Methods and compositions for modulating TH-GM cell function

Abstract

Disclosed herein is a T-helper cell (“TH-GM” cell) that is regulated by IL-7/STAT5 and which secrete GM-CSF/IL-3. Also disclosed are methods and compositions for modulating TH-GM function for the treatment of, e.g., inflammatory disorders. Diagnostic and prognostic methods for specifically identifying TH-GM-mediated inflammatory disorders (e.g., rheumatoid arthritis), as distinct from and/or in addition to non-TH-GM-mediated (e.g., TNF-α-mediated) inflammatory disorders, are also provided.

Description

RELATED APPLICATION

This application claims the benefit of Singapore Patent Application No. 10201406130P, filed Sep. 26, 2014. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

A significant body of research has led to the current model of immunity and inflammation, as well as the dysregulation in immune and inflammatory disorders. It is currently understood that CD4⁺ helper T (T_H) cells play a crucial role in host defense against various pathogens by orchestrating adaptive and innate immune responses. Upon T-cell receptor (TCR) activation by cognate antigen, naïve CD4⁺ T cells are committed to differentiate into at least five major subsets: T_H1, T_H2, T_H17, iT_reg and T_FH, which are modulated by cytokine milieus. T_H1 and T_H17 cells are known to be the primary effectors of inflammation. However, the pathogenic roles of either T_H1 or T_H17 in various inflammatory disorders remain unclear. For example, recent studies conflict with previously understood paradigm of T_H17 in multiple sclerosis (MS) pathogenicity (Haak et al., 2009), making it more challenging to identify potential drug targets for MS therapy. Similarly, while rheumatoid arthritis (RA) is traditionally understood to be a disorder mediated by tumor necrosis factor α (TNF-α), up to 40% of RA patients fail to respond to anti-TNF-α treatment.

Accordingly, there remains a significant unmet need for effective treatment methods for autoimmune and inflammatory disorders such as, e.g., MS and RA.

SUMMARY OF THE INVENTION

The present disclosure relates, in part, to the identification of an interleukin-7 (IL-7)/signal transducer and activator of transcription 5 (STAT5)-regulated granulocyte macrophage colony-stimulating factor (GM-CSF)/IL-3-producing T_H cells, termed T_H-GM, which represent a distinct helper T cell subset with unique developmental and functional characteristics. Identified herein is an inflammatory pathway mediated by T_H-GM cells (T_H-GM-mediated inflammatory pathway), which represents an independent inflammatory pathway apart from known non-T_H-GM-mediated inflammatory pathways (e.g., TNF-a, IL-6, and IL-1b pathways of inflammation). The present disclosure provides methods and compositions for diagnosing inflammatory disorders that are T_H-GM-mediated, and modulating T_H-GM cell function for the treatment of inflammatory disorders mediated by the T_H-GM pathway.

Accordingly, in one aspect, the present disclosure provides a method of diagnosing a T_H-GM-mediated inflammatory disorder in a patient suffering from an inflammatory disorder, comprising: a) contacting a sample collected from a patient suffering from an inflammatory disorder with a detecting agent that detects a polypeptide or nucleic acid level of STAT5 (e.g., phospho-STAT5 (Tyr694)), IL-7, GM-CSF or IL-3, or a combination thereof; and b) quantifying the polypeptide or nucleic acid level of STAT5 (e.g., phospho-STAT5 (Tyr694)), IL-7, GM-CSF or IL-3, or a combination thereof, wherein an increased level of STAT5 (e.g., phospho-STAT5 (Tyr694)), interleukin-7 (IL-7), GM-CSF or interleukin-3 (IL-3), or a combination thereof relative to a reference level indicates that the patient suffers from a T_H-GM-mediated inflammatory disorder.

In another aspect, the present disclosure provides an isolated population of GM-CSF-secreting T-helper cells (T_H-GM), wherein the T_H-GM cells are differentiated from cluster of differentiation 4 (CD4+) precursor cells in the presence of IL-7 and activated STAT5, and wherein the T_H-GM cells express GM-CSF and IL-3.

In another aspect, the present disclosure provides a method of modulating T_H-GM function, comprising contacting the T_H-GM, or CD4+ precursor cells, or both, with a modulating agent that modulates T_H-GM function.

In some aspects, the present disclosure provides a method of treating a T_H-GM-mediated inflammatory disorder in a patient in need thereof, comprising administering to said patient an effective amount of a modulating agent that modulates T_H-GM cell function.

In other aspects, the present disclosure provides a method of treating rheumatoid arthritis in a patient who exhibits limited response to anti-tumor necrosis factor alpha (TNF-α) therapy, comprising administering to said patient an effective amount of a modulating agent that modulates T_H-GM function.

In another aspect, the present disclosure provides a method of treating a STAT5-mediated inflammatory disorder in a patient in need thereof, comprising administering to the patient an effective amount of an agent that modulates STAT5 function.

In further aspects, the present disclosure provides a method of screening to identify a modulator of T_H-GM cell function, comprising contacting an isolated population of T_H-GM cells, or an isolated population of CD4+ precursor cells, with a candidate agent, and determining a readout of T_H-GM function in the presence or absence of the candidate agent, wherein a change in the readout of T_H-GM function indicates that the candidate agent is a modulator of T_H-GM function.

The present disclosure enables the identification or classification between inflammatory disorders that are either primarily T_H-GM-mediated, or primarily non-T_H-GM-mediated (e.g., mediated by TNF-α, IL-6, and/or IL-1β), or both. Thus, using the methods described herein, it is possible to determine whether a patient suffering from, e.g., RA, suffers from an RA that is primarily T_H-GM-mediated, or primarily non-T_H-GM-mediated, or both. This differentiation allows for a more targeted and tailored method of treating inflammatory disorders such as RA, for which current treatments are only 40% effective. Further, the present disclosure provides methods and compositions for prognosing the progression of an inflammatory disorder so as to tailor the treatment according to the stage of the disease. Also provided herein are compositions and methods for and the treatment of inflammatory disorders, particularly those that are T_H-GM-mediated.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particular description of example embodiments of the invention.

FIGS. 1A-1D depict Stat5-conditional mutant mice are resistant to EAE. Clinical EAE scores (FIG. 1A) and incidence (FIG. 1B) of Stat5^+/+ and Stat5^−/− mice immunized twice with MOG_35-55/CFA. Data are representative of three independent experiments (FIG. 1A) or pooled from three experiments (FIG. 1B, n=18 per group). Clinical scores of EAE mice immunized once with MOG_35-55/CFA (FIG. 1C, n=5 per group) or immunized twice with MOG_35-55/LPS (FIG. 1D). Data are representative of two independent experiments.

FIGS. 2A-2D depict reduced neuroinflammation in Stat5 conditional mutant mice. Histology of spinal cord sections obtained from EAE mice at day 9 after 2^nd immunization (FIG. 1A). Images shown are representative of two independent experiments with three mice per group. Scale bars, 200 μm (top), 50 μm (bottom). CD4⁺ and CD11b⁺ cells in spinal cord sections were stained by immunofluorescence (FIG. 1B). Images shown are representative of two independent experiments with three mice per group. Scale bars, 200 μm. CNS mononuclear cells were analyzed by flow cytometry at peak of disease (FIGS. 2C and 2D). Right panels are cell proportions (FIG. 2C, right) or cell numbers (FIG. 2D, right) pooled from two experiments (n=9).

FIGS. 3A and 3B depict the resistance to EAE in Stat5-deficient mice is independent of T_H1, T_H17 or T_reg cells. Flow cytometric analysis of IL-17 and IFN-γ expression by CNS-infiltrating CD4⁺ T cells at peak of disease (FIG. 3A). Data are representative of three independent experiments. Percentage of CD25⁺ among CD4⁺ T cells in the CNS of Stat5^+/+ and Stat5^−/− EAE mice at peak of disease were analyzed by flow cytometry (FIG. 3B).

FIGS. 4A-4C depict conditional Stat5 mutant mice have no defect in CD4⁺ T cell generation in periphery. Spleens were obtained from MOG_35-55/CFA-immunized Stat5^+/+ and Stat5^−/− mice at day 7 (FIG. 4A) and day 21 (FIG. 4B). The proportions of CD4⁺ and CD8⁺ T cells were analyzed by flow cytometry. The absolute number of CD4⁺ T cells was calculated (right panels). Data are representative of two independent experiments (FIG. 4A) or pooled from two independent experiments (FIG. 4B). IL-17 and IFN-γ expression by splenic CD4⁺ T cells of Stat5^+/+ and Stat5^−/− EAE mice was determined by intracellular cytokine staining (FIG. 4C). Data are representative of three independent experiments. *p<0.5, **p<0.005, ***p<0.0005.

FIGS. 5A-5D depict Stat5-deficient CD4⁺ T cells can infiltrate CNS but fail to induce effective neuroinflammation. CCR6, CXCR3 and CD69 expression by splenic CD4⁺ T cells of Stat5^+/+ and Stat5^−/− EAE mice was measured. Data are representative of two independent experiments with three to five mice per group (FIG. 5A). CNS-infiltrating CD4⁺ T cells were analyzed at day 7, 9 and 21 after first MOG_35-55/CFA immunization (FIGS. 5B-5D). Cell numbers were calculated (FIG. 5D). Data are representative of two independent experiments with three mice per group. *p<0.5.

FIGS. 6A-6C show resistance to EAE in Stat5^−/− mice is not caused by any defect in the survival of CD4⁺ T cells in the absence of STAT5. CD4⁺ T cell infiltration (FIG. 6A) and clinical scores (FIG. 6B) of Rag2^−/− recipient mice transferred with different numbers of Stat5^+/+ and Stat5^−/− CD4⁺ T cells. Clinical scores and frequencies of CD4⁺ T cells in the CNS at day 21 (disease peak) of EAE induction (FIG. 6C). *p<0.05, ***p<0.0005.

FIGS. 7A-7C depict the intrinsic defect of Stat5-deficient CD4⁺ T cells in encephalitogenicity. Clinical EAE scores (FIG. 7A) and incidence (FIG. 7B) of Rag2^−/− mice (n=5 per group) after adoptive transfer of 2 million MOG_35-55-specific Stat5^+/+ or Stat5^−/− CD4⁺ T cells respectively. IL-17 and IFN-γ expression by CNS-infiltrating CD4⁺ T cells was measured at peak of disease (FIG. 7C). Data represent two independent experiments. *p<0.05.

FIGS. 8A-8D depict the diminished induction of GM-CSF in splenic Stat5^−/− CD4⁺ T cells. In FIGS. 8A-8D, splenocytes were obtained from MOG_35-55/CFA-immunized Stat5^+/+ and Stat5^−/− mice (n=3 per group) before disease onset and challenged with MOG_35-55 at various concentrations for 24 h. GM-CSF secretion was measured by ELISA (FIG. 8A). Golgiplug was added in the last 4 h of MOG_35-55 (20 μg/ml) challenge and the frequencies of IL-17⁺ and GM-CSF⁺ cells among CD4⁺CD44^hiT cells were measured (FIG. 8B). In FIGS. 8C and 8C, splenocytes were obtained from MOG_35-55/CFA-immunized Stat5^−/−, Stat3^−/− or wild-type control mice and stimulated with PMA/Ionomycin in the presence of Golgiplug for 4 h. The frequencies of IL-17⁺ and GM-CSF⁺ cells among splenic CD4⁺CD44^hi T cells were measured by intracellular cytokine staining. *p<0.05, ***p<0.001.

FIGS. 9A-9C depict the diminished induction of GM-CSF in CNS-infiltrating Stat5^−/− CD4⁺ T cells. In FIG. 9A, IL-17, IFN-γ and GM-CSF expression by CNS-infiltrating CD4⁺ T cells of Stat5^+/+ and Stat5^−/− mice was measured at peak of disease. The percentage of GM-CSF⁺ cells among IL-17⁺ or IFN-γ⁺ cells was calculated (bottom right, FIG. 9A). IL-17, IFN-γ and GM-CSF expression by CNS-infiltrating CD4⁺ T cells of Rag2^−/− recipient mice at peak of disease in adoptive transfer EAE (FIG. 9B). Time-course analysis of cytokine mRNA expression in the CNS of naïve and MOG_35-55/CFA-immunized Stat5^+/+ and Stat5^−/− mice (n=3 per group at each time point). The RT-PCR data were normalized to Rn18S, and expression in naïve mice was set to 1 (FIG. 9C). Data represent two independent experiments. *p<0.05.

FIGS. 10A-10C show STAT5-mediated GM-CSF induction is independent of IL-23 or IL-1β signaling. In FIG. 10A, purified CD4⁺ T cells were cultured with TGF-β and IL-6 for 3 days, followed by starvation for 6 h. Then cells were treated with various cytokines for 30 min, and pSTAT3 and pSTAT5 was determined by immunoblotting. STAT3 and STAT5 were further detected after stripping. FIG. 10B shows the mRNA expression of IL-23R and IL-1R1 in splenic CD4⁺ T cells of Stat5^+/+ and Stat5^−/− EAE mice (n=3 per group). The RT-PCR data were normalized to β-Actin. In FIG. 10C, splenocytes were obtained from MOG_35-55/CFA-immunized WT mice before disease onset and challenged with MOG_35-55 (20 μg/ml) in the absence or presence of IL-2 for 48 h. The frequencies of GM-CSF⁺ and IL-17⁺ cells among CD4⁺CD44^hi T cells were measured by flow cytometry. *p<0.05.

FIGS. 11A-11C depict IL-7-induced STAT5 activation promotes GM-CSF expression in autoreactive CD4⁺ T cells. Splenocytes were obtained from MOG_35-55/CFA-immunized Stat5^+/+ and Stat5^−/− mice before disease onset and challenged with MOG_35-55 (20 μg/ml) in the absence or presence of IL-7 for 48 h. Frequencies of GM-CSF⁺ and IL-17⁺ cells among CD4⁺CD44^hi T cells were measured by flow cytometry (FIG. 11A). GM-CSF secretion was measured by ELISA (FIG. 11B). Data represent two independent experiments with two to three mice per group. Splenic CD62L^hiCD44^lo and CD62L^loCD44^hi T cells from MOG_35-55/CFA-immunized mice were sorted out. Cells were stimulated with anti-CD3 and anti-CD28 in the absence or presence of IL-7 for 4 h and then harvested for the analysis of GM-CSF expression by RT-PCR (FIG. 11C). *p<0.05

FIGS. 12A-12F depict IL-7Rα neutralization attenuates GM-CSF expression and ameliorates EAE. Clinical scores of EAE mice (n=5) treated with anti-IL-7Rα or normal IgG given every other day from day 5 after 2^nd immunization, as indicated by arrows. Data represent two independent experiments (FIG. 12A). Spinal cord sections were obtained from EAE mice at day 11 after 2^nd immunization. Immune cell infiltration was assessed histologically. Images shown are representative of three individuals per group. Scale bars, 200 μm (top), 50 μm (FIG. 12B, bottom). The percentages of CD4⁺ and CD8⁺ T cells in spleens of EAE mice. Data represent two independent experiments (FIG. 12C). FIGS. 12D and 12E illustrate the frequencies of GM-CSF⁺, IL-17⁺ and IFN-γ⁺ cells among CD4⁺ T cells in the CNS of EAE mice receiving different treatment. The mRNA expression of IFN-γ, IL-17 and GM-CSF in the CNS of EAE mice (FIG. 12F). *p<0.05

FIGS. 13A and 13B depict the differentiation of GM-CSF-expressing T_H cells is distinct from T_H17 and T_H1. Naïve CD4⁺ T cells were primed with plate-bound anti-CD3 and soluble anti-CD28 in the presence of a combination of various cytokines and neutralizing antibodies as indicated. GM-CSF, IL-17 and IFN-γ expression was analyzed by intracellular staining (FIG. 13A) or RT-PCR (FIG. 13B)

FIGS. 14A-14D show the effect of IL-2 and IL-6 on T_H-GM differentiation from naïve T cells. GM-CSF and IFN-γ expression in naive CD4⁺ T cells activated for 72 h with anti-CD3 alone or plus anti-CD28 (FIG. 14A). In FIG. 14B, sorted naïve CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 in the presence of neutralizing antibodies against IL-12 and IFN-γ without or with the addition of IL-6. The frequencies of GM-CSF⁺ and IL-17⁺ cells were measured by intracellular staining (FIG. 14B). In FIG. 14C, naïve CD4⁺ T cells from Stat3^+/+ and Stat3^−/− mice were polarized under conditions as indicated for 72 h. The frequencies of GM-CSF⁺ and IL-17⁺ cells were analyzed. In FIG. 14D, naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 in the presence of IL-2 or anti-IL-2. The frequencies of GM-CSF⁺, IL-17⁺ and IFN-γ⁺ cells were analyzed.

FIGS. 15A-15F depict IL-7-STAT5 signaling programs T_H-GM differentiation from naïve precursor cells. Naïve CD4⁺ T cells were primed with plate-bound anti-CD3 and soluble anti-CD28 in the presence of various concentration of IL-7 as indicated. GM-CSF and IFN-γ expression was analyzed by intracellular staining (FIG. 15A) or ELISA (FIG. 15B). In FIGS. 15C and 15D, Stat5^+/+ and Stat5^−/− naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 in the presence IL-7 for 3 days. GM-CSF, IL-17 and IFN-γ expression was analyzed by intracellular cytokine staining (FIG. 15C). GM-CSF secretion was measured by ELISA (FIG. 15D). Immunoblotting of pSTAT5 and STAT5 in IL-7-stimulated CD4⁺ T cells isolated from Stat5^−/− or control mice (FIG. 15E). The ChIP assay was performed with Stat5^+/+ and Stat5^−/− CD4⁺ T cells using normal IgG or STAT5-specific antibody. The binding of antibodies to Csf2 promoter region was detected by RT-PCR (FIG. 15F).

FIGS. 16A and 16B depict the differentiation conditions for T_H-GM subset. Naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 in the presence of IL-7 or/and anti-IFN-γ as indicated. GM-CSF, IL-17 and IFN-γ expression was analyzed (FIG. 16A). The mRNA expression of T-bet and RORγt in naïve, T_H1 (IL-12+anti-IL-4), T_H17 (TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and T_H-GM cells (IL-7+anti-IFN-γ) (FIG. 16B). The RT-PCR data were normalized to Gapdh, and expression in naïve T cells was set to 1.

FIGS. 17A-17E illustrate that IL-7 but not IL-2 induces STAT5 activation and GM-CSF expression in naïve CD4⁺ T cells. FIGS. 17A-17C show flow cytometry of CD25 and CD127 on the surface of naïve CD4⁺ T cells or cells activated with anti-CD3 and anti-CD28 at various time points as indicated. Activation of STAT5 in naïve CD4⁺ T cells stimulated with IL-2 or IL-7 for 30 min (FIG. 17D). FIG. 17E shows the mRNA expression of GM-CSF in naïve CD4⁺ T cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 or IL-7. The RT-PCR data were normalized to β-Actin, and expression in naïve T cells activated for 2 h without cytokine was set to 1.

FIGS. 18A-18C show that both IL-2 and IL-7 can induce STAT5 activation and GM-CSF expression in activated CD4⁺ T cells. As shown in FIGS. 18A and 18B, CD4⁺ T cells were activated with anti-CD3 and anti-CD28 for 3 days. After resting in fresh medium, cells were stimulated with IL-2 or IL-7 at various time points. The pTyr-STAT5 and β-Actin were detected by immunoblotting (FIG. 18A). GM-CSF mRNA expression was measured by RT-PCR (FIG. 18B). The RT-PCR data were normalized to β-Actin, and expression in cells without cytokine stimulation was set to 1. The ChIP assay shown in FIG. 18C was performed with normal IgG or STAT5-specific antibody. The binding of antibodies to Csf2 promoter region was detected by RT-PCR.

FIG. 19 depicts surface molecules selectively expressed at high level or low level in T_H-GM subset as characterized by microarray analysis. These surface molecules specific for each lineage serves as markers, signatures and potential targets for novel diagnosis, treatment and prevention of autoimmune inflammation including, but not limited to multiple sclerosis and rheumatoid arthritis. These cell surface molecules are listed in detail in Table 1. The order of naïve, Th1, Th17, and Th-GM as indicated in the figure insert is the same as that appears for the bars in each graph.

