting IL-17 ⁺ / IFNy ^" and IFNy ⁺ / IL-17 ^" cell populations (representing 2.0% and 1.94% of total sorted cells, respectively). Data are representative of three independent experiments. Error bars in all graphs are SEM. Data are presented at FIG. IB for Prdml (left plot) and Bcl6 (right plot).

[00129] These experiments demonstrate that Prdml expression is specific to Thl 7 cells as compared to Thl cells, which have reduced Prdml, but increased Bcl6 expression (an antagonistic regulator of Blimp-1). These results indicate that Blimp-1 expression is specifically elevated in Thl 7 cells and not Thl cells. [00130] To determine whether IL-23 signaling can directly induce Prdml expression in

Thl7 cells, draining lymph node cells were collected from IL-17-GFP Knockin reporter mice at day seven post immunization with MOG ₃₅ _55 peptide in CFA and allowed to rest briefly before stimulating with cytokine (IL-6 or IL-23). About 55-60% of the IL-17. GFP ⁺ cells responded to IL-23 and IL-6 stimulation as measured by phosphorylation of STAT-3 (data not shown). To assess gene expression, CD4 ⁺ IL-17.eGFP ⁺ cells from untreated, IL-23 treated, and IL-6 treated were sorted two hours after cytokine stimulation and TAQMAN ^® gene expression analysis was performed. Results are presented at FIG. 1C for Prdml (left plot) and 112 (right plot).

[00131] Prdml expression was significantly upregulated in IL-17 ⁺ cells by IL-23 but not IL-6 stimulation indicating that Prdml gene transcription is specifically induced by IL-23 and is a proximal event following IL-23 stimulation. In contrast, 112 expression was greatly diminished in IL-17 ⁺ cells as compared to IL-17 ^" cells. These results demonstrate that Prdml expression is significantly upregulated in IL-17 ⁺ cells by IL-23 but not IL-6, indicating that Prdml gene transcription is directly induced by IL-23 and is a proximal event following IL-23 stimulation.

Example 3

Blimp- 1 Promotes Development of Pathogenic Thl7 cells that

Co-express IL-17, GM-CSF, IFNy and T-bet

[00132] Experiments were performed to determine the role of Blimp- 1 in the development of Thl7 cells, as follows. To assess the function of upregulated Blimp-1 in Thl7 cells we generated a conditional knockout mouse by crossing Prdml floxed (Prdml^) mice to distal lck-cre mice (Dlck ^cre ). Distal-lck promoter drives cre-mediated recombination specifically in the peripheral mature T cell compartment thereby avoiding any thymic T cell development defects. Furthermore, Prdml^ x Dlckcre mice were bred to ROSA-flox- STOP-flox-YFP (Rosa-YFP^ ¹ ) mice to allow tracking of Blimp-1 deleted cells. Dlck-cre mediated YFP expression in mature single positive T cells in thymus and in about 65% of CD4 ⁺ T cells from lymph nodes (data not shown). By PCR analysis, Prdml expression was significantly reduced in YFP ⁺ CD4 ⁺ T cells from Prdml ^{m '} Rosa-YFP ^fl Dlck ^cre (Prdml ⁷ ) mice (data not shown). Interestingly, even YFP- CD4 ⁺ T cells expressed lower levels of Prdml mRNA compared to control CD4 ⁺ T cells (data not shown), suggesting that the Prdml locus is more accessible to the action of ere than the Rosa-YFP locus. To control for this potential difference, YFP ⁺ cells from Prdml ^"7" were compared to YFP ⁺ cells from either Prdml^ ^/wt Rosa-YF≠Dlck ^cre (Prdml ^"7+ ) mice or Prdml ^wt/wt Rosa-YF≠Dlck ^cre (Prdml ^+/+ ) mice.

[00133] Intracellular GM-CSF and IFNy expression was determined in IL-17 ⁺ cells from flow cytometry of intracellular IL-17-producing CD4 ⁺ T cells from draining lymph nodes of MOG ₃₅ _ ₅ 5 immunized Prdml ^'1' after ex vivo stimulation with PMA and ionomycin at day seven post immunization. Results are provided at FIG. 2A.

[00134] Intracellular T-bet expression was determined in CD4 ⁺ T cells and IL-17 ⁺

CD4 ⁺ T cells at days seven and 12 post immunization, respectively. Results are provided at FIG. 2B.

[00135] Intracellular IL-2 and IL-17 expression was determined in CD4 ⁺ T cells at seven days post immunization. Results are provided at FIG. 2C.