FIGS. 20A-20D show that IL-3 is preferentially expressed in T_H-GM cells. In FIGS. 20A and 20B, naïve CD4⁺ T cells were activated with anti-CD3 and anti-CD28 under T_H1-(IL-12+anti-IL-4), T_H17-(TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and T_H-GM-(GM-CSF⁺ T_H, IL-7+anti-IFN-γ+anti-IL-4) polarizing conditions. GM-CSF and IL-3 expression was analyzed by intracellular staining (FIG. 20A). The mRNA expression of IL-3, EBI-3, PENK or RANKL cytokines was measured by RT-PCR (FIG. 20B). Frequency of IL-3+ cells differentiated without or with IL-7 (FIG. 20C). GM-CSF and IL-3 expression by WT or STAT5-deficient GM-CSF-producing TH cells (FIG. 20D).

FIG. 21 depicts clinical EAE scores of Rag2^−/− mice (n=3˜6 mice per group) after adoptive transfer of 6×10⁵ various MOG_35-55-specific T_H subsets.

FIGS. 22A-27E depict inhibition of STAT5 activation suppresses T_H-GM cell differentiation in vitro. CD4⁺ T cells were pre-incubated with STAT5 inhibitor (Calbiochem) (FIG. 22A) or JAK3 inhibitor (FIG. 22B) at indicated concentrations or vehicle (−) for 1 h before stimulation with IL-7 for 30 min. Activation (Tyr694 phosphorylation) of STAT5 was determined by immunoblotting. CD4⁺ T cells were pre-incubated with STAT5 inhibitor at indicated concentrations or vehicle (−) for 1 h before stimulation with IL-6 for 30 min. Activation (Tyr705 phosphorylation) of STAT3 was determined by immunoblotting (FIG. 22C). In FIG. 22D, CD4⁺ T cells were pre-incubated with STAT5 inhibitor at indicated concentrations or vehicle (−) for 1 h before stimulation with IFN-γ for 30 min. Activation (Tyr701 phosphorylation) of STAT1 was determined by immunoblotting. In FIG. 22E, naïve CD4⁺ T cells were isolated and activated under neutral condition or T_H-GM cell-favoring condition with the addition of different concentrations of STAT5 inhibitor for 3 days. GM-CSF and IFN-γ expression was analyzed by intracellular cytokine staining and flow cytometry.

FIGS. 23A-23D depict targeting STAT5 activation by chemical inhibitor ameliorates EAE. (FIG. 23A) Clinical EAE scores of wild-type control mice (n=5) or administrated with STAT5 inhibitor (Calbiochem). Arrow indicates the treatment points. (FIG. 23B) Histology of spinal cords at day 18 of EAE mice receiving different treatments. (FIG. 23C) Intracellular staining and flow cytometry of CNS-infiltrating CD4⁺ T cells at peak of disease. (FIG. 23D) Whole CNS was harvest for RNA extraction. GM-CSF mRNA expression was analyzed by RT-PCR. Data represent two independent experiments. *p<0.05.

FIGS. 24A-24E depict GM-CSF-producing T_H cells are in association with human RA. Plasma concentrations of GM-CSF and TNF-α in healthy control HC (n=32) and RA (n=47) were quantified by ELISA (FIG. 24A). In FIGS. 24B and 24C, peripheral blood mononuclear cells (PBMCs) were collected from healthy control (HC) and rheumatoid arthritis (RA) patients, and were stimulated for 4 h with PMA/Ionomycin in the presence of Golgiplug, followed by intracellular cytokine staining. Representative flow cytometry of GM-CSF, IFN-γ and IL-17 in CD4⁺ T cells (FIG. 24B) and statistics of n>=9 per group (FIG. 24C) are shown. FIG. 24D shows the correlation between the frequency of GM-CSF⁺IFN-γ⁻ T_H cells and the level of plasma GM-CSF in peripheral blood of RA patients (n=18). Cytokine expression by CD4⁺ T cells derived from synovial fluid of RA patients was analyzed by intracellular cytokine staining and flow cytometry (FIG. 24E). A representative image of three cases was shown. *p<0.05, **p<0.01, ***p<0.001; ns, not significant.

FIGS. 25A-25E depict distinguishable effects of GM-CSF and TNF-α in mouse AIA. FIG. 25A shows knee joint swelling of wild-type mice over 7 days after intraarticular injection of 100 μg mBSA in AIA model, receiving treatment with control IgG, GM-CSF-specific and TNF-α-specific neutralizing antibodies separately or in combination (n=5 per group) at indicated times (arrows). FIG. 25B shows knee joint swelling of Stat5^+/+ and Stat5^−/− mice (n=6 per group) over 7 days after arthritis induction. Data are representative of more than three independent experiments. Representative images of joint sections stained with H&E (FIG. 25C) or Safranin-O/Fast Green (FIG. 25D) at day 7 after arthritis induction as in FIG. 25C. Bars, 500 μm (FIG. 25C upper panels and FIG. 25D) or 100 μm (FIG. 25C lower panels). Arrow in upper panels (FIG. 25C) indicated bone destruction. In FIG. 25E, serum concentrations of GM-CSF, IFN-γ and TNF-α in Stat5^+/+ and Stat5−/− AIA mice were quantified by ELISA. Statistics of n>=8 per group were shown. *p<0.05, **p<0.01, ***p<0.001.

FIGS. 26A-26D depicts mice with Stat5 deletion in T cells are resistant to CIA. (FIG. 26A) Representative images of paw swelling of Stat5^+/+ and Stat5^−/− mice at day 40 after collagen II/CFA immunization in CIA model. (FIG. 26B) Clinical score of Stat5^+/+ and Stat5^−/− mice (n=5 per group) over 40 days after collagen II/CFA immunization. Data are representative of two independent experiments. (FIG. 26C) Representative images of paw sections stained with H&E at day 40. (FIG. 26D) Serum concentrations of TNF-α (n=8 per group) were quantified by ELISA. *p<0.05, **p<0.01, ***p<0.001.

FIGS. 27A-27E depicts STAT5-deficient CD4⁺ T cells are defective in arthritogenic potential. (FIGS. 27A and 27B) Representative flow cytometry of CD4⁺ T cells in spleens (FIG. 27A) and inguinal lymph nodes (FIG. 27B) of Stat5^+/+ and Stat5^−/− mice at day 7 after AIA induction. (FIGS. 27C and 27D) Synovial tissues were harvested from Stat5^+/+ and Stat5^−/− mice at day 7 after AIA induction, and dissociated into single cells. Cell numbers of CD45⁺ leukocytes were calculated (FIG. 27C). The percentages of CD4⁺ T cells among CD45⁺ fraction were analyzed by flow cytometry, and cell numbers were calculated (FIG. 27D). (FIG. 27E) Histological analysis of joint sections from wild-type naïve mice at day 7 after being transferred with in vitro-expanded antigen-reactive CD4⁺ T cells and followed with intraarticular injection of mBSA. Bars, 100 μm. Data represent two independent experiments. *p<0.05; ns, not significant.

FIGS. 28A-28G depicts STAT5-regulated GM-CSF-producing T_H cells are crucial for AIA. Spleens and synovial tissues were collected from Stat5^+/+ and Stat5^−/− mice at day 7 after arthritis induction. (FIG. 28A) Splenic fractions of wild-type AIA mice (n=3) were stimulated under indicated conditions for 18 h. GM-CSF levels in supernatant were quantified by ELISA. (FIGS. 28B-28D) Intracellular staining and flow cytometry of GM-CSF, IL-17 and IFN-γ in splenic CD4⁺CD44^hi effector T cells (FIG. 28B) or in synovial infiltrating CD4⁺ T cells (FIGS. 28C and 28D) after restimulation for 4 h with PMA/Ionomycin in the presence of Golgiplug. Representative images and statistics of n=5 (FIG. 28B, right panels) or n=3 (FIG. 28D, right panels) per group were shown. Data represent two independent experiments. (FIG. 28E) Protein expression of several proinflammatory cytokines in synovial tissues was measured by ELISA. (FIGS. 28F and 28G) Representative images of joint sections stained with H&E (FIG. 28F) or Safranin-O/Fast Green (FIG. 28G) at day 7 after intraarticular injection of mBSA alone to the right knee joints and mBSA supplemented with GM-CSF to the left knee joints. Bars, 500, 50 or 200 μm as indicated. Data represent two independent experiments.*p<0.05, **p<0.01, p<0.05, **p<0.01, ***p<0.001; ns, not significant. “Splenocytes” represent the left-most bars in each group, “splenocytes depleted of CD4+ T cells” represent the middle bars in each group, and “CD4+ T cells” represent the right-most bars in each group.

FIGS. 29A-29C depicts loss of STAT5 results in impaired GM-CSF production by antigen-specific CD4⁺ T cells. Spleens and inguinal LNs were collected from Stat5^+/+ and Stat5^−/− mice at day 7 after arthritis induction, and dissociated into single cell suspensions. Then, cells were stimulated with mBSA (20 μg/ml) for 24 h. (FIG. 29A) Golgiplug was added in the last 4 h of culture, followed by intracellular staining and flow cytometry. Representative plots of GM-CSF, IL-17 and IFN-γ expression in CD4⁺CD44^hi effector T cells was shown, representing two independent experiments. (FIGS. 29B and 29C) Secreted cytokines in the supernatant (n=3 per group) were quantified by ELISA. Data represent two independent experiments. *p<0.05; ns, not significant.

FIGS. 30A-30C depicts loss of STAT5 impairs IL-6 and IL-1β expression in synovial tissues of arthritic mice. (FIGS. 30A-30C) The mRNA (FIGS. 30A and 30 C) and protein (FIG. 30B) expression of several proinflammatory cytokines in synovial tissues of Stat5^+/+ and Stat5^−/− mice (n>=3 per group) at day 5 or 7 after arthritis induction was measured by qPCR and ELISA. The qPCR data were normalized to Rn18S.

FIGS. 31A-31D depicts SAT5-induced GM-CSF expression mediates CD11b⁺ cell accumulation in inflamed synovial tissues. (FIG. 31A) The frequencies of CD11b⁺ cells in spleens of Stat5^+/+ and Stat5^−/− AIA mice were analyzed by flow cytometry. Statistics of n=3 per group (right panel) were shown. (FIG. 31B) Synovial tissues were harvested from Stat5^+/+ and Stat5^−/− mice at day 7 after arthritis induction, and dissociated into single cell suspensions. The percentage of CD11b⁺ myeloid cells among CD45⁺ fraction was analyzed by flow cytometry. Statistics of n=5 per group were shown in right panel. (FIG. 31C) Representative flow cytometry of CD11b⁺ and CD4⁺ cells gated on synovial CD45⁺ fraction over 7 days after arthritis induction. (FIG. 31D) Flow cytometric analysis of CD4⁺, CD11b⁺ and B220⁺ cell infiltration in synovial tissues of Stat5^+/+ and Stat5^−/− mice at day 7 after intraarticular injection of mBSA alone to the right knee joints and mBSA supplemented with GM-CSF to the left knee joints. Representative images were shown. All data shown are representative of two independent experiments. **p<0.01; ns, not significant.

FIGS. 32A-32D depicts GM-CSF mediates neutrophil accumulation in arthritic mice. (FIG. 32A) Flow cytometric analysis of Ly6C and Ly6G expression gated on synovial CD45⁺CD11b⁺ fraction over 7 days after arthritis induction. (FIG. 32B) Giemsa stain of sorted Ly6C^hiLy6G⁻ and Ly6C^loLy6G^hi cells from synovial tissues of AIA mice. Scale bar, 100 μm (left) or 20 μm (right). (FIG. 32C) Flow cytometric analysis of Ly6C^hiLy6G⁻ and Ly6C^loLy6G^hi populations in synovial tissues of Stat5^+/+ and Stat5^−/− mice at day 7 after intraarticular injection of mBSA alone to the right knee joints and mBSA supplemented with GM-CSF to the left knee joints. (FIG. 32D) Knee joint swelling of wild-type mice treated with Ly6G-specific neutralizing antibody (1A8) or IgG control (n=5 per group) over 3 days after intraarticular injection of mBSA in AIA model. Arrows indicate time points of antibody administration. *p<0.05.

FIGS. 33A-33C depicts GM-CSF enhances neutrophil transmigration and delay apoptosis in vitro. (FIG. 33A) Percentages of migrated neutrophils with or without GM-CSF as chemoattractant in transmigration assay at 3 h post start. (FIG. 33B) Microscopic images of CFSE-labeled neutrophils in the bottom of the lower chamber in transmigration assay. (FIG. 33C) Sorted neutrophils were cultured in vitro with or without GM-CSF (20 ng/ml) for 24 h. Neutrophils undergoing apoptosis were examined by Annexin V and propidium iodide (PI) co-staining. A representative flow cytometry of three repeats was shown. *p<0.05.

FIGS. 34A-34I depicts GM-CSF mediates proinflammatory cytokine expression by myeloid cells and synovial fibroblasts in arthritic mice. Synovial tissues were dissected from wild-type AIA mice and dissociated into single cell suspensions. (FIG. 34A) Flow cytometry plots depicting the fractionation into different populations based on differential expression of surface markers. (FIG. 34B) The mRNA expression of several proinflammatory cytokines in sorted CD45⁺ TCRβ⁺ (TCRβ⁺ in short), CD45⁺ TCRβ⁻CD11c⁻CD11b⁺ (CD11b⁺) and CD45⁺ TCRβ⁻CD11c⁺ (CD11c⁺) populations was measured by qPCR. The qPCR data were normalized to GAPDH. (FIG. 34C) The mRNA expression of IL-6, IL-1β and TNF-α in sorted Ly6C^hiLy6G⁻ and Ly6C^loLy6G^hi populations (gated on CD11b⁺ cells). The qPCR data were normalized to GAPDH. (FIGS. 34D and 34E) The mRNA expression of IL-6 and IL-1β by BMDMs (FIG. 34D) and BMDCs (FIG. 34E) upon stimulation with 20 ng/ml GM-CSF for 1 h. The qPCR data were normalized to GAPDH. (FIGS. 34F and 34G) BMDMs (FIG. 34F) and BMDCs (FIG. 34G) were stimulated with GM-CSF at indicated concentrations (n=3 per group) for 18 h. The secretion of IL-6 in the culture supernatant was quantified by ELISA. (FIG. 34H) BMDMs were primed with LPS (100 μg/ml) in the presence of GM-CSF at indicated concentrations (n=3 per group) for 6 h, followed by stimulation with ATP (5 mM) for 30 min. The secretion of IL-1β in the culture supernatant was quantified by ELISA. (FIG. 34I) Cells were cultured in DMEM medium supplemented with 10% FBS for over 20 days with more than 5 passages to obtain synovial fibroblasts. Synovial fibroblasts were stimulated with GM-CSF (20 ng/ml) for 1 h and harvested for RNA extraction. The mRNA expression of IL-1β was measured by qPCR. The qPCR data were normalized to GAPDH. All data shown represent two independent experiments. *p<0.05, **p<0.01.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

The present disclosure relates, in part, to the identification of a granulocyte macrophage colony stimulating factor (GM-CSF)-secreting T helper cell, termed “T_H-GM”. As detailed herein, IL-7/STAT5 signaling programs the differentiation of precursor CD4+ cells to T_H-GM, a process which is further modulated by IL-2 and IL-23 signaling. T_H-GM cells are characterized by, e.g., GM-CSF and IL-3 production. T_H-GM cells are distinct from the known helper T cells T_H1 and T_H17, with respect to, e.g., differentiation conditions, transcriptional regulation and effector cytokine expression. For example, IL-12/IFN-γ and TGF-β/IL-6, which mediate (e.g., promote the development of) T_H1 and T_H17, respectively, potently suppress the development of T_H-GM from naïve CD4⁺ precursor cells, establishing that T_H-GM cells develop via a lineage distinct from T_H1 and T_H17. Thus, the present disclosure provides a distinct network of factors, unique from factors known to mediate T_H1 or T_H17, that mediate T_H-GM function (e.g., its differentiation and pathogenicity).

As shown herein, T_H-GM cells preferentially induce EAE as compared with T_H1 and T_H17 cells, indicating that T_H-GM cells represent the primary effectors in the pathogenesis of autoimmune neuroinflammation in humans. Moreover, blockade of IL-7 signaling and/or inhibition of STAT5 function (e.g., abrogation of expression or inhibition of STAT5 activity) attenuates autoimmune neuroinflammation associated with diminished GM-CSF production by T_H-GM cells. Further, blockade of T_H-GM cell-secreted GM-CSF ameliorates experimental arthritis in a TNF-α-independent manner, indicating an approach for the treatment of, e.g., rheumatoid arthritis patients who are unresponsive to TNF-α antagonistic drugs. Thus, the present disclosure enables one to distinguish between an inflammatory disorder (e.g., RA) that is mediated by the T_H-GM pathway (e.g., a disorder that results from T_H-GM pathogenicity through the action of, e.g., GM-CSF and/or IL-3, or any factor associated with the T_H-GM pathway), or an inflammatory disorder that is mediated by, e.g., TNF-α, IL-6, and/or IL-1β pathways (i.e., non-T_H-GM-mediated pathway). For example, a patient who has, e.g., RA may be afflicted with a type of RA that is primarily T_H-GM-mediated, or primarily non-T_H-GM-mediated (e.g., TNF-α-mediated or IL-6 mediated). The present disclosure enables the classification between T_H-GM-mediated and non-T_H-GM-mediated inflammation, allowing for a more precise diagnosis, prognosis, and treatment in an individual who is afflicted with an inflammatory disorder such as RA or MS.

As demonstrated herein, the present disclosure identifies a helper T cell subset (T-_H-GM), provides the molecular basis for the commitment and development of this subset from naïve precursor cells in vitro and in vivo, and demonstrates T_H-GM cells as the primary pathogenic cells in autoimmune diseases and inflammatory disorders, for example, MS and RA. Thus, provided herein are compositions and methods for diagnosing inflammatory conditions primarily mediated by T_H-GM cells, thereby enabling the identification of, e.g., RA patients who are non-responsive to TNF-α therapy (e.g., TNF-α inhibitor based therapy), as well as compositions and methods for modulating T_H-GM function to treat autoimmune and inflammatory disorders. The methods of modulating T_H-GM function include, e.g., administering agents to modulate the function (e.g., signaling, expression or activity) of the network of factors (e.g., IL-2/IL-7/STAT5/GM-CSF/IL-3) that mediate T_H-GM function in an effective amount to modulate the function (e.g., development and pathogenicity) of T_H-GM cells. In particular, the disclosure provides methods and composition for differentiating and diagnosing an inflammatory disorder, e.g., multiple sclerosis (MS), rheumatoid arthritis (RA) as primarily mediated by either T_H-GM cells (i.e., T_H-GM pathway mediated) or by non-T_H-GM mechanism (e.g., TNF-α, IL-6, and/or IL-1β pathways), or both. Also provided herein are compositions and methods for and the treatment of inflammatory disorders, particularly those that are T_H-GM-mediated.

Accordingly, in one aspect, the present disclosure provides a method of diagnosing a T_H-GM-mediated inflammatory disorder in a patient suffering from an inflammatory disorder. In some embodiments, the method comprises contacting a sample collected from a patient suffering from an inflammatory disorder with a detecting agent that detects a polypeptide or nucleic acid level of a T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof; and quantifying the polypeptide or nucleic acid level of the T_H-GM-mediating factor (e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof), wherein an increased level of a T_H-GM-mediating factor (e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof) relative to a reference level indicates that the patient suffers from a T_H-GM-mediated inflammatory disorder, thereby diagnosing a T_H-GM-mediated inflammatory disorder in a patient suffering from an inflammatory disorder.

As used herein, a “T_H-GM-mediated” inflammatory disorder refers to a subtype of an inflammatory disorder (e.g., a subtype of RA or MS) that results from the physiological action of any one or more of the network of factors in the pathway that modulate T_H-GM function (a “T_H-GM-mediating factor”), as described herein. Such factors include, e.g., GM-CSF, activated STAT5, IL-7, IL-2, and IL-3. In a particular embodiment, STAT5 is activated STAT5, wherein tyrosine at position 694 is phosphorylated.

In some embodiments, the level of a T_H-GM-mediating factor (e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof) that is not increased relative to a reference level indicates that the patient suffers from a non-T_H-GM-mediated inflammatory disorder.

In certain embodiments, the method further comprises administering to the patient a TNF-α therapy, as described herein, if the level of a T_H-GM-mediating factor (e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof) is not increased relative to a reference level.

As used herein, a “non-T_H-GM-mediated” inflammatory disorder refers to an inflammatory disorder (e.g., RA or MS) that is primarily caused by, e.g., TNF-α, IL-6, or IL-1β (and/or factors in the TNF-α, IL-6, or IL-1β pathway). As such, a “T_H-GM-mediated” inflammatory disorder results primarily (or exclusively) from a pathway that is distinct from one or more of the pathways that leads to a “non-T_H-GM-mediated” inflammatory disorder (e.g., the pathways associated with TNF-α, IL-6, or IL-1β).

However, as those of skill in the art would appreciate, a T_H-GM-mediated inflammatory disorder does not necessarily exclude the possibility that the inflammatory disorder could also be partially non-T_H-GM-mediated (e.g., mediated by TNF-α, IL-6, or IL-1β, and/or factors in the TNF-α, IL-6, or IL-1β pathway). Thus, a classification or diagnosis as “T_H-GM-mediated” is synonymous with “primarily/predominantly T_H-GM-mediated”, and a classification as “non-T_H-GM-mediated” is synonymous with “primarily/predominantly non-T_H-GM-mediated.” For example, without wishing to be bound by any particular theory, an inflammatory disorder in its early stage may be T_H-GM-mediated. As the inflammatory condition advances to a late stage characterized by, e.g., tissue damage, the inflammatory disorder becomes progressively non-T_H-GM-mediated. In some embodiments, a T_H-GM-mediated inflammatory disorder is a condition that is responsive to modulation of T_H-GM function, as determined by clinical standards; a non-T_H-GM-mediated inflammatory disorder is a condition that is responsive to, e.g., TNF-α, IL-6, or IL-1β therapy, as determined by clinical standards. In certain embodiments, an inflammatory disorder can be responsive to modulation of T_H-GM function as well as TNF-α, IL-6, and/or IL-1β therapy.

In some embodiments, the sample can be e.g., peripheral blood, cerebrospinal fluid, synovial fluid, or synovial membrane, or a combination thereof.

In some embodiments, the inflammatory disorder is an autoimmune disorder. In certain embodiments, the inflammatory disorder can be any inflammatory disorder mediated by T_H-GM cells, and includes, but is not limited to rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, Crohn's disease, diabetes, Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism, Irritable Bowel Syndrome (IBS), lupus erythematosus, polymyalgia rheumatic, psoriasis, psoriatic arthritis, Raynaud's syndrome/phenomenon, reactive arthritis (Reiter syndrome), sarcoidosis, scleroderma, Sjögren's syndrome, ulcerative colitis, uveitis, or vasculitis.

As used herein, a “detecting agent” refers to, e.g., an antibody, a peptide, a small molecule, or a nucleic acid that binds to a polypeptide or nucleic acid to be detected (e.g., STAT5 (e.g., phospho-STAT5 (Tyr694)), IL-7, GM-CSF or IL-3), and enables the quantification of the polypeptide or nucleic acid to be detected. The detecting agent can be detectably labeled, or quantifiable by other means known in the art.

In some embodiments, the detecting agent is an antibody that binds to the polypeptide of STAT5, IL-7, GM-CSF or IL-3. In one embodiment, the antibody is one that binds to an activated STAT5 (e.g., phosphorylated STAT5), as described herein. Antibodies to STAT5 (e.g., phospho-STAT5 (Tyr694)), IL-7, GM-CSF or IL-3 suitable for use in the present method are known and commercially available in the art (e.g., STAT5 Ab: C-17 from Santa Cruz Biotech; Phospho-STAT5 (Tyr694) Ab: #9351 or #9359 from Cell Signaling; IL-7 Ab: clone BVD10-40F6 from BD Pharmingen; IL-7R Ab: clone SB/14 from BD Pharmingen; GM-CSF Ab: clone MP1-22E9 from BD Pharmingen; IL-3 Ab: clone MP2-8F8 from BD Pharmingen.

In other embodiments, the detecting agent is a nucleic acid that binds to the nucleic acid of STAT5, IL-7, GM-CSF and/or IL-3. Nucleic acid molecules encoding a, e.g., STAT5, IL-7, GM-CSF and/or IL-3 sequence, or fragments or oligonucleotides thereof, that hybridize to a nucleic acid molecule encoding a e.g., STAT5, IL-7, GM-CSF and/or IL-3 polypeptide sequence at high stringency may be used as a probe to monitor expression of nucleic acid levels of STAT5, IL-7, GM-CSF and/or IL-3 in a sample for use in the diagnostic methods of the disclosure. Methods of quantifying nucleic acid levels are routine and available in the art.

In some embodiments, the method further comprises contacting the sample with a detecting agent that detects a polypeptide or nucleic acid level of one or more genes (as well as the gene product) listed in Table 1. As described herein, Table 1 lists genes that are differentially expressed in T_H-GM cells as well as genes that are differentially expressed on the surface of T_H-GM cells, as compared to T_H1 or T_H17 cells.

In a particular embodiment, the method further comprises contacting the sample with a detecting agent that detects the polypeptide or nucleic acid level of basic helix-loop-helix family member e40 (BHLHe40), chemokine (C-C Motif) Receptor 4 (CCR4), and/or CCR6.

Standard methods may be used to quantify polypeptide levels in any sample. Such methods include, e.g., ELISA, Western blotting, immunohistochemistry, fluorescence activated cells sorting (FACS) using antibodies directed to a polypeptide, and quantitative enzyme immunoassay techniques known in the art. Such methods are routine and available in the art. Similarly, methods for quantifying nucleic acid levels (e.g., mRNA) are known in the art.

In the diagnostic method of the present disclosure, an increased level of STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF and/or IL-3 relative to a reference level indicates that the patient suffers from a T_H-GM-mediated inflammatory disorder.

In some embodiments, a STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF and/or IL-3 level that is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, or at least 600% relative to a reference level indicates that the patient suffers from a T_H-GM-mediated inflammatory disorder. In a particular embodiment, an increase of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 150% relative to a reference level indicates that the patient suffers from a T_H-GM-mediated inflammatory disorder.

In some embodiments, a STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF and/or IL-3 level that is not increased by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 150% relative to a reference level indicates that the patient suffers from a non-T_H-GM-mediated inflammatory disorder.

In certain embodiments, a STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF and/or IL-3 level that is comparable (or unchanged) relative to a reference level indicates that the patient suffers from a non-T_H-GM-mediated disorder. As used herein, a level that is “comparable” to that of a reference level refers to a level that is unchanged, or a change relative to the reference level that is statistically insignificant according to clinical standards. In certain embodiments, a comparable level (or unchanged level) can include a level that is not increased by at least 40%, at least 50%, at least 60%, or at least 70% relative to a reference level as, for example, it may not indicate a clinically significant change. In some embodiments, a level of a T_H-GM-mediating factor (e.g., STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF, and/or IL-3) that is decreased relative to a reference level can also indicate that the patient suffers from a non-T_H-GM-mediated disorder.

In some embodiments, the reference level is a level that is used for comparison purposes, and may be obtained from, for example, a prior sample taken from the same patient; a normal healthy subject; a sample from a subject not having an autoimmune disease or an inflammatory disorder; a subject that is diagnosed with a propensity to develop an autoimmune disease but does not yet show symptoms of the disease; a patient that has been treated for an autoimmune disease; or a sample of a purified reference polypeptide or nucleic acid molecule of the disclosure (e.g., STAT5) at a known normal concentration. By “reference standard or level” is meant a value or number derived from a reference sample, or a value or range accepted in the art as indicative of being healthy (e.g., an individual that does not have an inflammatory disorder). A normal reference standard or level can also be a value or number derived from a normal subject who does not have an autoimmune disease. In one embodiment, the reference sample, standard, or level is matched to the sample subject by at least one of the following criteria: age, weight, body mass index (BMI), disease stage, and overall health. A standard curve of levels of purified DNA, RNA or mRNA within the normal reference range can also be used as a reference. A standard curve of levels of purified protein within the normal reference range can also be used as a reference.

In some embodiments, the patient afflicted with an inflammatory disorder who has been diagnosed or classified as having a T_H-GM-mediated inflammatory disorder does not have a non-T_H-GM-mediated inflammatory disorder (i.e., does not have a TNF-α, IL-6, or IL-1β-mediated inflammatory disorder). That is, the patient diagnosed as suffering from a T_H-GM-mediated inflammatory disorder responds to modulation of T_H-GM function (e.g., inhibition of STAT5, IL-7, GM-CSF and/or IL-3), but does not respond (or exhibits a limited response) to TNF-α therapy, as determined by clinical standards. However, as described herein, a T_H-GM-mediated inflammatory disorder does not exclude the possibility that the inflammatory disorder is also partially (though not primarily) contributed by a non-T_H-GM-mediated pathway (e.g., TNF-α, IL-6, IL-1β).

In some embodiments, the methods of the present disclosure further comprise administering an effective amount of a modulating agent that modulates T_H-GM cell function to the patient diagnosed or classified as having a T_H-GM-mediated inflammatory disorder. As described herein, in some embodiments, the modulating agent inhibits T_H-GM function.

In some embodiments, the methods of the present disclosure further comprise administering an effective amount of, e.g., a TNF-α therapy, an IL-6 therapy, or an IL-1β therapy to a patient diagnosed or classified as having a non-T_H-GM-mediated inflammatory disorder, as described herein.

In some aspects, the present disclosure also provides a method of classifying a patient suffering from an inflammatory disorder as having a T_H-GM-mediated inflammatory disorder or a non-T_H-GM-mediated inflammatory disorder. In some embodiments, the method comprises contacting a sample collected from a patient suffering from an inflammatory disorder with a detecting agent that detects a polypeptide or nucleic acid level of a T_H-GM-mediating factor, such as, e.g., STAT5 (e.g., phosphorylated STAT5, Tyr694), IL-7, GM-CSF or IL-3, or a combination thereof. In certain aspects, the method further comprises quantifying the polypeptide or nucleic acid level of the T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof, wherein an increased level of the T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof relative to a reference level indicates that the patient suffers from a T_H-GM-mediated inflammatory disorder; or a comparable level of the T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof relative to a reference level indicates that the patient suffers from a non-T_H-GM-mediated inflammatory disorder, thereby classifying the patient suffering from an inflammatory disorder as a T_H-GM-mediated inflammatory disorder or a non-T_H-GM-mediated inflammatory disorder.

In other aspects of the present disclosure, the methods disclosed herein can further comprise measuring the polypeptide or nucleic acid level of a factor that mediates a non-T_H-GM-mediated inflammatory disorder. Such factors include, e.g., TNF-α, IL-6, and IL-1β.

For example, in some aspects, the present disclosure provides a method of determining a treatment regimen in a patient suffering from an inflammatory disorder. To illustrate, the method comprises quantifying a polypeptide or nucleic acid level of, e.g., activated STAT5 or GM-CSF in a sample collected from a patient suffering from an inflammatory disorder, and quantifying the polypeptide or nucleic acid level of, e.g., TNF-α in a sample collected from the patient. At least four scenarios can be considered.

In the first scenario, if the activated STAT5 or GM-CSF level is increased (e.g., by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 150%) relative to a first reference level and the TNF-α level is comparable to a second reference level, then the patient is classified as having a T_H-GM-mediated inflammatory disorder and the patient can be treated with an agent that modulates T_H-GM function, as described herein.

In a second scenario, if the activated STAT5 or GM-CSF level is comparable to the first reference level and the TNF-α level is increased (e.g., by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or at least 150%) relative to the second reference level, then the patient is classified as having a non-T_H-GM-mediated inflammatory disorder and the patient can be treated with, e.g., a TNF-α therapy.

In a third scenario, if the activated STAT5 or GM-CSF level is increased relative to the first reference level and the TNF-α level is also increased relative to the second reference level, and the increase is equivalent within clinical and/or statistical standards (e.g. both GM-CSF and TNF-α are at least 50% increased relative to the respective reference levels), then the patient is classified as having an inflammatory disorder that is equally T_H-GM-mediated and non-T_H-GM mediated (e.g., TNF-α-mediated). In such a case, the patient can be treated with an effective amount of an agent that modulates T_H-GM function and an effective amount of, e.g., a TNF-α therapy. As demonstrated herein, the combination of both agents can have a synergistic effect.

In a fourth scenario, if the activated STAT5 or GM-CSF level is increased relative to the first reference level and the TNF-α level is also increased relative to the second reference level, but one is increased more than the other, then the inflammatory disorder is primarily mediated by the pathway that shows a greater increase. For example, if GM-CSF is increased by 40% relative to a reference level, and TNF-α is increased by 90% relative to a reference level, then the inflammatory disorder is primarily non-T_H-GM-mediated. However, in this scenario, the patient may receive a combined treatment with an agent that modulates T_H-GM function as well a TNF-α therapy (e.g., anti-TNF-α therapy), since GM-CSF is increased by, e.g., at least 40% relative to a reference level.

In some embodiments, the first and second reference levels are obtained from the same reference sample.

In a related aspect, the disclosure also provides a method of tailoring the treatment of a patient suffering from an inflammatory disorder according to the progression of a patient's inflammatory disorder. In the above illustrative example, the first scenario (increased T_H-GM-mediating factor, e.g. STAT5 or GM-CSF but TNF-α level is comparable to a reference level) may indicate that the patient is in an early stage of an inflammatory disorder. Without wishing to be bound by any particular theory, during, for example, the early stages of an inflammatory disorder, naïve T cells are stimulated by antigen and programmed by IL-7/STAT5 to differentiate into GM-CSF/IL-3 producing T_H-GM cells. During, for example, the late stages of an inflammatory disorder, T_H-GM cytokines (e, g, IL-3 and GM-CSF) progressively stimulate more inflammatory cells such as macrophages and neutrophils resulting in the production of, e.g., TNF-α, IL-6, IL-1β, resulting in full-scale inflammation. Thus, in the above illustrative example, the second scenario (activated STAT5 or GM-CSF level is comparable to the first reference level and the TNF-α level is increased) may indicate that the patient is in a late stage of an inflammatory disorder characterized by, e.g., tissue damage. Accordingly, the present disclosure enables the prognosis of a patient depending on the quantifiable level of one or more T_H-GM-mediating factor (e.g., STAT5 (e.g., activated phospho-STAT5 (Tyr694)), IL-7, GM-CSF, and/or IL-3) and one or more non-T_H-GM-mediating factor (e.g., TNF-α, IL-6, IL-1β), thereby tailoring the treatment according to the progression of the disease. Accordingly, as would be appreciated by those of skill in the art, a patient suffering from an inflammatory disorder can be monitored for disease progression to ensure effective and tailored treatment according to the level of one or more T_H-GM-mediating factor, as described herein, and one or more non-T_H-GM-mediating factor (e.g., TNF-α, IL-6, IL-1β).

In related aspects, the present disclosure also provides a method of prognosing progression of an inflammatory disorder in a patient in need thereof. In some embodiments, the method comprises a) quantifying a polypeptide or nucleic acid level of a T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof, in a first sample collected from a patient suffering from an inflammatory disorder, and b) quantifying a polypeptide or nucleic acid level of, e.g., TNF-α, IL-6, or IL-1β, or a combination thereof, in a second sample collected from the patient, wherein i) an increased level of the T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof relative to a first reference level and an unchanged level of TNF-α, IL-6, or IL-1β, or a combination thereof relative to a second reference level indicates that the patient is in an early stage of the inflammatory disorder, as described herein; or ii) an unchanged level of the T_H-GM-mediating factor, such as, e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof relative to the first reference level and an increased level of TNF-α, IL-6, or IL-1β, or a combination thereof relative to the second reference level indicates that the patient is in a late stage of the inflammatory disorder, as described herein. In some embodiments, the method further comprises administering an effective amount of an agent that modulates T_H-GM function and/or, e.g., a TNF-α therapy, as described herein.

In some embodiments, the first sample and the second sample are the same.

In various aspects, the present disclosure also provides an isolated population of GM-CSF-secreting T-helper cells (T_H-GM). In one embodiment, the T_H-GM cells are differentiated from a precursor cell (e.g., CD4+ cells) in the presence of signal transducer and activator of transcription 5 (STAT5) and/or IL-7, and wherein the T_H-GM cells express GM-CSF and IL-3.

In some embodiments, the T_H-GM cells are differentiated from a precursor cell (e.g., CD4+ cells) in the presence of an agent that inhibits IL-12, IFN-γ, TGF-β, and/or IL-6. Similarly, the differentiation of a precursor cell (e.g., CD4+ precursor cell) into a T_H-GM cell is inhibited by IL-12, IFN-γ, TFG-β, and/or IL-6.

In some embodiments, the T_H-GM cells are differentiated from a precursor cell in vitro, under artificial conditions, but wherein the T_H-GM cells retain physiological properties as described herein.

In some embodiments, the T_H-GM cells are further characterized by an overexpression of one or more genes listed in Table 1. For example, the T_H-GM cells are further characterized by an overexpression of, for example, basic helix-loop-helix family, member e40 (BHLHe40), preproenkephalin (PENK), IL-2, serine (or cysteine) peptidase inhibitor, clade B member 6b (Serpinb6b), neuritin 1 (Nrn1), stearoyl-Coenzyme A desaturase 1 (Scd1), or phosphotriesterase related C1q-like 3 (Pter), or a combination thereof.

In some embodiments, the T_H-GM cells are further characterized by an underexpression of one or more genes listed in Table 1. For example, the T_H-GM cells are further characterized an underexpression of lymphocyte antigen 6 complex, locus A (Ly6a); CD27; or selectin, lymphocyte (Sell).

As described herein, the identification of a distinct network of factors (unique from factors known to mediate T_H1 or T_H17) that mediate T_H-GM function (e.g., its differentiation and pathogenicity) enables targeted modulation of T_H-GM function to treat T_H-GM-mediated disorders, e.g., disorders that result from aberrant T_H-GM function. Thus, in some aspects, the present disclosure provides a method of modulating T_H-GM function, comprising contacting the T_H-GM, or cluster of differentiation 4 (CD4+) precursor cells, or both, with a modulating agent that modulates T_H-GM function. In one embodiment, the modulating agent is contacted with the T_H-GM cells or CD4+ precursor cells in vitro or in vivo.

As used herein, “T_H-GM function” refers to the commitment, development, maintenance, survival, proliferation, or activity, or a combination thereof, of T_H-GM cells. Thus, an agent that modulates (e.g., enhances or inhibits) T_H-GM function is one that modulates T_H-GM commitment, development, survival, proliferation, or activity, or combination thereof, of T_H-GM cells. For example, T_H-GM function can be modulated by modulating its: commitment from a CD4⁺ precursor T cell; development of a CD4⁺ precursor cell that has been committed to the T_H-GM developmental pathway; maintenance of a T_H-GM phenotype; survival or proliferation under development or effector T_H-GM cells; and/or activity of effector T_H-GM cells (e.g., modulating function of a secreted factor such as GM-CSF or IL-3). For example, a modulation in T_H-GM function includes, but is not limited to, a modulation in: the number of T_H-GM cells; the survival of T_H-GM cells; the proliferation of T_H-GM cells; and/or the activity of T_H-GM cells. The activity of T_H-GM cells herein includes the activity induced by the cytokines, chemokines, growth factors, enzymes and other factors secreted by T_H-GM cells, as described herein, and the activity induced by direct contact with T_H-GM cells.

As used herein, a T helper subset cell “T_H-GM” refers to a cell that, similar to T_H1 and T_H17 cells, differentiates from precursor CD4+ precursor cells, but which commits and develops through a pathway that is mediated by a subset of factors (the T_H-GM-mediating factors) that is distinct and unique from the known subset of factors that commit and develop T_H1 or T_H17 cell subtypes, as described herein. In some embodiments, a T_H-GM cell produces a distinct and unique set of genes (see, e.g., Table 1) and effects pathogenicity through a different mechanism and pathway than the known factors that mediate pathogenicity of T_H1 or T_H17 cell subtypes. For example, a T_H-GM cell commits and develops by IL-7/STAT5 function (its regulators), and effects pathogenicity by GM-CSF/IL-3 (its effectors).

In some aspects, the present disclosure provides a method of treating a T_H-GM-mediated inflammatory disorder in a patient in need thereof, comprising administering to said patient an effective amount of a modulating agent that modulates T_H-GM cell function. In certain embodiments, the patient is previously diagnosed as having a T_H-GM-mediated inflammatory disorder, as described herein.

In some aspects, the present disclosure also provides a method of treating rheumatoid arthritis in a patient who exhibits limited response to TNF-α therapy, comprising administering to said patient an effective amount of a modulating agent that modulates T_H-GM function.

As used herein, “limited response” refers to no response or insignificant response such that a patient is not treated by the therapy, as determined by clinical standards.