[00136] The data show that the proportion of YFP ⁺ CD4 ⁺ T cells producing only IL-17 is reduced by Prdml deficiency. More importantly, there is a significant defect in the ability of Prdml ^"7" IL-17 producers to co-express GM-CSF and IFNy. The absence of Prdml expression also prevented T-bet upregulation in CD4 ⁺ T cells and in IL-17 CD4 ⁺ T cells. Furthermore, in the absence of IL-23 signal nearly all the Thl7 cells are trapped at the early activation stage and are unable to downregulate their IL-2 expression. Together these results indicate that IL-23 mediates functional maturation of Thl7 cells through induction of

Blimp- 1 that promotes co-expression of GM-CSF, IFNy and T-bet and inhibits IL-2 expression by IL-17-producing cells.

Example 4

T cell-Specific Blimp- 1 Deficiency Renders Mice Resistant to

Experimental Autoimmune Encephalomyelitis (EAE)

[00137] Experiments were performed to determine the role of Blimp- 1 in EAE, an experimental mouse model of multiple sclerosis, as follows. The experiments were performed in mice with T cell-restricted deletion of Blimp- 1 gene Prdml^ Rosa-YFP^ ¹ Dlck ^cre ). Blimp- 1 conditional knockout mice {Prdml^Rosa-YFP^Dlck ^cre ) were immunized in four sites on the back with 100 μg MOG ₃₅ -55 in 200 ml CFA and also received 100 ng pertussis toxin intraperitoneally on days 0 and 2 to induce EAE. Clinical scores for EAE in these Prdml conditional knockout mice are provided at FIG. 3A. FIG. 3B provides a summary of maximal clinical scores post EAE induction from three experiments. [00138] The frequency and absolute number of CD4 ⁺ T cells in the CNS of Prdml conditional knockout mice were determined 12 days after EAE induction, and results are shown at FIG. 3C.

[00139] Intracellular staining was used to sort for cells expressing IL-17, GM-CSF and

IFNy from the CNS of Prdml conditional knockout mice 12 days after EAE induction.

Results are provided at FIGS. 3D, 3E and 3F. Data are representative of three independent experiments. Error bars in all graphs are SEM.

[00140] The results show that Prdml ^'1' mice {Prdml ^m Rosa-YFP ^fl Dlck ^cre ) are completely protected from EAE and have significantly lower percent CD4 ⁺ T cells accumulated in the central nervous system (CNS) as compared to Prdml ^~/+ (Prdml^ ^/+ Rosa- YFP ^fl Dlck ^cre ) and Prdml ^+/+ (Prdml ^+/+ Rosa-YFP^Dlck ⁰ ) mice. Furthermore, the few Prdml ^' ' ^' YFP ⁺ CD4 ⁺ T cells that infiltrate the CNS are functionally defective. They have reduced IL-17, GM-CSF and IFNy production.

Example 5

Blimp- 1 Promotes Optimal Expansion and Expression of

Homing Receptors on Thl7 cells

[00141] Further experiments were performed to characterize the effects of Blimp-1 on

Thl7 cells, as follows. Flow cytometry of CCR6 and CXCR3 expressing CD4 ⁺ T cells was performed on cells from draining lymph nodes of MOG ₃₅ _55 immunized Prdml conditional knockout mice at day seven post immunization. Results are shown in FIG. 4A. Intracellular Ki-67 expression was measured in CD4 ⁺ T cells (left panel) and IL-17 ⁺ CD4 ⁺ T cells (right panel) from draining lymph nodes at day seven post immunization. Results are shown in FIG. 4B. Intracellular Bcl-2 expression was measured in CD4 ⁺ T cells (top panel) and IL-17 ⁺ CD4 ⁺ T cells (bottom panel) from draining lymph nodes at day seven post immunization. Data are representative of three independent experiments. Error bars in all graphs are SEM.

[00142] These results demonstrate that the proportion of cells expressing CCR6 is reduced slightly but there is a significant reduction in CXCR3 expressing cells in the absence of Blimp-1, suggesting that Blimp-1 promotes expression of chemokine receptors on Thl7 cells required for trafficking to sites of inflammation. They further show that in the absence of Blimp-1, fewer Thl7 cells were Ki67 (a cellular proliferation marker) positive, suggesting that Blimp-1 promotes optimal expansion of Thl7 cells. Finally, the results demonstrate that the proportion of CD4 ⁺ T cells, and specifically Thl7 cells, expressing Bcl-2 (cell survival molecule) did not differ between Prdml ^'1' and Prdml ^~/+ mice. [00143] Together these results indicate that Blimp-1 is required for optimal Thl7 cell differentiation and expansion, thereby allowing Thl7 cells to migrate to the site of inflammation and mediate pathogenic functions.

[00144] Table 1 provides a brief description of the sequences in the Sequence Listing.

Table 1

Sequence Identifiers

SEQ ID NO: Description

1 PRDM1 isoform 1 (nucleic acid)

2 PRDM1 isoform 1 (polypeptide)

3 PRDM1 isoform 2 (nucleic acid)

4 PPvDMl isoform 2 (polypeptide)

5 muPRDMl (nucleic acid)

6 muPRDMl (polypeptide)