“Treatment” or “treating” refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to reduce the extent of or likelihood of occurrence of the condition or event in the instance where the patient is afflicted. It also refers to reduction in the severity of one or more symptoms associated with the disease or condition. In the present application, it may refer to amelioration of one or more of the following: pain, swelling, redness or inflammation associated with an inflammatory condition or an autoimmune disease. As used herein, and as well-understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, and/or amelioration or palliation of the disease state. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

An “effective amount” of an agent is that amount sufficient to effect beneficial or desired results, including clinical results. An “effective amount” depends upon the context in which it is being applied. In the context of administering a composition that modulates an autoimmune response, an effective amount of an agent which is a modulator of T_H-GM function is an amount sufficient to achieve such a modulation as compared to the response obtained when there is no agent administered. An effective amount can confer immediate, short term or long term benefits of disease modification, such as suppression and/or inhibition of T_H-GM function, as defined herein. An effective amount can be administered in one or more administrations. An “effective amount” as used herein, is intended to mean an amount sufficient to reduce by at least 10%, at least 25%, at least 50%, at least 75%, or an amount that is sufficient to cause an improvement in one or more clinically significant symptoms in the patient.

In some embodiments, the modulating agent inhibits T_H-GM function to, e.g., reduce inflammation. The inhibition conferred by the modulating agent (the inhibitor) does not imply a specific mechanism of biological action. Indeed, the term “antagonist” or “inhibitor” as used herein includes all possible pharmacological, physiological, and biochemical interactions with factors that mediate T_H-GM function (e.g., IL-7, IL-7 receptor, STAT5, GM-CSF, IL-3, IL-2, IL-2 receptor, PENK, RANKL, JAK1/3, or any of the genes that are differentially expressed in T_H-GM cells, e.g., genes in Tables 1 and 2), whether direct or indirect, and includes interaction with a factor (or its active fragment) that mediates T_H-GM function at the protein and/or nucleic acid level, or through another mechanism.

In certain embodiments, a modulating agent that inhibits T_H-GM function includes an antibody, a polypeptide (e.g., a soluble receptor that binds and inhibits, for example, IL-7), a small molecule, a nucleic acid (e.g., antisense, small interfering RNA molecule, short hairpin RNA, microRNA), or a protein (e.g., cytokine), or a combination thereof that prevents the function (e.g., expression and/or activity) of a factor that mediates T_H-GM function. Methods of designing, producing, and using such inhibitors are known and available in the art.

As used herein, “binds” is used interchangeably with “specifically binds,” which means a polypeptide (e.g., a soluble receptor) or antibody which recognizes and binds a polypeptide of the present disclosure, but that does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the present disclosure. In one example, an antibody specifically binds an activated STAT5 polypeptide does not bind a non-STAT5 polypeptide.

As used herein, “antibody” refers to an intact antibody or antigen-binding fragment of an antibody, including an intact antibody or antigen-binding fragment that has been modified or engineered, or that is a human antibody.

In a particular embodiment, the antibody binds to and inhibits the function of any one or more of the factors that mediate T_H-GM function. For example, the antibody binds to and inhibits the function of IL-7, IL-7 receptor (IL-7R), IL-2, IL-2 receptor (IL-2R), STAT5 or janus kinase 1/3 (JAK1/3), or a combination thereof. In other examples, the antibody binds to and inhibits the function of GM-CSF (or its receptor), IL-3, PENK, or RANKL, or a combination thereof. In some embodiments, the antibody binds to and inhibits the function of a gene listed in Table 1. In some embodiments, the antibody binds to and inhibits the protein or any functional fragment thereof. Methods of designing, producing and using suitable antibodies are known and available to those of skill in the art. Examples of antibodies suitable for use in the present disclosure include, e.g., daclizumab, basiliximab, mavrilimumab, MOR103, KB003, namilumab, and MOR Ab-022.

The terms “protein” and “polypeptide” are used interchangeably, and can include full-length polypeptide or functional fragments thereof (e.g., degradation products, alternatively spliced isoforms of the polypeptide, enzymatic cleavage products of the polypeptide), the polypeptide bound to a substrate or ligand, or free (unbound) forms of the polypeptide. The term “functional fragment”, refers to a portion of a full-length protein that retains some or all of the activity (e.g., biological activity, such as the ability to bind a cognate ligand or receptor) of the full-length polypeptide.

In some embodiments, the modulating agent that inhibits T_H-GM function can be a particular biological protein (e.g., cytokines) that inhibits, directly or indirectly, one or more of the factors that mediate T_H-GM function. Such cytokines include, e.g., IL-12, IFN-γ, TGF-β, and IL-6.

In some embodiments, the modulating agent that inhibits T_H-GM function can be a small molecule that inhibits, directly or indirectly, one or more of the factors that mediate T_H-GM function. As used herein a “small molecule” is an organic compound or such a compound complexed with an inorganic compound (e.g., metal) that has biological activity and is not a polymer. A small molecule generally has a molecular weight of less than about 3 kilodaltons. Examples of known small molecules include CAS 285986-31-4 (Calbiochem), pimozide, and tofacitinib.

In other embodiments, the modulating agent enhances T_H-GM function in disorders such as, e.g., viral, fungal and bacterial infections, cancers and/or conditions associated with therewith. In one embodiment, modulating agents that enhance T_H-GM function include, e.g., CD28 activator; IL-7 and/or IL-2 on naïve (precursor) CD4⁺ T cells; activator of STAT5; or effectors of T_H-GM cells (e.g., GM-CSF, IL-3).

In another aspect, the present disclosure provides a method of treating a STAT5-mediated inflammatory disorder in a patient in need thereof, comprising administering to the patient an effective amount of an agent that modulates STAT5 function.

As used herein, “STAT5-mediated” inflammatory disorder refers to an inflammatory disorder that is caused by aberrant STAT5 function (aberrantly enhanced or inhibited), and which is responsive to modulation of STAT5 function, as determined by clinical standards. In some embodiments, the STAT5 is activated STAT5 (e.g., phospho-STAT5, Tyr694).

In some embodiments, the inflammatory disorder is an autoimmune disorder. In certain embodiments, the inflammatory disorder can be any inflammatory disorder mediated by STAT5 (e.g., activated STAT5), and includes, but is not limited to rheumatoid arthritis, multiple sclerosis, ankylosing spondylitis, Crohn's disease, diabetes, Hashimoto's thyroiditis, hyperthyroidism, hypothyroidism, Irritable Bowel Syndrome (IBS), lupus erythematosus, polymyalgia rheumatic, psoriasis, psoriatic arthritis, Raynaud's syndrome/phenomenon, reactive arthritis (Reiter syndrome), sarcoidosis, scleroderma, Sjögren's syndrome, ulcerative colitis, uveitis, or vasculitis.

In some embodiments, the term “patient” refers to a mammal, preferably human, but can also include an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like), and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

In some embodiments, the agent inhibits STAT5 function (e.g., expression and/or activity). Examples of agents that inhibit STAT5 (e.g., activated STAT5, Tyr694) are described herein.

In certain embodiments, the methods of the present disclosure further comprise administering to the patient a TNF-α therapy. In certain embodiments, TNF-α therapy is administered in a patient determined to have an inflammatory condition that is non-T_H-GM-mediated. As described herein, in certain embodiments, a TNF-α therapy is administered if a quantified TNF-α level is increased by, e.g., at least 40% relative to a reference level.

Examples of TNF-α therapy include those that are TNF-α-inhibitor based, and those that are non-TNF-α-inhibitor based. In particular, TNF-α-inhibitor based therapy includes etanercept, adalimumab, infliximab, golimumab, and certolizumab pegol. Examples of non-TNF-α-inhibitor based therapy includes corticosteroid medications (e.g., prednisone), nonsteroidal anti-inflammatory drugs (e.g., methotrexate), and JAK inhibitors (e.g., tofacitinib). Other examples of non-TNF-α-inhibitor based therapy include anakinra, abatacept, rituximab and tocilizumab.

The TNF-α therapy can be administered before, simultaneously with, or after the administration of an effective amount of an agent that modulates T_H-GM function. Accordingly, an agent that modulates T_H-GM function and the TNF-α therapy can be administered together in a single dose, or can be administered in separate doses, e.g., either simultaneously or sequentially, or both. The duration of time between the administration of an agent that modulates T_H-GM function and a TNF-α therapy will depend on the nature of the therapeutic agent(s). In addition, an agent that modulates T_H-GM function and a TNF-α therapy may or may not be administered on similar dosing schedules. For example, the agent that modulates T_H-GM function and the TNF-α therapy may have different half-lives and/or act on different time-scales such that the agent that modulates T_H-GM function is administered with greater frequency than the TNF-α therapy, or vice-versa. The number of days in between administration of therapeutic agents can be appropriately determined by persons of ordinary skill in the art according to the safety and pharmacodynamics of each drug.

The identification of the T_H-GM cells as well as the identification of genes differentially produced by T_H-GM cells relative to T_H1 or T_H17 enables the use of T_H-GM cells to identify novel therapeutics for modulating T_H-GM function, thereby enabling new therapeutics for treating T_H-GM-mediated disorders (e.g., inflammatory disorders). Thus, in further aspects, the present disclosure provides a method of screening to identify a modulator of T_H-GM cell function, comprising contacting an isolated population of T_H-GM cells, or an isolated population of CD4+ precursor cells, with a candidate agent, and measuring a readout of T_H-GM function in the presence or absence of the candidate agent, wherein a change in the readout of T_H-GM function indicates that the candidate agent is a modulator of T_H-GM function.

As used herein, a candidate agent refers to an agent that may modulate T_H-GM function by modulating the function (e.g., expression and/or activity) of a factor that mediates T_H-GM function. Such candidate agents include, e.g., an antibody, a peptide, a small molecule, a nucleic acid (e.g., antisense, small interfering RNA molecule), or a protein (e.g., cytokine), or a combination thereof. A candidate agent can be designed to target any of the factors (at the protein and/or nucleic acid level) that mediate T_H-GM function, as described herein, including the genes listed in Table 1 (e.g., genes preferentially upregulated in T_H-GM cells, genes preferentially overexpressed/underexpressed on the surface of T_H-GM cells).

As used herein, “readout” refers to any change (or lack of change) in T_H-GM function that can be measured or quantified. For example, a candidate agent can be assessed for its effect on, e.g., GM-CSF secretion by T_H-GM cells, or its effect on the abundance of T_H-GM cells (through an effect on the commitment/development/proliferation of T_H-GM cells), as described herein. Assays for determining such readouts are known and available in the art, and are exemplified herein.

In some embodiments, the change in the presence of the candidate agent is a reduction in the measurement of the readout, indicating an inhibition of T_H-GM function (e.g., decrease in GM-CSF or IL-3 production, or decrease in the abundance of T_H-GM cells), thereby identifying the candidate agent as an inhibitor of T_H-GM function.

In certain embodiments, the change in the presence of the candidate agent is an increase in the measurement of the readout, indicating an enhancement of T_H-GM function (e.g., increase in GM-CSF or IL-3 production, or increase in the abundance of T_H-GM cells), thereby identifying the candidate agent as an enhancer of T_H-GM function.

In some embodiments, the readout can be any one or more of the genes listed in Tables 1 and 2 which are preferentially upregulated or downregulated in T_H-GM cells. Thus, a candidate agent that downregulates a gene that is preferentially upregulated in a T_H-GM cell is a inhibitor of T_H-GM function. Similarly, a candidate agent that upregulates a gene that is preferentially downregulated in a T_H-GM cell is an enhancer of T_H-GM function.

In certain aspects, the method of screening, if performed with precursor CD4+ cells, is performed under T_H-GM polarizing conditions, as described herein. For example, the method can be performed in the presence of IL-7/STAT5, TCR activation, CD28 co-stimulation, in combination with the blockade of IFN-gamma and IL-4.

Unless indicated otherwise, the definitions of terms described herein apply to all aspects and embodiments of the present disclosure

The practice of the present disclosure includes use of conventional techniques of molecular biology such as recombinant DNA, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology as described for example in: Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001), jointly and individually referred to herein as “Sambrook”); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture (R. I. Freshney, ed., 1987); Handbook of Experimental Immunology (D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); The Immunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996); Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993), Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, and Harlow and Lane (1999) Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. jointly and individually referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry (John Wiley & Sons, Inc., New York, 2000); and Agrawal, ed., Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Humana Press Inc., New Jersey, 1993).

Exemplification

Methods

Mice

Stat5^f/f mice were provided by L. Hennighausen (National Institute of Diabetes and Digestive and Kidney Diseases). Stat3^f/f mice were generated as described². Cd4-Cre transgenic mice were purchased from Taconic Farms. Rag2^−/− mice were obtained from Jean-Pierre Abastado (Singapore Immunology Network). All mice are on a C57BL/6 genetic background and housed under specific-pathogen-free conditions at National University of Singapore. All experiments were performed with mice 6˜8 weeks old and approved by the Institutional Animal Care and Use Committee of NUS.

Patients and Controls

Blood samples (n=47) and synovial fluid samples (n=3) were collected from RA patients admitted to the Department of Rheumatology and Immunology, the Affiliated Drum Tower Hospital of Nanjing University Medical School. All patients fulfilled the American College of Rheumatology criteria for the classification of RA. Age and gender matched healthy controls (n=32) were obtained from Medical Examination Center of the Affiliated Drum Tower Hospital. The study protocol was approved by the Ethics Committee of the Affiliate Drum Tower Hospital of Nanjing University Medical School.

In Vitro T Cell Differentiation

CD4⁺ T cells were obtained from spleens and lymph nodes by positive selection and magnetic separation (Miltenyi Biotech), followed by purification of naïve CD4⁺ T cell population (CD4⁺CD25⁻CD62L^hiCD44^lo) sorted with FACS Aria. Naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 (3 μg/ml; BD Pharmingen) and anti-CD28 (1 μg/ml; BD Pharmingen) in presence of different combinations of neutralizing antibodies and cytokines for 3˜4 days: for neutral conditions, no addition of any cytokine or neutralizing antibody; for T_H1 conditions, IL-12 (10 ng/ml), and anti-IL-4 (10 μg/ml, BD Pharmingen); for T_H17 conditions, hTGF-β (3 ng/ml), IL-6 (20 ng/ml), anti-IFN-γ (10 μg/ml, eBioscience), and anti-IL-4 (10 μg/ml); for an alternative T_H17 conditions, IL-6 (20 ng/ml), IL-23 (10 ng/ml), IL-1β (10 ng/ml), anti-IFN-γ (10 μg/ml), and anti-IL-4 (10 μg/ml). For GM-CSF-expressing cell differentiation, naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 (2 μg/ml) and soluble anti-D28 (1 μg/ml) with the addition of IL-7 and/or anti-IFN-γ (10 μg/ml) as indicated. All cytokines were obtained from R&D Systems. All cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 1 mM sodium pyruvate, 0.1 mM nonessential amino acid and 5 μM beta-mercaptoethanol. After 3˜4 days polarization, cells were washed and restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in presence of Golgiplug for 4-5 h, followed by fixation and intracellular staining with a Cytofix/Cytoperm kit from BD Pharmingen. Foxp3 staining was done with a kit from eBioscience. Cells were acquired on the LSR II (BD Biosciences) and analyzed with FlowJo software (Tree Star).

EAE Induction

EAE induction procedures were modified from previous report³. For active EAE induction, mice were immunized in two sites on the hind flanks with 300 μg MOG_35-55 in 100 μl CFA containing 5 mg/ml heat-killed M. tuberculosis strain H37Ra (Difco) at day 0 and day 7. Pertussis toxin (List Bio Lab) was administrated intraperitoneally at the dosage of 500 ng per mouse at day 1 and day 8. For single MOG_35-55/CFA immunization, the similar procedure was performed at day 0 and day 1 only. In an alternative active EAE induction, LPS (600 μg/ml in IFA, O111:B4 from Sigma) was used as adjuvant. For active EAE induction in Rag2^−/− mice, CD4⁺ T cells derived from Stat5^f/f or Cd4-Cre; Stat5^f/f mice were transferred, followed by MOG_35-55/CFA immunization as described above. Clinical symptoms were scored as follows: 0, no clinical sign; 1, loss of tail tone; 2, wobbly gait; 3, hind limb paralysis; 4, hind and fore limb paralysis; 5, death. IL-7Rα neutralizing antibody (SB/14, BD Pharmingen) and isotype control was administrated intraperitoneally at 200 μg per mouse every other day. For analysis of CNS-infiltrating cells, both spinal cord and brain were collected and minced from perfused mice, and mononuclear cells were isolated by gradient centrifuge with Percoll (GE Healthcare).

For passive EAE induction with Stat5^+/+ or Stat5^−/− CD4⁺ T cells, splenocytes and LNs were harvested 10-14 days post-immunization and passed through a 70 μm cell strainer (BD Falcon). Cells were cultured in vitro for 3 days with MOG_35-55 (20 μg/ml) in the presence of IL-23 (5 ng/ml) and IL-1β (2 ng/ml). After harvesting, CD4⁺ T cells were purified by positive selection to a purity >90%. CD4⁺ T cells (2 million in sterile PBS) were injected intraperitoneally into Rag2^−/− mice, followed by Pertussis toxin administration on the following day. Mice were observed daily for the signs of EAE as described above. For EAE induction by transferring various T_H subsets, similar procedures was performed as described above. Different subsets skewing conditions were as follows: Non-skewed, MOG_35-55 only; T_H1: MOG_35-55 plus IL-12 (10 ng/ml) and anti-IL-4 (5 μg/ml); T_H17: MOG_35-55 plus TGF-β (3 ng/ml), IL-6 (10 ng/ml), anti-IFN-γ (5 μg/ml) and anti-IL-4 (5 μg/ml); GM-CSF-expressing T_H: MOG_35-55 plus IL-7 (2 ng/ml) and anti-IFN-γ (5 μg/ml). 6×10⁵ CD4⁺ T cells were transferred per recipient mouse.

Antigen-Induced Arthritis (AIA)

Briefly, mice were immunized subcutaneously in two sites on the hind flanks with 100 μg methylated bovine serum albumin (mBSA, Sigma) in 100 μl complete Freund's adjuvant (CFA) containing 5 mg/ml heat-killed M. tuberculosis strain H37Ra (Difco) at day 0. Pertussis toxin (List Bio Lab) was administrated intraperitoneally at the dosage of 250 ng per mouse at day 1. Arthritis was induced by intraarticular injection of 100 μg mBSA (in 10 μl saline) into the hind right knee joint at day 7 after immunization. The hind left knee joint was injected with same volume of saline as control. Joint swelling was recorded by measuring the difference between right and left knee joint diameters with a caliper over 7 days after arthritis induction. To assess the effect of GM-CSF administration, AIA was induced by intraarticular injection of mBSA alone to the right knee joint or mBSA supplemented with 100 ng GM-CSF (ImmunoTools) to the left knee joint. To assess the effect of GM-CSF and/or TNF-α blockade, mice were administrated intraperitoneally with neutralizing antibodies (100 μg for each antibody per mouse) specific for GM-CSF (MP1-22E9, BD Pharmingen) and/or TNF-α (MP6-XT3, BD Pharmingen) at indicated times.

For AIA induction by adoptive transfer, splenocytes and inguinal LN cells were isolated from mBSA/CFA-immunized mice at day 7, and cultured in vitro with mBSA (10 μg/ml) in the presence of IL-7 (2 ng/ml) for 3 days. After harvesting, CD4⁺ T cells were purified by positive selection (Miltenyi Biotec) to a purity >90%. Then CD4⁺ T cells (1 million in sterile PBS) were transferred into WT naïve mice, followed by intraarticular injection of mBSA on the next day.

Collagen-Induced Arthritis (CIA)

CIA was induced in a similar procedure as AIA as described above, by immunizing mice with chicken collagen II/CFA emulsion (purchased from Chondrex, Inc), followed with pertussis toxin injection. Mice were monitored and scored for arthritis: 0, normal; 1, mild swelling of ankle or wrist, or apparent swelling limited to individual digits; 2, moderate swelling of entire paw; 3, severe swelling of entire paw with ankylosis. Scores for four limbs were summed for each mouse.

Histological Analysis

For paraffin-embedded tissues, spinal cords were fixed in 4% PFA. Knee joints or paws were removed, fixed in 10% formalin and decalcified in 5% formic acid before dehydration and embedding. Sections (5 μm) were stained with hematoxylin and eosin (H&E) to assess immune cell infiltration and inflammation, or with Safranin-O/Fast Green to assess cartilage depletion. For frozen tissues, spinal cords were embedded in OCT (Tissue-Tek) and snap frozen on dry ice. Sections (10 μm) were fixed in ice-cold acetone and stained with primary anti-CD4 (Biolegend) and anti-CD11b (eBioscience), followed by incubation with fluorescence-conjugated secondary antibodies (Invitrogen). For AIA experiments, knee joint were fixed in 10% formalin for 5 days, followed by decalcification in 5% formic acid for 5 days. Sections (10 μm) were stained with hematoxylin and eosin (H&E) to assess immune cell infiltration and inflammation, or stained with Safranin-O/fast green to access cartilage destruction.

Cell Sorting and May Grünwald-Giemsa Staining

Monocytes/macrophages (Ly6C^hiLy6G⁻) and neutrophils (Ly6C^loLy6G^hi) gated on CD45⁺CD11b⁺ were sorted with FACS Aria from spleens or synovial single cell suspensions. Sorted cells were cytospun onto glass slides and subsequently stained with May Grünwald and Giemsa dye following a standard procedure.

Real-Time PCR

Total RNA was extracted from cells with RNeasy kit (Qiagen) according to the manufacturer's instruction. Complementary DNA (cDNA) was synthesized with Superscript reverse transcriptase (Invitrogen). Gene expressions were measured by 7500 real-time PCR system (Applied Biosystems) with SYBR qPCR kit (KAPA). Actinb, Gapdh or Rn18S was used as internal control. The primer sequences are available upon request.

ELISA

TNF-α, IL-6, IL-1β, IFN-γ, GM-CSF and IL-2 levels were assayed by Ready-SET-Go ELISA kit (eBioscience), and IL-17 level was measured by DuoSet ELISA kit (R&D Systems) according to the manufactures' instructions.

Chromatin Immunoprecipitation Assays

CD4⁺ T cells isolated from Stat5^f/f or Cd4-Cre; Stat5^f/f mice were activated with plate-bound anti-CD3 and anti-CD28 for 3 days. Cells were stimulated with IL-7 (20 ng/ml) or IL-2 (25 ng/ml) for 45 min. Crosslink was performed by addition of formaldehyde at final concentration of 1% for 10 min followed by quenching with Glycine. Cell lysates were fragmented by sonication and precleared with protein G Dynabeads, and subsequently precipitated with anti-STAT5 antibody (Santa Cruz) or normal rabbit IgG (Santa Cruz) overnight at 4° C. After washing and elution, crosslink reversal was done by incubating at 65° C. for 8 hr. The eluted DNA was purified and analyzed by RT-PCR with primers specific to Csf2 promoter as described previously⁵.

Statistics

Statistical significance was determined by Student's t test using GraphPad Prism 6.01. The p value<0.05 was considered significant. The p values of clinical scores were determined by two-way multiple-range analysis of variance (ANOVA) for multiple comparisons. Unless otherwise specified, data were presented as mean and the standard error of the mean (mean±SEM).

Example 1. Stat5 Conditional Knockout Mice are Resistant to EAE

STAT5 negatively regulates T_H17 differentiation by restraining IL-17 production (Laurence et al., 2007; Yang et al., 2011). However, the function of STAT5 in T_H17-mediated pathogenesis is not well understood. To explore this question, EAE was induced in Cd4-Cre; Stat5^f/f (Stat5^−/−) mice, where Stat5 was specifically deleted in T cell compartment, and in littermate controls by immunizing the mice with MOG_35-55/CFA at day 0 and day 7. Development of paralysis was assessed by daily assignment of clinical scores. Surprisingly, diminished occurrence and severity of clinical disease in Stat5^−/− mice was observed (FIGS. 1A and 1B), a result that was opposite to expectations based on an antagonistic role for STAT5 in T_H17 generation. Similar results were observed when a single MOG_35-55/CFA immunization was performed or replaced CFA with LPS as the adjuvant to induce EAE (FIGS. 1C and 1D). Consistent with reduced EAE disease in Stat5^−/− mice, a remarkable reduction of immune cell infiltration in the spinal cord of Stat5^−/− mice was observed (FIG. 2A). Furthermore, the infiltration of various immune cell populations, including CD4⁺, CD8⁺, B220⁺ and CD11b⁺ cells was reduced in Stat5^−/− mice (FIGS. 2B-D and data not shown). However, the frequencies of IL-17⁺ and IFN-γ⁺ cells among CD4⁺ T cells in the CNS were comparable between Stat5^+/+ and Stat5^−/− mice (FIG. 3A), suggesting the resistance to EAE in Stat5^−/− mice is independent of T_H1 and T_H17 cell development. Nevertheless, decreased CD4⁺CD25⁺ T_reg population in the CNS of Stat5^−/− mice was detected (FIG. 3B), indicating the resistance to EAE in Stat5^−/− mice was unlikely due to altered T_reg cells.

Example 2. Resistance to EAE in Stat5-Mutant Mice is Due to an Intrinsic Defect of Antigen Specific CD4⁺ T Cells Independent of T_H1 and T_H17 Generation

Stat5 deletion (Cd4-cre; Stat5^f/−) mice was reported to develop peripheral lymphopenia, with a reduction of both CD4⁺ and CD8⁺ T cells (Yao et al., 2006). However, another study showed that Stat5 deletion (Cd4-cre; Stat5^f/f) did not affect the proportion of peripheral CD4⁺ T cells (Burchill et al., 2007). In the experimental setting, a change in the absolute number of peripheral CD4⁺ T cells was not detected by Stat5 deletion during EAE development (FIGS. 4A and 4B), suggesting the resistance to EAE in Stat5^−/− mice was not caused by peripheral lymphopenia. Furthermore, increased frequencies of IL-17⁺ and IFN-γ⁺ cells were detected among CD4⁺ T cells in spleens of Stat5^−/− mice (FIG. 4C), which further support the idea that the resistance to EAE in Stat5^−/− mice is likely independent of T_H1 and T_H17 generation. To validate the function of STAT5 in T_H1 and T_H17 generation, the in vitro differentiation was performed by activating naïve CD4⁺ T cells under T_H1- and T_H17-polarizing conditions. In agreement with previous reports, that STAT5 mediated the suppressive effect of IL-2 on T_H17 differentiation (data not shown). Interestingly, IL-7, which also signals through STAT5, was not observed to have a demonstrable effect on T_H17 differentiation (data not shown). Nevertheless, a slight decrease of IFN-γ⁺ cells under T_H1-polarizing condition was observed when STAT5 was deleted (data not shown).

To confirm if the resistance of EAE in Stat5^−/− mice is mediated by CD4⁺ T cells, Rag2^−/− mice were reconstituted with Stat5^+/+ or Stat5^−/− CD4⁺ T cells followed by EAE induction. We found that Rag2^−/− mice that received Stat5^−/− CD4⁺ T cells were resistant to the disease compared with mice receiving wild-type cells (data not shown), demonstrating that Stat5^−/− CD4⁺ T cells were impaired in their ability to promote EAE development.

Next, whether the lack of encephalitogenicity was caused by defects in migration of Stat5^−/− CD4⁺ T cells to the CNS was examined. It has been shown that the chemokine receptor CCR6 is essential for T_H17 cell entry into the CNS through the choroid plexus (Reboldi et al., 2009). Thus, CCR6 expression in both Stat5^−/− and Stat5^+/+ CD4⁺ T cells was examined. Increased CD4⁺CCR6⁺ cells in spleens of Stat5^−/− mice compared with Stat5^+/+ controls (FIG. 5A) was observed. CXCR3 and CD69 expression was also examined, which showed increased expression of both molecules in CD4⁺ T cells in the absence of STAT5 (FIG. 5A). These results indicate that Stat5^−/− CD4⁺ T cells can infiltrate CNS. Furthermore, comparable number of CD4⁺ T cells present in the CNS of Stat5^+/+ and Stat5^−/− mice during EAE induction was observed (at day 7 and day 9) (FIG. 5B). However, CD4⁺ T cells in CNS of Stat5^−/− mice dropped dramatically during disease onset (Day 21) (FIGS. 5C and 5D). Together, these results demonstrate that Stat5^−/− CD4⁺ T cells can infiltrate CNS, but fail to induce effective inflammation in the CNS in EAE.

To further exclude the possibility that the resistance of Stat5-deficient mice to EAE was caused by any potential defect in the survival of autoreactive CD4⁺ T in the CNS, increased numbers of Stat5^−/− CD4⁺ T cells than wild-type cells were transferred into Rag2^−/− mice respectively to make sure comparable numbers of autoreactive CD4⁺ T cells were present in the CNS during EAE development. As shown in FIGS. 6A and 6B, despite similar numbers of CD4⁺ T cells in the CNS between two groups of mice, reduced disease severity was nevertheless observed in mice receiving Stat5-deficient CD4⁺ T cells. Additionally, certain numbers of Stat5-deficient mice containing similar numbers of CD4⁺ T cells in the CNS as wild-type mice at peak of EAE disease were observed, yet, they were relatively resistant to EAE compared with those wild-type mice (FIG. 6C), further suggesting that the resistance to EAE disease in Stat5-deficient mice was unlikely due to impaired CD4⁺ T cell survival in the CNS.

To further develop a causal link between these observations and the intrinsic impairment of Stat5^−/− CD4⁺ T cells, MOG_35-55-specific Stat5^+/+ and Stat5^−/− CD4⁺ T cells were transferred into Rag2^−/− mice separately without further immunization to test if these cells were able to mediate EAE development. As shown in FIGS. 7A and 7B, mice receiving Stat5^+/+ CD4⁺ T cells spontaneously developed EAE disease 1 week after transfer. In contrast, mice receiving Stat5^−/− CD4⁺ T cells had significantly reduced disease severity and incidence. The frequencies of IL-17⁺ and IFN-γ+ cells among CD4⁺ T cells in the CNS of Rag2^−/− mice were comparable between two groups (FIG. 7C), further suggesting that the intrinsic defect in encephalitogenicity of Stat5^−/− CD4⁺ T cells is independent of T_H1 and T_H17. To exclude the possible role of CD8⁺ T cells in the resistance to EAE observed in Stat5^−/− mice, Rag2^−/− mice were reconstituted with MOG_35-55-specific Stat5^+/+ or Stat5^−/− CD4⁺ T cells together with equal numbers of Stat5^+/+ CD8⁺ T cells. The transfer of Stat5^−/− CD4⁺ together with Stat5^+/+ CD8⁺ T cells still failed to induce EAE (data not shown). Together, these data demonstrate that Stat5^−/− CD4⁺ T cells have intrinsic defect in encephalitogenicity. The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

Example 3. Diminished Expression of GM-CSF in Stat5^−/− CD4⁺ T Cells

To test whether GM-CSF production was impaired by Stat5 deletion, its expression was examined in MOG_35-55-specific Stat5^+/+ and Stat5^−/− CD4⁺ T cells. Splenocytes derived from MOG_35-55/CFA-immunized Stat5^+/+ and Stat5^−/− mice were challenged with various concentrations of MOG_35-55 for 24 h, to examine the secretion of GM-CSF. GM-CSF production was observed to increase in a MOG_35-55 dose-dependent manner in Stat5^+/+ cells (FIG. 8A). In contrast, GM-CSF production was severely diminished in Stat5^−/− cells in all conditions. To further validate this, splenocytes derived from mice were stimulated during the development of EAE with PMA/Ionomycin in the presence of GolgiPlug for GM-CSF and IL-17 intracellular staining. Although IL-17 expression was enhanced in Stat5^−/− cells, a significantly reduced proportion of GM-CSF⁺IL-17⁻ and GM-CSF⁺ IL-17⁺ cells was observed among CD4⁺CD44^hi cells in the absence of STAT5 (FIG. 8B). Moreover, the frequency of MOG_35-55-specific GM-CSF⁺ T cells was also significantly reduced in spleens of Stat5^−/− mice (FIG. 8C). Together, these results indicate that STAT5 is required for GM-CSF expression in autoreactive CD4⁺ T cells. However, STAT3, an important transcription factor in T_H17 differentiation, was required for GM-CSF expression (FIG. 8D).

Next, GM-CSF induction in the CNS during EAE development was examined. Although IL-17 and IFN-γ production by CNS-infiltrating CD4⁺ T cells was not impaired by Stat5 deficiency, a diminished frequency of CD4⁺GM-CSF⁺ cells in the CNS of Stat5^−/− mice was detected compared with control mice (FIG. 9A). Further analysis showed a reduced GM-CSF⁺ percentage among CD4⁺IL-17⁺ cells and among CD4⁺IFN-γ⁺ cells (FIG. 9A). Similarly, Rag2^−/− mice transferred with MOG_35-55-specific Stat5^−/− CD4⁺ T cells also showed a reduced frequency of CD4⁺GM-CSF⁺ T cells in the CNS compared with control mice (FIG. 9B). GM-CSF mRNA expression in the CNS of Stat5^−/− mice was markedly lower than that of Stat5^+/+ mice at day 8 after EAE induction (FIG. 9C), when comparable CD4⁺ T cell infiltration was detected in Stat5^−/− and Stat5^+/+ mice (FIGS. 5C and 5D). Meanwhile, no significant difference of IL-17 and IFN-γ expression was detected between Stat5^−/− and Stat5^+/+ mice (FIG. 9C). The impaired cytokine induction (IL-17 and IFN-γ) in the CNS of Stat5^−/− mice at later stage (day 14, FIG. 9C) could be explained by the inability of Stat5^−/− CD4⁺ T to sustain neuroinflammation (FIGS. 5C and 5D). Interestingly, GM-CSF induction in the CNS preceded IL-23 induction (FIG. 9C), suggesting IL-23 might not be required for GM-CSF expression in the induction phase of EAE. In summary, the results suggest that GM-CSF expression in autoreactive CD4⁺ T cells is regulated by STAT5 and the impaired GM-CSF production in the absence of STAT5 caused the resistance of the mice to EAE.

Example 4. IL-7-STAT5 Signaling Induces GM-CSF Expression in Autoreactive CD4⁺ T Cells and Contributes to Neuroinflammation

Next, the mechanism by which STAT5 regulates GM-CSF expression was investigated. As the present disclosure indicates, neither IL-23 nor IL-1β seemed to be potent STAT5 stimulators (FIG. 10A). Furthermore, IL-1R1 expression was not changed, whereas IL-23Rα expression was increased in Stat5^−/− CD4⁺ T cells (FIG. 10B). These data suggest that the ability of STAT5 to induce GM-CSF expression is likely independent of IL-23 and IL-1β signaling. In contrast, both IL-2 and IL-7 potently activated STAT5 by inducing tyrosine phosphorylation (FIG. 10A). Therefore, the effect of these two cytokines on GM-CSF induction in autoreactive T cells was further examined. Splenocytes derived from MOG_35-55-immunized wild-type mice were challenged with MOG_35-55 alone or plus IL-2. GM-CSF and IL-17 production by CD4⁺ T cells were analyzed by intracellular cytokine staining. As shown in FIG. 10C, IL-2 showed modest effects on the frequency of GM-CSF⁺ T cells. In contrast, IL-7 significantly promoted GM-CSF expression in both IL-17⁻ and IL-17⁺ CD4⁺CD44^hi T cells (FIG. 11A). Furthermore, IL-7 carried out this function in a STAT5-dependent manner, as Stat5 deletion abrogated its effect on GM-CSF expression as assessed by intracellular cytokine staining and ELISA (FIG. 11A, lower panels, and FIG. 11B).

IL-7Rα is expressed in both CD62L^hiCD44^loT cells and CD62L^loCD44^hi T cells, suggesting IL-7 may directly act on CD4⁺ T cells to regulate GM-CSF expression. Thus, CD62L^hiCD44^lo and CD62L^loCD44^hi T cells were sorted from Stat5^−/− mice and littermate controls during EAE development, and then activated cells in the presence or absence of IL-7. As shown in FIG. 11C, CD62L^loCD44^hi T cells potently expressed GM-CSF, while CD62L^hiCD44^lo T cells expressed 30-fold lower GM-CSF amounts. STAT5 deletion resulted in reduced basal GM-CSF production in CD62L^loCD44^hi T cells. As expected, IL-7 promoted GM-CSF expression in both subsets of CD4⁺ T cells in a STAT5-dependent manner (FIG. 11C).

To examine the contribution of IL-7-induced GM-CSF expression in autoreactive CD4⁺ T cells to EAE development, mice were treated with IL-7Rα-specific antibody (clone SB/14) during EAE development. The treatment resulted in a significant reduction of disease severity, which was accompanied with reduced CNS inflammation (FIGS. 12A and 12B). In agreement with previous report (Lee et al., 2012), this neutralizing antibody did not have T cell depleting activity (FIG. 12C). Notably, the blocking of IL-7 signaling resulted in decreased GM-CSF expression in CNS-infiltrating CD4⁺ T cells (FIGS. 12D-12F). In summary, the present findings indicate that IL-7 induces STAT5-dependent GM-CSF expression in autoreactive CD4⁺ T cells, which contributes to the development of neuroinflammation.

Example 5. GM-CSF-Expressing T_H Cells are Distinct from T_H17 and T_H1

Since both T_H17 and T_H1 can produce GM-CSF, it was determined if the IL-7-stimulated phenotype was related to either of these subsets. To further understand the characteristics of GM-CSF-expressing CD4⁺ cells, naïve CD4⁺ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 under T_H1- or T_H17-polarizing conditions. It was observed that anti-CD3 together with anti-CD28 induced the expression of GM-CSF (FIG. 14A). However, while T_H1 differentiation conditions promoted IFN-γ expression and T_H17 conditions promoted IL-17 expression as expected, both T_H1 and T_H17 differentiation conditions greatly suppressed the production of GM-CSF (FIGS. 13A and 13B). Conversely, IL-12 and IFN-γ neutralization promoted GM-CSF-expressing cell generation (FIG. 13A), consistent with a previous report (Codarri et al., 2011). IL-23 and IL-1β did not increase GM-CSF-expressing cell differentiation from naïve CD4⁺ T cells (FIG. 13A), which was consistent with the finding that naïve CD4⁺ T cells did not express their receptors. TGF-β inhibits GM-CSF expression (El-Behi et al., 2011). IL-6, an essential cytokine for T_H17 differentiation, had a profound inhibitory effect on GM-CSF expression (FIG. 14B), indicating STAT3 could be a negative regulator. To address this, naïve CD4⁺ T cells were purified from Stat3^−/− mice for cell differentiation. Strikingly, in the absence of STAT3, cells polarized under T_H17 condition expressed GM-CSF (FIG. 14C). Interestingly, even without exogenous IL-6, STAT3 still had a moderate suppressive effect on GM-CSF-expressing cell differentiation (FIG. 14C). In addition, RORγt and T-bet have been reported unnecessary for GM-CSF expression in CD4⁺ T cells (El-Behi et al., 2011). Thus, the present datasupport a model wherein GM-CSF-expressing CD4⁺ T cells develop via a lineage distinct from T_H17 and T_H1.

Example 6. IL-7-STAT5 Programs GM-CSF-Expressing T_H Cell Differentiation

The present findings disclosed herein (including. g., diminished GM-CSF expression in Stat5^−/− CD4⁺ T cells in vivo, IL-7/STAT5-mediated induction of GM-CSF expression in naïve CD4⁺ T cells, and the distinct features of GM-CSF-expressing T_H cells versus T_H1 and T_H17 cells) indicates a distinct T_H cell subset that is regulated by IL-7-STAT5 signaling. This finding was further explored by examining GM-CSF-expressing T_H cell differentiation in vitro by activating naïve CD4⁺ T cells with anti-CD3 and anti-CD28 in the presence of different concentrations of IL-7. As shown in FIGS. 15A and 15B, IL-7 strongly promoted the generation of GM-CSF-expressing cells and GM-CSF secretion. Moreover, the generation of GM-CSF-expressing T_H by IL-7 was mediated by STAT5. Without STAT5, IL-7 was unable to promote the generation of GM-CSF-expressing cells (FIGS. 15C and 15D). Further investigation showed that IL-7-induced STAT5 activation directly bound promoter regions of the Csf2 gene (FIGS. 15E and 15F).

Small proportions of IFN-γ-expressing cells were generated during GM-CSF-expressing T_H differentiation (FIG. 15A). Thus, the effect of blocking IFN-γ on GM-CSF-expressing cell generation was tested, which showed that the combination of IL-7 and IFN-γ neutralization induced the highest frequency of GM-CSF⁺ cells, where few IL-17⁺ or IFN-γ⁺ cells were detected (FIG. 16A). Moreover, the expression of subset defining transcriptional factors in GM-CSF-expressing T_H was examined and observed that the expression of RORγt or T-bet in GM-CSF-expressing T_H was significantly lower than those in T_H17 or T_H1 cells, respectively (FIG. 16B), confirming that the GM-CSF-expressing T_H cells are distinct from T_H1 and T_H17 cells. Together, these data suggest that IL-7-STAT5 signaling direct the differentiation of a novel GM-CSF-expressing helper T cell subset, termed T_H-GM.

Next, it was determined whether IL-2 signaling could influence T_H-GM differentiation from naïve CD4⁺ T cells. The addition of IL-2 or antibody against IL-2 only had modest effect on the frequency of GM-CSF⁺ cells (FIG. 14D), indicating a minimal effect of IL-2 on T_H-GM differentiation. Unlike IL-7Rα, IL-2 high-affinity receptor IL-2Rα was not expressed in naïve CD4⁺ T cells, but its expression was gradually induced by TCR activation (FIGS. 17A-17C). Thus, the minimal effect of IL-2 at least in part is due to the unresponsiveness of naïve CD4⁺ T cells to IL-2 stimulation. In support of this view, IL-7, but not IL-2, induced STAT5 activation and upregulated GM-CSF mRNA expression in naïve CD4⁺ T cells (FIGS. 17D and 17E). To further confirm this idea, activated CD4⁺ T cells were stimulated with IL-2 or IL-7, which showed that both cytokines induced STAT5 activation, Csf2 promoter binding and GM-CSF mRNA upregulation (FIGS. 18A-18C). Notably, IL-2 induced a prolonged STAT5 activation compared with IL-7 (FIG. 18A).

Example 7. Distinct Gene Expression Profile of T_H-GM

To demonstrate T_H-GM as distinct from known T cell subsets (e.g., T_H1 and T_H17), a whole transcriptome analysis was performed by microarray to validate its specificity compared with known T cell subsets, in particular T_H17 cells. Naïve CD4⁺ T cells were differentiated into T_H1, T_H17 and T_H-GM. Microarray analysis was performed to examine their gene expression profiles. Whole transcriptome clustering indicates T_H-GM cells as representing a novel subset distinct from T_H1 or T_H17 cells. T cell lineage-specific gene expression is shown in Table 1. A list of 202 genes preferentially expressed in T_H1 cells were identified, compared with naïve, T_H17 or T_H-GM cells (fold change >1.7), among which IFN-γ and T-bet are on the top of the list (Table 1). Similarly, T_H17-feature genes, such as IL-17, IL-17F, RORγt and RORα, were identified in the list including 411 genes specific to T_H17 cells (Table 1). The T_H-GM cell-specific gene list (“Genes preferentially upregulated in T_H-GM”—the T_H-GM signature genes) contains 210 genes including the gene encoding GM-CSF as the top gene in the list (Table 1). A set of surface molecules which were selectively expressed at high level in T_H-GM subset, and another set of surface molecules which were selectively expressed at low level in T_H-GM subset compared with other subsets were identified (FIG. 19 and Table 1). These molecules (also T_H-GM signature genes) can be used for further characterization by surface markers to identify the T_H-GM subset of T cells. Several other genes of interest were also identified, including genes encoding cytokines and transcriptional factors, in particular IL-3. Various helper T cells were differentiated in vitro and confirmed that T_H-GM cells are potent IL-3 producers as compared with T_H1 and T_H17 cells (FIGS. 20A, 20C and 20D). In addition, several other cytokines, including EBI-3, PENL and RANKL were found preferentially expressed in T_H-GM cells (FIG. 20B), indicating diverse biological functions of T_H-GM cells.

Example 8. T_H-GM Cells are the Primary Pathogenic Population

To test the hypothesis that GM-CSF-expressing T_H subset (T_H-GM) was the primary encephalitogenic effector cells, adoptive transfer of different subsets of MOG_35-55-specific CD4⁺ T cells was performed into Rag2^−/− mice for EAE induction. As shown in FIG. 21, GM-CSF-expressing T_H cells were preferentially able to induce EAE compared with T_H17 and T_H1 subsets.

Example 9. The Suppression of STAT5 Activity by Chemical Inhibitor Attenuates GM-CSF Expression by T_H-GM and Ameliorates EAE

The effect of disrupting STAT5 activation by chemical inhibitor was examined to explore possible methods of treating autoimmune neuroinflammation. The phosphorylation on the key tyrosine residue in SH2 domain is crucial for STAT5 activation and function. A commercial STAT5 inhibitor (CAS 285986-31-4, Calbiochem) has been reported to selectively disrupt tyrosine phosphorylation and DNA binding of STAT5 (Muller et al., 2008). First, the inhibitory effect of this inhibitor on STAT5 activation upon IL-7 stimulation in CD4⁺ T cells was tested. At a concentration of 50 μM, the inhibitor had about 50% inhibitory effect, which was further enhanced with the increase of concentration (FIG. 22A). STAT5 inhibitor had low affinity and thus required a high concentration to fully block STAT5 activation, whereas JAK3 inhibitor showed potent inhibitory effect even at low concentration (FIG. 22B). The specificity of STAT5 inhibitor was next tested by examining its effect on the activation of STAT3 and STAT1. As shown in FIGS. 22C and 22D, this STAT5 inhibitor at relatively lower concentration (50 or 100 μM) showed minimal inhibitory effect on both STAT3 and STAT1 activation.

The effect of STAT5 inhibition on T_H-GM differentiation was examined. As shown, STAT5 inhibitor suppressed T_H-GM differentiation in a dosage-dependent manner (FIG. 22E). Reduced T_H1 differentiation upon STAT5 inhibitor treatment was observed (data not shown), but T_H17 differentiation was not suppressed by STAT5 inhibitor (data not shown).

To explore the therapeutic effect of targeting STAT5 activation in EAE disease, the commercial STAT5 inhibitor was administered to wild-type mice intraperitoneally every other day after disease onset. Development of paralysis was assessed by daily assignment of clinical scores. STAT5 inhibition ameliorated EAE severity, associated with reduced immune cell infiltration in the CNS (FIGS. 23A and 23B). In contrast, although JAK3 inhibitor can potently block STAT5 activation (FIG. 22B), it showed detrimental effect on EAE (FIG. 23B). Of note, STAT5 inhibitor resulted in reduced GM-CSF production in CNS-infiltrating CD4⁺ T cells (FIGS. 23C and 23D). This study indicates that targeting STAT5 by chemical inhibitor is useful in therapeutic intervention in MS.

Example 10. GM-CSF-Producing T_H Cells are Associated with Human RA

Plasma concentrations of GM-CSF and TNF-α in peripheral blood of RA patients were examined in comparison with gender/age-matched healthy control (HC), and found that both cytokines were elevated in RA (FIG. 24A). Ex vivo frequencies of IFN-γ-, IL-17- or GM-CSF-producing T_H cells were quantified in RA and HC. High frequencies of IFN-γ- and/or GM-CSF-producing T_H cells were detected in all samples, but observed low frequency (<1%) of IL-17-producing T_H cells (FIG. 24B). GM-CSF-single-producing (GM-CSF⁺IFN-γ⁻) T_H cells represented a substantial population in both RA and HC (FIG. 24B). More importantly, the frequency of this population in peripheral blood of RA was significantly higher than that of HC (FIG. 24C). In contrast, neither GM-CSF/IFN-γ-double-producing nor IFN-γ-single-producing T_H cells showed any significant difference in their frequencies between RA and HC (FIG. 24C). Therefore, the frequency of GM-CSF-single-producing T_H cells in peripheral blood is selectively elevated in RA, suggesting a functional association of T_H-cell-secreted GM-CSF with RA. Moreover, a significant correlation between plasma GM-CSF concentration and GM-CSF-single-producing T_H cell frequency was observed in RA (FIG. 24D).

To further evaluate the association of GM-CSF-producing T_H cells with RA, mononuclear cells were isolated from synovial fluid of RA patients and analyzed the abundance of these cells. A marked elevation of GM-CSF-producing T_H cell frequency was observed in synovial fluid compared with peripheral blood, but most of these cells co-expressed IFN-γ (FIG. 24E). Similarly, both T_H1 and T_H17 frequencies were also increased in synovial fluid, with T_H17 remaining to be a minor population compared with T_H1 (FIG. 24E).

Example 11. GM-CSF Mediates Experimental Arthritis in a TNF-α-Independent Manner

The elevation of GM-CSF and TNF-α level in plasma of RA in comparison to HC may suggest a therapeutic approach by targeting these two cytokines. The efficacy of blocking both GM-CSF and TNF-α was tested in treating arthritic mice in antigen-induced arthritis (AIA) model, which is a T-cell driven RA model and is easily inducible in C57BL/6 strain with a rapid and synchronized disease onset, facilitating the exploration of RA pathogenesis. Either GM-CSF or TNF-α individual blockade attenuated AIA development (FIG. 25A). Interestingly, the combination of GM-CSF− and TNF-α-specific neutralizing antibodies showed better efficacy in controlling arthritis development than individual treatments (FIG. 25A). That is, targeting GM-CSF may have beneficial efficacy in treating arthritis in a way independent of TNF-α activity. To further study the distinguishable effects of GM-CSF and TNF-α in mediating arthritis development, a mouse strain (Cd4-Cre; Stat5^f/f, or Stat5^−/− in short) with conditional Stat5 deletion was used in T cells for AIA induction. These conditional Stat5-knockout mice resisted arthritis development, as exemplified by milder joint selling, fewer immune cell infiltration in synovia, and reduced joint destruction (FIGS. 25B-25D), even though they had markedly increased level of serum TNF-α as well as IFN-γ (FIG. 25E). In contrast, serum level of GM-CSF was significantly reduced in knockout mice (FIG. 25E), which was likely the causal factor of the resistance to arthritis development as further supported by results described below. Consistent results were also observed in collagen-induced arthritis (CIA) model (FIGS. 26A-26D). Together, these findings suggest that GM-CSF is an important pathogenic mediator in RA and also indicate the promise of developing anti-GM-CSF drugs to treat RA patients who are anti-TNFα drugs unresponsive, marking GM-CSF-producing T_H cells as a new biomarker for RA diagnosis.

Example 12. STAT5-Regulated GM-CSF Secretion by Autoreactive T_H Cells Mediates Synovial Inflammation

On the basis of association of GM-CSF with RA, the cellular producers of GM-CSF and the regulatory mechanism underlying GM-CSF expression in arthritic mice were examined. Splenocytes were collected from wild-type AIA mice and separated cells into three fractions: splenocytes, splenocytes depleted of CD4⁺ T cells and CD4⁺ T cells; and stimulated each fraction at same cell numbers under various conditions. Splenocytes produced low but detectable level of GM-CSF without stimulation, which was markedly increased by PMA/Ionomycin or mBSA antigen stimulation (FIG. 28A). Under all conditions, splenocytes depleted of CD4⁺ T cells almost completely abrogated GM-CSF production (FIG. 28A). In contrast, CD4⁺ T cells produced dramatically elevated GM-CSF in comparison to splenocytes under all conditions (FIG. 28A). These results strongly support that CD4⁺ T cells are predominant producers of GM-CSF at least in spleens of arthritic mice, which is somehow consistent the observed correlation of plasma GM-CSF concentration with GM-CSF-single-producing T_H cell frequency in RA (FIG. 24D). Thus, the functional significance of T_H-cell-secreted GM-CSF was examined in arthritis development. Given T-cell-specific Csf2-knockout mice is not available and STAT5 is a key regulator of GM-CSF expression in T_H cells, conditional Stat5-knockout mice was used, which showed decreased GM-CSF level and resistance to arthritis development as described above.

Consistent with a previous study (Burchill et al., 2007), similar frequencies of CD4⁺ T cells were observed in peripheral lymphoid tissues as well as in inflamed synovial tissues of STAT5-deficient mice compared with wild-type mice at day 7 after AIA induction (FIGS. 27A-27D), suggesting loss of STAT5 seems to not impair CD4⁺ T-cell generation in periphery and infiltration in synovial tissues. To determine the requirement of STAT5 for arthritogenic potential of CD4⁺ T cells, ex vivo-expanded antigen-reactive CD4⁺ T cells, derived from Stat5^+/+ and Stat5^−/− AIA mice, were transferred into wild-type naïve mice separately, followed by intra-articular injection of mBSA. Mice receiving Stat5^+/+ CD4⁺ T cells displayed an abundant immune cell infiltration in synovial tissues at day 7 after AIA induction (FIG. 27E). In contrast, mice receiving Stat5^−/− CD4⁺ T cells had marked reduction of synovial infiltrates (FIG. 27E). Therefore, STAT5-deficient CD4⁺ T cells are defective in arthritogenic potential.

Multiple lines of evidence support a central role of T cells in RA. However, the pathogenic mechanism of T cells remains insufficiently understood. Although T_H1 is a predominant population among synovial infiltrating CD4⁺ T cells in human RA (Berner et al., 2000; Yamada et al., 2008), defective IFN-γ signaling results in increased disease susceptibility in animal models of arthritis (Guedez et al., 2001; Irmler et al., 2007; Manoury-Schwartz et al., 1997; Vermeire et al., 1997). In contrast, T_H17 cells are proven crucial in animal models of arthritis (Pernis, 2009), but predominance of T_H17 cells is limited in both peripheral blood and synovial compartment of human RA (Yamada et al., 2008) and (FIGS. 1B and 1E). As demonstrated herein, STAT5-regulated GM-CSF-producing T_H cells potentiate arthritis pathogenesis.

To validate the regulatory role of STAT5 in GM-CSF production, splenocytes derived from AIA mice were stimulated with PMA/Ionomycin plus Golgiplug ex vivo, followed by intracellular cytokine staining and flow cytometry. As expected, the frequency of GM-CSF-single-producing cells among CD4⁺CD44^hi population was significantly decreased in Stat5^−/− mice (FIG. 2834B). Notably, no significant differences were observed in frequencies of IL-17-single-producing (T_H17) or IFN-γ-single-producing (T_H1) cells between two groups (FIG. 28B). Further study by combining mBSA restimulation and intracellular cytokine staining showed that the frequency of mBSA-specific GM-CSF-producing effector T cells was much lower in spleens of Stat5^−/− mice than those in controls (FIG. 29A). In addition, AIA mice-derived splenocytes and inguinal lymph nodes (LNs) were restimulated with mBSA ex vivo to measure cytokine concentrations in culture supernatants and found a significant reduction of GM-CSF with deletion of STAT5, but comparable levels of both IL-17 and IFN-γ between two groups (FIGS. 29B and 29C). Together, the results indicate that loss of STAT5 may specifically suppress GM-CSF-producing effector Th cells but not T_H17 or T_H1 cells in experimental arthritis.

To investigate the involvement of GM-CSF-producing T_H cells and their regulation by STAT5 in synovial inflammation, synovial tissues were dissected from AIA mice and examined cytokine production by T_H cells. In spite of multiple cellular sources of GM-CSF (Cornish et al., 2009), CD4⁺ T_H cells were prominent producers of GM-CSF in synovial tissues of AIA mice (FIG. 28C), consistent with the observation in spleens (FIG. 28A). Moreover, a significantly lower percentage of synovial GM-CSF-producing T_H cells was detected in Stat5^−/− mice than Stat5^+/+ mice (FIG. 28D). On the other hand, both T_H1 and T_H17 cells exhibited similar percentages between two groups (FIG. 28C). A decrease in GM-CSF level in synovial compartments of Stat5^−/− mice in comparison to controls was expected. To address this, inflamed synovial tissues were harvested from AIA mice for RNA and protein extraction to examine cytokine level by qPCR and ELISA. Indeed, lower synovial GM-CSF but not IFN-γ or IL-17 was detected in Stat5^−/− mice than Stat5^+/+ mice at day 5 or 7 after arthritis induction (FIGS. 28E and 30A-30C). In addition, two important proinflammatory cytokines IL-6 and IL-1β were also found persistently and significantly reduced in STAT5-deficient mice (FIGS. 28E and 30A-30C), indicating the attenuated synovial inflammation. Notably, TNF-α production was reduced at day 7 but not at day 5 in STAT5-deficient mice (FIGS. 28E and 30A-C). Together, these results indicate that STAT5-regulated GM-CSF expression by arthritogenic T_H cells is crucial for evoking synovial inflammation.

To determine the critical role of STAT5-regulated GM-CSF production by T_H cells in mediating synovitis and arthritis development, GM-CSF was administered via intra-articular injection in mixture with mBSA to the left knee joints of mBSA/CFA-immunized mice, whereas mBSA was injected alone to the right knee joints. Injection with mBSA alone was sufficient to induce abundant immune cell infiltration in the synovial compartments of Stat5^+/+ mice but failed to do so in Stat5^−/− mice (FIG. 28F). Administration of GM-CSF together with mBSA efficiently restored synovial inflammation in Stat5^−/− mice (FIG. 34F). Consistently, the Safranin-O/Fast Green staining revealed severe cartilage depletion upon GM-CSF/mBSA injection, but not mBSA alone, in Stat5^−/− mice (FIG. 28G). These results therefore provide support for the notion that STAT5-regulated GM-CSF production by arthritogenic T_H cells is essential for mediating arthritis pathogenesis.

Example 13. Th-Cell-Derived GM-CSF Mediates Neutrophil Accumulation in Synovial Tissues

The mechanism by which GM-CSF-producing Th cells evoke synovial inflammation and drive arthritis development was examined. Myeloid lineage-derived cells, including neutrophils, DCs and macrophages, express GM-CSF receptor and are common targets of GM-CSF (Hamilton, 2008). Importantly, those cells invade synovial compartments in RA patients and mouse arthritis models, and contribute to synovitis (McInnes and Schett, 2011). The infiltration of myeloid lineage-derived cells in synovial compartments of AIA mice was examined. CD11b⁺ myeloid cells represented a predominant population (˜70%) among synovial infiltrating leukocytes (FIG. 31B). Although CD4⁺ T_H cell infiltration was not altered by STAT5 deletion, synovial CD11b⁺ cell infiltration was significantly reduced in Stat5^−/− mice compared with Stat5^+/+ mice when examined at day 7 after arthritis induction (FIG. 31B). This reduction is unlikely due to defective hematopoiesis, as similar frequencies of CD11b⁺ cells were detected in spleens of two group (FIG. 31A). Further, CD11b⁺ cells continuously increased in synovial tissues of wild-type mice, but not STAT5-deficient mice, over a 7-day time course (FIG. 31C). Notably, the selective ablation of synovial CD11b⁺ cell accumulation in STAT5-deficient mice can be partially restored by local administration of GM-CSF during arthritis induction (FIG. 31D). Together, these results indicate that myeloid cell accumulation in synovial compartments may be specifically dampened by T-cell-specific STAT5 deletion and resultant GM-CSF insufficiency.

Next, different populations of CD11b⁺ cells, including DCs, macrophages and neutrophils were analyzed. Monocyte-derived dendritic cells (MoDCs), characterized as CD11c^intCD11b^hiLy6C^+/hiMHCII^hi, were recently reported to be involved in the mBSA/IL-1β arthritis model (Campbell et al., 2011). In the AIA model of the present study, MoDCs were identified at low abundance in spleens and synovial tissues (data not shown). Furthermore, comparable frequencies of MoDCs were detected in both peripheral lymphoid tissues and synovial tissues between Stat5^+/+ and Stat5^−/− mice (data not shown). These results are in agreement with a previous study showing a dispensable role of GM-CSF in MoDC differentiation (Greter et al., 2012).

Neutrophils have great cytotoxic potential and contribute to the RA initiation and progression in multiple ways (Wright et al., 2014). It has been suggested that RA disease activity and joint destruction directly correlates with neutrophil influx to joints (Wright et al., 2014). Based on the differential expression of Ly6C and Ly6G, CD11b⁺ myeloid cells can be classified into Ly6C^loLy6G^hi population (neutrophils) and Ly6C^hiLy6G⁻ population (monocytes/macrophages). The present study shows that Ly6C^loLy6G^hi population continued to accumulate in synovial tissues over a 7-day time course, and represented a predominant population among synovial CD11b⁺ cells in wild-type mice at day 7 after AIA induction, whereas this population was persistently and dramatically diminished in STAT5-deficient mice (FIG. 32A). Using Giemsa stain, it was validated that synovial-infiltrating Ly6C^loLy6G^hi population were neutrophils, which displayed typical polymorphonuclear characteristics with ring-shaped nuclei (FIG. 32B). In contrast, synovial-infiltrating Ly6C^hiLy6G⁻ population had mononuclear morphology and were likely monocytes/macrophages (FIG. 32B). Importantly, intraarticular administration of GM-CSF during arthritis induction efficiently restored neutrophil accumulation in synovial compartments of STAT5-deficient mice (FIG. 32C), suggesting a critical role of T_H-cell-derived GM-CSF in mediating neutrophil accumulation to inflamed joints.

Neutrophils are recruited during inflammation, in which complex interactions between neutrophils and vascular endothelial cells direct neutrophil adhesion and transmigration from circulation to inflamed tissues (Kolaczkowska and Kubes, 2013). In an in vitro transmigration assay, neutrophil adhesion and migration across monolayers of endothelial cells was significantly enhanced by GM-CSF as chemoattractant (FIGS. 33A and 33B), suggesting GM-CSF may mediate neutrophil recruitment to inflamed joints in AIA. Effective neutrophil apoptosis is crucial for the resolution of inflammation. However, in synovitis, neutrophil apoptosis is delayed with a result of extended survival and persistent inflammation (Wright et al., 2014). Thus, the effect of GM-CSF on neutrophil survival was tested and found that GM-CSF had profound efficacy in delaying neutrophil apoptosis (FIG. 33C). Together, these results indicate that GM-CSF may mediate neutrophil recruitment and sustain neutrophil survival in synovial compartments and contribute to persistent synovitis. To determine the critical role of neutrophils in AIA, \ neutralizing antibody (1A8) specific for Ly6G was used to deplete neutrophils in vivo. The administration of neutralizing antibody resulted in significant improvement of joint swelling in AIA (FIG. 32D). Thus, neutrophils accumulation mediated by T_H-cell-derived GM-CSF are important for AIA development.

Example 14. GM-CSF Enhances Proinflammatory Cytokine Production by Myeloid Cells and Synovial Fibroblasts

Cytokines are important mediators in the cross-talk between innate and adaptive immunity. As shown herein, several proinflammatory cytokines (IL-6, IL-1β and TNF-α), which are in association with RA pathogenesis (Choy and Panayi, 2001), were significantly reduced in synovial tissues of STAT5-deficient AIA mice (FIGS. 28E and 30A-30C). To gain insights into the mechanism underlying the observed cytokine reduction, the cellular sources of these proinflammatory cytokines were examined by fractionating synovial cells into different populations based the differential expression of surface markers (FIG. 34A). Cytokine mRNA expression level in CD45⁺ TCRβ⁺ population (T cells), CD45⁺ TCRβ⁻CD11c⁻ CD11b⁺ population (mostly monocytes/macrophages and neutrophils) and CD45⁺ TCRβ⁻ CD11c⁺ population (dendritic cells) was assessed by RT-PCR. GM-CSF, as similar to IL-17 (as a control), was predominantly produced by synovial T cells (FIG. 34B), further reinforcing the importance of GM-CSF-producing T_H cells. In contrast, IL-6 and IL-1β were mainly produced by myeloid cells, e.g. CD11b⁺ population and CD11c⁺ population (FIG. 34B). TNF-α was expressed by all three populations, with relatively lower abundance in T cells (FIG. 34B). Based on the differential expression of Ly6C and Ly6G in CD11b⁺ population as discussed above, Ly6C^loLy6G^hi population (neutrophils) and Ly6C^hiLy6G⁻ population (monocytes/macrophages) were further analyzed, which showed that monocytes/macrophages were likely the major IL-6 producers whereas neutrophils seemed to be better producers of IL-1β and TNF-α (FIG. 34C). These results, together with the findings above (FIGS. 28E and 30A-30C), indicate a link that T_H-cell-secreted GM-CSF elicits proinflammatory cytokine expression from myeloid cells in synovitis.

To test the regulatory role of GM-CSF in the expression of IL-6 and IL-1β, bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs) were cultured, and stimulated with GM-CSF. Indeed, GM-CSF stimulation quickly upregulated mRNA expression of both IL-6 and IL-1β within 1 hour (FIGS. 34D and 34E). In addition, GM-CSF markedly increased the secretion of IL-6 from BMDMs in a dosage-dependent manner (FIG. 34F), and from BMDCs even at low dosage (FIG. 34G). To induce mature IL-1β secretion, BMDMs were primed with LPS for 6 h during which different concentrations of GM-CSF was added, followed by ATP stimulation. The addition of GM-CSF significantly enhanced the secretion of IL-1β into culture supernatant as measured by ELISA (FIG. 34H). Synovial fibroblasts, the active players in synovial inflammation (Muller-Ladner et al., 2007), also showed increased IL-1β mRNA expression upon GM-CSF stimulation (FIG. 34I). An inducible effect of GM-CSF on TNF-α expression was not observed in BMDMs, BMDCs or synovial fibroblasts (data not shown). Given the functional importance of IL-6 and IL-1β in arthritis development (Choy and Panayi, 2001), T_H-cell-secreted GM-CSF may mediate synovial inflammation also via eliciting the expression of IL-6 and IL-1β from myeloid cells and synovial fibroblasts.

TABLE 1
Summary of genes differentially expressed in TH1, TH17, and TH-GM cells
Genes differentially	Genes differentially	Genes differentially	Genes upregulated on	Genes downregulated on
expressed in TH1	expressed in TH17	upregulated in TH-GM cells	TH-GM surface	TH-GM cells surface
Gene ID	Gene Title	Gene ID	Gene Title	Gene ID	Gene Title	Gene ID	Gene Title	Gene Symbol	Gene Title
10366586	interferon gamma	10353415	interleukin 17F	10385918	interleukin 3	10435704	CD80 antigen	Ly6a	lymphocyte
									antigen 6 complex,
									locus A
10598013	chemokine (C-C	10511779	ATPase, H+	10511363	preproenkephalin	10548409	killer cell lectin-	Cd27	CD27 antigen
	motif) receptor 5 ///		transporting,				like receptor
	chemokine (C-C		lysosomal VO				subfamily C,
	motif) receptor 2		subunit D2				member 1
10523717	secreted	10345762	interleukin 1	10497878	interleukin 2	10421737	tumor necrosis	Sell	selectin,
	phosphoprotein 1		receptor, type I				factor (ligand)		lymphocyte
							superfamily,
							member 11
10420308	granzyme B	10359697	chemokine (C	10385912	colony	10597420	chemokine (C-C	Ctsw	cathepsin W
			motif) ligand 1		stimulating factor		motif) receptor 4
					2 (granulocyte-
					macrophage)
10545135	interleukin 12	10587639	5′ nucleotidase,	10404422	serine (or	10441633	chemokine (C-C	Ltb	lymphotoxin B
	receptor, beta 2		ecto		cysteine)		motif) receptor 6
					peptidase
					inhibitor, clade
					B, member 6b
10531724	placenta-specific 8	10501860	formin binding	10408689	neuritin 1	10365933	early endosome	Gngt2	guanine nucleotide
			protein 1-like				antigen 1		binding protein (G
									protein), gamma
									transducing activity
									polypeptide 2 ///
									ABI gene family,
									member 3
10363070	glycoprotein 49 A	10345032	interleukin 17A	10467979	stearoyl-	10404840	CD83 antigen	Gpr18	G protein-coupled
	/// leukocyte				Coenzyme A				receptor 18
	immunoglobulin-				desaturase 1
	like receptor,
	subfamily B,
	member 4
10363082	leukocyte	10446965	RAS, guanyl	10469312	phosphotriesterase	10359434	Fas ligand (TNF	Igfbp4	insulin-like growth
	immunoglobulin-		releasing protein 3		related ///		superfamily,		factor binding
	like receptor,				C1q-like 3		member 6)		protein 4
	subfamily B,
	member 4
10424683	lymphocyte antigen	10565990	ADP-	10435704	CD80 antigen	10344966	lymphocyte	Il17ra	interleukin 17
	6 complex, locus G		ribosyltransferase				antigen 96		receptor A
			2a
10552406	natural killer cell	10465059	cathepsin W	10502655	cysteine rich	10345752	interleukin 1	Il18r1	interleukin 18
	group 7 sequence				protein 61		receptor,		receptor 1
							type II
10603151	glycoprotein m6b	10358476	proteoglycan 4	10350159	ladinin	10439527	T cell	Klrd1	killer cell lectin-
			(megakaryocyte				immunoreceptor		like receptor,
			stimulating factor,				with Ig and ITIM		subfamily D,
			articular				domains		member 1
			superficial zone
			protein)
10360173	SLAM family	10471953	activin receptor	10548409	killer cell lectin-	10494595	Notch gene	Mctp2	multiple C2
	member 7		IIA		like receptor		homolog 2		domains,
					subfamily C,		(Drosophila)		transmembrane 2
					member 1
10455961	interferon inducible	10400006	aryl-hydrocarbon	10571399	zinc finger,	10597279	chemokine (C-C	Ms4a6b	membrane-
	GTPase 1		receptor		DHHC domain		motif) receptor-		spanning 4-
					containing 2		like 2		domains, subfamily
									A, member 6B
10400304	EGL nine homolog	10409876	cytotoxic T	10538791	TNFAIP3	10485405	CD44 antigen	Pld3	phospholipase D
	3 (C. elegans)		lymphocyte-		interacting				family, member 3
			associated protein		protein 3
			2 alpha
10574023	metallothionein 2	10388591	carboxypeptidase	10407126	polo-like kinase	10561104	AXL receptor	Pyhin1	pyrin and HIN
			D		2 (Drosophila)		tyrosin kinase		domain family,
									member 1
10493108	cellular retinoic	10390640	IKAROS family	10355984	serine (or	10585048	cell adhesion	S1pr1	sphingosine-1-
	acid binding protein		zinc finger 3		cysteine)		molecule 1		phosphate receptor
	II				peptidase				1
					inhibitor, clade
					E, member 2
10375436	family with	10590623	chemokine (C-X-	10421737	tumor necrosis			Slc44a2	solute carrier
	sequence similarity		C motif) receptor		factor (ligand)				family 44, member
	71, member B		6		superfamily,				2
					member 11
10398039	serine (or cysteine)	10367734	uronyl-2-	10503023	cystathionase
	peptidase inhibitor,		sulfotransferase		(cystathionine
	clade A, member 3F				gamma-lyase)
	/// serine (or
	cysteine) peptidase
	inhibitor, clade A,
	member 3G
10349108	serine (or cysteine)	10500656	CD101 antigen	10389207	chemokine (C-C
	peptidase inhibitor,				motif) ligand 5
	clade B, member 5
10607738	carbonic anhydrase	10347895	WD repeat	10361887	PERP, TP53
	5b, mitochondrial		domain 69		apoptosis
					effector
10496539	guanylate binding	10495854	protease, serine,	10530841	insulin-like
	protein 5		12 neurotrypsin		growth factor
			(motopsin)		binding protein 7
10373918	leukemia inhibitory	10425049	apolipoprotein	10504838	nuclear receptor
	factor		L9b ///		subfamily 4,
			apolipoprotein		group A, member
			L9a		3
10455954	predicted gene 4951	10378286	integrin alpha E,	10482762	isopentenyl-
			epithelial-		diphosphate delta
			associated		isomerase
10598976	tissue inhibitor of	10362896	CD24a antigen	10597420	chemokine (C-C
	metalloproteinase 1				motif) receptor 4
10492136	doublecortin-like	10409866	cytotoxic T	10441633	chemokine (C-C
	kinase 1		lymphocyte-		motif) receptor 6
			associated protein
			2 beta
10405211	growth arrest and	10400989	potassium	10595402	family with
	DNA-damage-		voltage-gated		sequence
	inducible 45 gamma		channel,		similarity 46,
			subfamily H (eag-		member A
			related), member
			5
10503202	chromodomain	10590242	chemokine (C-C	10480139	C1q-like 3 ///
	helicase DNA		motif) receptor 8		phosphotriesterase
	binding protein 7				related
10542275	ets variant gene 6	10407435	aldo-keto	10540472	basic helix-loop-
	(TEL oncogene)		reductase family		helix family,
			1, member C18		member e40
10556820	transmembrane	10592023	amyloid beta	10404429	serine (or
	protein 159		(A4) precursor-		cysteine)
			like protein 2		peptidase
					inhibitor, clade
					B, member 9
10444291	histocompatibility	10359480	dynamin 3	10595404	family with
	2, class II antigen A,				sequence
	beta 1				similarity 46,
					member A
10439299	stefin A3	10475544	sema domain,	10365933	early endosome
			transmembrane		antigen 1
			domain (TM), and
			cytoplasmic
			domain,
			(semaphorin) 6D
10547641	solute carrier family	10409767	golgi membrane	10384373	fidgetin-like 1
	2 (facilitated glucose		protein 1
	transporter),
	member 3
10503200	chromodomain	10392464	family with	10400072	scinderin
	helicase DNA		sequence
	binding protein 7		similarity 20,
			member A
10544320	RIKEN cDNA	10504891	transmembrane	10377938	enolase 3, beta
	1810009J06 gene ///		protein with EGF-		muscle
	predicted gene 2663		like and two
			follistatin-like
			domains 1
10503218	chromodomain	10504817	transforming	10589994	eomesodermin
	helicase DNA		growth factor,		homolog
	binding protein 7		beta receptor I		(Xenopus laevis)
10503198	chromodomain	10393559	tissue inhibitor of	10404840	CD83 antigen
	helicase DNA		metalloproteinase
	binding protein 7		2
10507594	solute carrier family	10474419	leucine-rich	10485624	proline rich Gla
	2 (facilitated glucose		repeat-containing		(G-
	transporter),		G protein-coupled		carboxyglutamic
	member 1		receptor 4		acid) 4
					(transmembrane)
10438626	ets variant gene 5	10456492	DNA segment,	10369102	predicted gene
			Chr 18, ERATO		9766
			Doi 653,
			expressed
10390328	T-box 21	10345241	dystonin	10505030	fibronectin type
					III and SPRY
					domain
					containing 1-like
10574027	metallothionein 1	10471555	angiopoietin-like	10606868	brain expressed
			2		gene 1
10493820	S100 calcium	10494821	tetraspanin 2	10501832	ATP-binding
	binding protein A6				cassette, sub-
	(calcyclin)				family D (ALD),
					member 3
10376324	predicted gene	10542355	epithelial	10457225	mitogen-
	12250		membrane protein		activated protein
			1		kinase kinase
					kinase 8
10406852	calponin 3, acidic	10500295	pleckstrin	10554521	phosphodiesterase
			homology domain		8A
			containing, family
			O member 1
10412076	gem (nuclear	10375402	a disintegrin and	10446229	tumor necrosis
	organelle)		metallopeptidase		factor (ligand)
	associated protein 8		domain 19		superfamily,
			(meltrin beta)		member 9
10496555	guanylate binding	10484227	SEC14 and	10593842	tetraspanin 3
	protein 1 ///		spectrin domains
	guanylate binding		1
	protein 5
10345074	centrin 4	10472097	formin-like 2	10407211	phosphatidic
					acid phosphatase
					type 2A
10503194	chromodomain	10587829	procollagen	10488655	BCL2-like 1
	helicase DNA		lysine, 2-
	binding protein 7		oxoglutarate 5-
			dioxygenase 2
10537561	RIKEN cDNA	10530536	tec protein	10470182	brain expressed
	1810009J06 gene ///		tyrosine kinase		myelocytomatosis
	predicted gene 2663				oncogene
10439895	activated leukocyte	10586700	RAR-related	10445977	Epstein-Barr
	cell adhesion		orphan receptor		virus induced
	molecule		alpha		gene 3
10459772	lipase, endothelial	10354191	ring finger	10587495	interleukin-1
			protein 149		receptor-
					associated kinase
					1 binding protein
					1
10439762	S-	10438738	B-cell	10419082	RIKEN cDNA
	adenosylhomocysteine		leukemia/lymphoma		5730469M10
	hydrolase		6		gene
10482030	stomatin	10347888	chemokine (C-C	10472212	plakophilin 4
			motif) ligand 20
10459905	SET binding	10440131	G protein-	10487588	interleukin 1
	protein 1		coupled receptor		alpha
			15
10357833	ATPase, Ca++	10453057	cytochrome P450,	10359434	Fas ligand (TNF
	transporting, plasma		family 1,		superfamily,
	membrane 4		subfamily b,		member 6)
			polypeptide 1 ///
			RIKEN cDNA
			1700038P13 gene
10475517	expressed sequence	10542140	killer cell lectin-	10351015	serine (or
	AA467197 ///		like receptor		cysteine)
	microRNA 147		subfamily B		peptidase
			member 1F		inhibitor, clade C
					(antithrombin),
					member 1
10585778	sema domain,	10471880	microRNA 181b-	10344966	lymphocyte
	immunoglobulin		2		antigen 96
	domain (Ig), and
	GPI membrane
	anchor,
	(semaphorin) 7A
10354529	RIKEN cDNA	10542791	PTPRF	10488415	cystatin C
	1700019D03 gene		interacting
			protein, binding
			protein 1 (liprin
			beta 1)
10582275	solute carrier family	10583242	sestrin 3	10598771	monoamine
	7 (cationic amino				oxidase A
	acid transporter, y+
	system), member 5
10576034	interferon	10489569	phospholipid	10345752	interleukin 1
	regulatory factor 8		transfer protein ///		receptor, type II
			cathepsin A
10503222	chromodomain	10523297	cyclin G2	10588577	cytokine
	helicase DNA				inducible SH2-
	binding protein 7				containing
					protein
10503220	chromodomain	10381187	ATPase, H+	10439527	T cell
	helicase DNA		transporting,		immunoreceptor
	binding protein 7		lysosomal V0		with Ig and ITIM
			subunit A1		domains
10503210	chromodomain	10346651	bone	10511258	family with
	helicase DNA		morphogenic		sequence
	binding protein 7		protein receptor,		similarity 132,
			type II		member A
			(serine/threonine
			kinase)
10476945	cystatin F	10490159	prostate	10403584	nidogen 1
	(leukocystatin)		transmembrane
			protein, androgen
			induced 1
10503216	chromodomain	10389581	yippee-like 2	10399973	histone
	helicase DNA		(Drosophila)		deacetylase 9
	binding protein 7
10366983	transmembrane	10581992	avian	10494595	Notch gene
	protein 194		musculoaponeurotic		homolog 2
			fibrosarcoma		(Drosophila)
			(v-maf) AS42
			oncogene
			homolog
10495675	coagulation factor	10413250	cytoplasmic	10346168	signal transducer
	III		polyadenylated		and activator of
			homeobox		transcription 4
10421697	RIKEN cDNA	10555063	integrator	10350630	family with
	9030625A04 gene		complex subunit 4		sequence
					similarity 129,
					member A
10445112	ubiquitin D	10406982	a disintegrin-like	10564667	neurotrophic
			and		tyrosine kinase,
			metallopeptidase		receptor, type 3
			(reprolysin type)
			with
			thrombospondin
			type 1 motif, 6
10530627	leucine rich repeat	10596303	acid phosphatase,	10419288	GTP
	containing 66		prostate		cyclohydrolase 1
10440019	transmembrane	10357472	chemokine (C-X-	10407535	ribosomal
	protein 45a		C motif) receptor		protein L10A ///
			4		ribosomal protein
					L10A,
					pseudogene 2
10378783	ribosomal protein	10545130	growth arrest and	10468945	acyl-Coenzyme
	L36		DNA-damage-		A binding
			inducible 45 alpha		domain
					containing 7
10447341	ras homolog gene	10436402	claudin domain	10435271	HEG homolog 1
	family, member Q		containing 1		(zebrafish)
	///
	phosphatidylinositol
	glycan anchor
	biosynthesis, class F
10373452	predicted gene 129	10539135	capping protein	10576639	neuropilin 1
			(actin filament),
			gelsolin-like
10454286	microtubule-	10428534	trichorhinophalan-	10505059	T-cell acute
	associated protein,		geal syndrome I		lymphocytic
	RP/EB family,		(human)		leukemia 2
	member 2
10572497	interleukin 12	10368675	myristoylated	10457091	neuropilin
	receptor, beta 1		alanine rich		(NRP) and
			protein kinase C		tolloid (TLL)-
			substrate		like 1
10368060	epithelial cell	10531910	hydroxysteroid	10428081	heat-responsive
	transforming		(17-beta)		protein 12
	sequence 2		dehydrogenase 13
	oncogene-like
10471457	ST6 (alpha-N-	10370303	adenosine	10435712	CD80 antigen
	acetyl-neuraminyl-		deaminase, RNA-
	2,3-beta-galactosyl-		specific, B1
	1,3)-N-
	acetylgalactosaminide
	alpha-2,6-
	sialyltransferase 4
10374366	epidermal growth	10592888	chemochine (C-	10597279	chemokine (C-C
	factor receptor		X-C motif)		motif) receptor-
			receptor 5		like 2
10450501	tumor necrosis	10503259	transformation	10485405	CD44 antigen
	factor		related protein 53
			inducible nuclear
			protein 1
10347291	chemokine (C-X-C	10446771	lysocardiolipin	10436662	microRNA 155
	motif) receptor 2		acyltransferase 1
10553501	solute carrier family	10428579	exostoses	10562044	zinc finger and
	17 (sodium-		(multiple) 1		BTB domain
	dependent inorganic				containing 32
	phosphate
	cotransporter),
	member 6
10345824	interleukin 18	10476314	prion protein	10463599	nuclear factor of
	receptor accessory				kappa light
	protein				polypeptide gene
					enhancer in B-
					cells 2, p49/p100
10458314	transmembrane	10406598	serine	10456005	CD74 antigen
	protein 173		incorporator 5		(invariant
					polypeptide of
					major
					histocompatibility
					complex, class
					II antigen-
					associated)
10388430	serine (or cysteine)	10461765	leupaxin	10490903	carbonic
	peptidase inhibitor,				anhydrase 13
	clade F, member 1
10496015	phospholipase A2,	10428536	trichorhinophalangeal	10468762	RIKEN cDNA
	group XIIA		syndrome I		4930506M07
			(human)		gene
10510391	spermidine synthase	10362245	erythrocyte	10470316	na
			protein band 4.1-
			like 2
10486396	EH-domain	10604008	predicted gene	10363195	heat shock factor
	containing 4		10058 ///		2
			predicted gene
			10230 ///
			predicted gene
			10486 ///
			predicted gene
			14632 ///
			predicted gene
			14819 ///
			predicted gene
			4836 ///
			predicted gene
			2012 ///
			predicted gene
			5169 ///
			predicted gene
			6121 /// Sycp3
			like X-linked ///
			predicted gene
			5168 ///
			predicted gene
			10488 ///
			predicted gene
			14525 ///
			predicted gene
			5935
10368054	epithelial cell	10409857	RIKEN cDNA	10596652	HemK
	transforming		4930486L24 gene		methyltransferase
	sequence 2				family member 1
	oncogene-like
10608637	na	10522368	NIPA-like	10435693	cytochrome c
			domain containing		oxidase, subunit
			1		XVII assembly
					protein homolog
					(yeast)
10595718	carbohydrate	10368720	solute carrier	10544660	oxysterol
	sulfotransferase 2		family 16		binding protein-
			(monocarboxylic		like 3
			acid transporters),
			member 10
10496580	guanylate binding	10438639	diacylglycerol	10384725	reticuloendotheliosis
	protein 3		kinase, gamma		oncogene
10594053	promyelocytic	10499431	synaptotagmin XI	10408600	serine (or
	leukemia				cysteine)
					peptidase
					inhibitor, clade
					B, member 6a
10544829	JAZF zinc finger 1	10565840	neuraminidase 3	10391444	RUN domain
					containing 1 ///
					RIKEN cDNA
					1700113I22 gene
10601778	armadillo repeat	10494023	RAR-related	10561516	nuclear factor of
	containing, X-linked		orphan receptor		kappa light
	3		gamma		polypeptide gene
					enhancer in B-
					cells inhibitor,
					beta
10355967	adaptor-related	10391103	junction	10566846	DENN/MADD
	protein complex AP-		plakoglobin		domain
	1, sigma 3				containing 5A
10592503	cytotoxic and	10417053	muscleblind-like	10435048	Tctex1 domain
	regulatory T cell		2		containing 2
	molecule
10496023	caspase 6	10350341	microRNA 181b-	10470175	lipocalin 13
			1
10599192	LON peptidase N-	10459071	RIKEN cDNA	10586250	DENN/MADD
	terminal domain and		2010002N04 gene		domain
	ring finger 3				containing 4A
10467578	phosphoinositide-3-	10463476	Kazal-type serine	10512774	coronin, actin
	kinase adaptor		peptidase inhibitor		binding protein
	protein 1		domain 1		2A
10585703	ribonuclease P 25	10348537	receptor	10366546	carboxypeptidase
	subunit (human)		(calcitonin)		M
			activity modifying
			protein 1
10365482	tissue inhibitor of	10348432	ArfGAP with	10354286	KDEL (Lys-
	metalloproteinase 3		GTPase domain,		Asp-Glu-Leu)
			ankyrin repeat and		containing 1
			PH domain 1
10469151	inter-alpha	10576332	tubulin, beta 3 ///	10547621	apolipoprotein B
	(globulin) inhibitor		melanocortin 1		mRNA editing
	H5		receptor		enzyme, catalytic
					polypeptide 1
10503192	chromodomain	10554094	insulin-like	10440419	B-cell
	helicase DNA		growth factor I		translocation
	binding protein 7		receptor		gene 3 /// B-cell
					translocation
					gene 3
					pseudogene
10593050	interleukin 10	10495794	phosphodiesterase	10407467	aldo-keto
	receptor, alpha		5A, cGMP-		reductase family
			specific		1, member E1
10597648	myeloid	10569504	tumor necrosis	10558580	undifferentiated
	differentiation		factor receptor		embryonic cell
	primary response		superfamily,		transcription
	gene 88		member 23		factor 1
10538290	sorting nexin 10	10452516	ankyrin repeat	10544644	na
			domain 12
10503204	chromodomain	10534596	cut-like	10424543	WNT1 inducible
	helicase DNA		homeobox 1		signaling
	binding protein 7				pathway protein
					1
10353707	protein tyrosine	10362073	serum/glucocorticoid	10507137	PDZK1
	phosphatase 4a1 ///		regulated		interacting
	protein tyrosine		kinase 1		protein 1
	phosphatase 4a1-
	like
10377010	SCO cytochrome	10408331	acyl-CoA	10384691	RIKEN cDNA
	oxidase deficient		thioesterase 13		0610010F05
	homolog 1 (yeast)				gene
10440903	RIKEN cDNA	10415413	NYN domain and	10565315	fumarylacetoacetate
	4932438H23 gene		retroviral		hydrolase
			integrase
			containing
10521205	SH3-domain	10598359	synaptophysin	10586248	DENN/MADD
	binding protein 2				domain
					containing 4A
10604587	microRNA 363	10544114	homeodomain	10561104	AXL receptor
			interacting protein		tyrosine kinase
			kinase 2
10571958	SH3 domain	10436128	myosin, heavy	10385837	interleukin 13
	containing ring		chain 15
	finger 1
10357553	interleukin 24	10408450	SRY-box	10440393	SAM domain,
			containing gene 4		SH3 domain and
					nuclear
					localization
					signals, 1
10606730	armadillo repeat	10487011	glycine	10401987	potassium
	containing, X-linked		amidinotransferase		channel,
	6		(L-		subfamily K,
			arginine:glycine		member 10
			amidinotransferase)
10564960	furin (paired basic	10378833	slingshot	10453715	RAB18, member
	amino acid cleaving		homolog 2		RAS oncogene
	enzyme)		(Drosophila)		family
10402585	tryptophanyl-tRNA	10521498	collapsin	10496466	alcohol
	synthetase		response mediator		dehydrogenase 4
			protein 1		(class II), pi
					polypeptide
10417095	FERM, RhoGEF	10538939	eukaryotic	10396712	fucosyltransferase
	(Arhgef) and		translation		8
	pleckstrin domain		initiation factor 2
	protein 1		alpha kinase 3
	(chondrocyte-
	derived)
10442435	ribonucleic acid	10585276	POU domain,	10603708	calcium/calmodulin-
	binding protein S1		class 2,		dependent
			associating factor		serine protein
			1		kinase (MAGUK
					family)
10394990	membrane bound	10512156	aquaporin 3	10352178	saccharopine
	O-acyltransferase				dehydrogenase
	domain containing 2				(putative) ///
					similar to
					Saccharopine
					dehydrogenase
					(putative)
10538753	atonal homolog 1	10469110	USP6 N-terminal	10349081	PH domain and
	(Drosophila)		like		leucine rich
					repeat protein
					phosphatase 1
10351667	signaling	10568392	regulator of G-	10364950	growth arrest
	lymphocytic		protein signalling		and DNA-
	activation molecule		10		damage-
	family member 1				inducible 45 beta
10461844	guanine nucleotide	10603346	proteolipid	10566877	SET binding
	binding protein,		protein 2		factor 2
	alpha q polypeptide
10422057	ribosomal protein	10353947	transmembrane	10575160	nuclear factor of
	L7A		protein 131		activated T-cells
					5
10572897	heme oxygenase	10452633	TGFB-induced	10458090	receptor
	(decycling) 1		factor homeobox		accessory protein
			1		5
10507784	palmitoyl-protein	10380289	monocyte to	10439845	predicted gene
	thioesterase 1		macrophage		5486
			differentiation-
			associated
10445702	ubiquitin specific	10521969	IMP1 inner	10461558	solute carrier
	peptidase 49		mitochondrial		family 15,
			membrane		member 3
			peptidase-like (S.
			cerevisiae)
10569057	ribonuclease/	10521678	CD38 antigen	10586254	DENN/MADD
	angiogenin				domain
	inhibitor 1				containing 4A
10370471	1-acylglycerol-3 -	10592515	ubiquitin	10574166	copine II
	phosphate O-		associated and
	acyltransferase 3		SH3 domain
			containing, B
10586591	carbonic	10512470	CD72 antigen	10598467	proviral
	anyhydrase 12				integration site 2
10512701	translocase of outer	10587085	cDNA sequence	10447084	galactose
	mitochondrial		BC031353		mutarotase
	membrane 5
	homolog (yeast)
10462702	HECT domain	10492689	platelet-derived	10366346	pleckstrin
	containing 2		growth factor, C		homology-like
			polypeptide		domain, family
					A, member 1
10552740	nucleoporin 62 ///	10514221	perilipin 2	10355567	transmembrane
	Nup62-Il4i1 protein				BAX inhibitor
					motif containing
					1
10581996	chromodomain	10458247	leucine rich	10407420	neuroepithelial
	protein, Y		repeat		cell transforming
	chromosome-like 2		transmembrane		gene 1
			neuronal 2
10363901	ets variant gene 5	10468898	lymphocyte	10411882	neurolysin
			transmembrane		(metallopeptidase
			adaptor 1		M3 family)
10520862	fos-like antigen 2	10555059	potassium	10585048	cell adhesion
			channel		molecule 1
			tetramerisation
			domain containing
			14
10526520	procollagen-lysine,	10408629	RIKEN cDNA	10538890	hypothetical
	2-oxoglutarate 5-		1300014I06 gene		protein
	dioxygenase 3				LOC641050
10571274	glutathione	10546510	leucine-rich	10406681	adaptor-related
	reductase		repeats and		protein complex
			immunoglobulin-		3, beta 1 subunit
			like domains 1
10351206	selectin, platelet	10544596	transmembrane	10455647	tumor necrosis
			protein 176B		factor, alpha-
					induced protein 8
10493474	mucin 1,	10361748	F-box protein 30	10447521	transcription
	transmembrane				factor B1,
					mitochondrial ///
					T-cell lymphoma
					invasion and
					metastasis 2
10370000	glutathione S-	10356291	RIKEN cDNA	10523772	leucine rich
	transferase, theta 1		A530040E14 gene		repeat containing
					8D
10500272	predicted gene 129	10581450	DEAD (Asp-Glu-	10417759	ubiquitin-
			Ala-Asp) box		conjugating
			polypeptide 28		enzyme E2E 2
					(UBC4/5
					homolog, yeast)
10452815	xanthine	10414417	pellino 2	10586244	DENN/MADD
	dehydrogenase				domain
					containing 4A
10393823	prolyl 4-	10372528	potassium large	10436500	glucan (1,4-
	hydroxylase, beta		conductance		alpha-),
	polypeptide		calcium-activated		branching
			channel,		enzyme 1
			subfamily M, beta
			member 4 ///
			RIKEN cDNA
			1700058G18 gene
10408280	leucine rich repeat	10408613	tubulin, beta 2B	10556297	adrenomedullin
	containing 16A
10575685	nudix (nucleoside	10411274	synaptic vesicle	10593492	zinc finger
	diphosphate linked		glycoprotein 2c		CCCH type
	moiety X)-type				containing 12C
	motif 7
10599174	interleukin 13	10456357	phorbol-12-	10373358	interleukin 23,
	receptor, alpha 1		myristate-13-		alpha subunit p19
			acetate-induced
			protein 1
10458940	zinc finger protein	10511498	pleckstrin	10358583	hemicentin 1
	608		homology domain
			containing, family
			F (with FYVE
			domain) member
			2
10476197	inosine	10402136	G protein-	10567995	nuclear protein 1
	triphosphatase		coupled receptor
	(nucleoside		68
	triphosphate
	pyrophosphatase)
10419790	ajuba	10549990	vomeronasal 1	10512030	RIKEN cDNA
			receptor, G10 ///		3110043O21
			vomernasal 1		gene
			receptor Vmn1r-
			ps4 ///
			vomeronasal 1
			receptor 3 ///
			vomeronasal 1
			receptor
			Vmn1r238 ///
			vomeronasal 1
			receptor 2
10364909	ornithine	10554789	cathepsin C	10594652	lactamase, beta
	decarboxylase
	antizyme 1 ///
	ornithine
	decarboxylase
	antizyme 1
	pseudogene
10503190	chromodomain	10427928	triple functional	10344960	transmembrane
	helicase DNA		domain (PTPRF		protein 70
	binding protein 7		interacting)
10516932	sestrin 2	10549162	ST8 alpha-N-	10399908	protein kinase,
			acetyl-		cAMP dependent
			neuraminide		regulatory, type
			alpha-2,8-		II beta
			sialyltransferase 1
10585338	KDEL (Lys-Asp-	10482109	mitochondrial	10605766	melanoma
	Glu-Leu) containing		ribosome		antigen, family
	2		recycling factor		D, 1
			/// RNA binding
			motif protein 18
10464425	G protein-coupled	10425092	cytohesin 4	10474141	solute carrier
	receptor kinase 5				family 1 (glial
					high affinity
					glutamate
					transporter),
					member 2
10441601	T-cell activation	10356866	programmed cell	10461909	cDNA sequence
	Rho GTPase-		death 1		BC016495
	activating protein
10482059	glycoprotein	10554204	ATP/GTP	10548030	CD9 antigen
	galactosyltransferase		binding protein-
	alpha 1,3		like 1
10522411	cell wall biogenesis	10403229	integrin beta 8	10525473	transmembrane
	43 C-terminal				protein 120B
	homolog (S.
	cerevisiae)
10369276	coiled-coil domain	10374529	expressed	10435266	HEG homolog 1
	containing 109A		sequence		(zebrafish)
			AV249152
10368970	PR domain	10565434	ribosomal protein	10593483	ferredoxin 1
	containing 1, with		S13
	ZNF domain
10369541	hexokinase 1	10431266	ceramide kinase	10476569	RIKEN cDNA
					2310003L22
					gene
10374236	uridine	10410124	cathepsin L	10526718	sperm motility
	phosphorylase 1				kinase 3A ///
					sperm motility
					kinase 3B ///
					sperm motility
					kinase 3C
10489660	engulfment and cell	10441003	runt related	10547613	ribosomal
	motility 2, ced-12		transcription		modification
	homolog (C.		factor 1		protein rimK-like
	elegans)				family member B
10488797	peroxisomal	10555303	phosphoglucomutase	10511446	aspartate-beta-
	membrane protein 4		2-like 1		hydroxylase
10558090	transforming, acidic	10530215	RIKEN cDNA	10375137	potassium large
	coiled-coil		1110003E01 gene		conductance
	containing protein 2				calcium-activated
					channel,
					subfamily M,
					beta member 1
10409265	AU RNA binding	10480275	nebulette	10528154	predicted gene
	protein/enoyl-				6455 /// RIKEN
	coenzyme A				cDNA
	hydratase				4933402N22
					gene
10374364	thymoma viral	10434302	kelch-like 24	10514173	ribosomal
	proto-oncogene 2		(Drosophila)		protein L34 ///
					predicted gene
					10154 ///
					predicted
					pseudogene
					10086 ///
					predicted gene
					6404
10598575	LanC lantibiotic	10565002	CREB regulated	10586227	DENN/MADD
	synthetase		transcription		domain
	component C-like 3		coactivator 3		containing 4A
	(bacterial)
10439514	growth associated	10413338	na	10402648	brain protein 44-
	protein 43				like
10497842	Bardet-Biedl	10523670	AF4/FMR2	10575745	ATM interactor
	syndrome 7 (human)		family, member 1
10462091	Kruppel-like factor	10478594	cathepsin A	10346255	ORMl-like 1 (S.
	9 /// predicted gene				cerevisiae)
	9971
10498024	solute carrier family	10514128	tetratricopeptide	10400405	nuclear factor of
	7 (cationic amino		repeat domain		kappa light
	acid transporter, y+		39B		polypeptide gene
	system), member 11				enhancer in B-
					cells inhibitor,
					alpha
10483719	chimerin	10535956	StAR-related	10528527	family with
	(chimaerin) 1		lipid transfer		sequence
			(START) domain		similarity 126,
			containing 13		member A
10606694	Bruton	10503695	BTB and CNC	10472738	DDB1 and
	agammaglobulinemia		homology 2		CUL4 associated
	tyrosine kinase				factor 17
10443110	synaptic Ras	10584334	sialic acid	10368534	nuclear receptor
	GTPase activating		acetylesterase		coactivator 7
	protein 1 homolog
	(rat)
10368062	epithelial cell	10502890	ST6 (alpha-N-	10407543	GTP binding
	transforming		acetyl-		protein 4
	sequence 2		neuraminyl-2,3-
	oncogene-like		beta-galactosyl-
			1,3)-N-
			acetylgalactosami
			nide alpha-2,6-
			sialyltransferase 3
10575693	vesicle amine	10564467	leucine rich	10376555	COP9
	transport protein 1		repeat containing		(constitutive
	homolog-like (T.		28		photomorphogenic)
	californica)				homolog,
					subunit 3
					(Arabidopsis
					thaliana)
10562897	zinc finger protein	10345715	mitogen-activated	10567297	inositol 1,4,5-
	473 /// vaccinia		protein kinase		triphosphate
	related kinase 3		kinase kinase		receptor
			kinase 4		interacting
					protein-like 2
10373709	eukaryotic	10568668	a disintegrin and	10589886	RIKEN cDNA
	translation initiation		metallopeptidase		4930520O04
	factor 4E nuclear		domain 12		gene
	import factor 1		(meltrin alpha)
10487238	histidine	10462406	RIKEN cDNA	10423593	lysosomal-
	decarboxylase		C030046E11 gene		associated
					protein
					transmembrane
					4B
10594988	mitogen-activated	10472649	myosin IIIB	10577954	RAB11 family
	protein kinase 6				interacting
					protein 1 (class I)
10422436	dedicator of	10363894	inositol	10604528	muscleblind-like
	cytokinesis 9		polyphosphate		3 (Drosophila)
			multikinase
10459084	synaptopodin	10606058	chemokine (C-X-	10432675	RIKEN cDNA
			C motif) receptor		I730030J21 gene
			3
10567450	dynein, axonemal,	10439955	family with	10385747	PHD finger
	heavy chain 3		sequence		protein 15
			similarity 55,
			member C
10604751	fibroblast growth	10530615	OCIA domain	10398240	echinoderm
	factor 13		containing 2		microtubule
					associated
					protein like 1
10584827	myelin protein	10528183	spermatogenesis	10511803	RIKEN cDNA
	zero-like 2		associated		2610029I01 gene
			glutamate (E)-rich
			protein 4d ///
			spermatogenesis
			associated
			glutamate (E)-rich
			protein 4c ///
			spermatogenesis
			associated
			glutamate (E)-rich
			protein 4e ///
			predicted gene
			9758 /// RIKEN
			cDNA
			4930572O03 gene
			///
			spermatogenesis
			associated
			glutamate (E)-rich
			protein 7,
			pseudogene 1 ///
			predicted gene
			7361
10473356	ubiquitin-	10488507	abhydrolase	10466606	annexin A1
	conjugating enzyme		domain containing
	E2L 6		12
10498350	purinergic receptor	10420668	microRNA 15a	10520304	ARP3 actin-
	P2Y, G-protein				related protein 3
	coupled, 14				homolog B
					(yeast)
10497862	transient receptor	10469951	ring finger	10425903	na
	potential cation		protein 208
	channel, subfamily
	C, member 3
10368056	epithelial cell	10501629	CDC14 cell	10488709	RIKEN cDNA
	transforming		division cycle 14		8430427H17
	sequence 2		homolog A (S.		gene
	oncogene-like		cerevisiae)
10425357	Smith-Magenis	10386789	Unc-51 like	10376096	acyl-CoA
	syndrome		kinase 2 (C.		synthetase long-
	chromosome region,		elegans)		chain family
	candidate 7-like				member 6
	(human)
10498952	guanylate cyclase 1,	10401138	ATPase, H+	10429491	activity
	soluble, alpha 3		transporting,		regulated
			lysosomal VI		cytoskeletal-
			subunit D		associated
					protein
10548905	epidermal growth	10554118	family with	10439710	pleckstrin
	factor receptor		sequence		homology-like
	pathway substrate 8		similarity 169,		domain, family
			member B		B, member 2
10579703	calcium	10603843	synapsin I	10467110	expressed
	homeostasis				sequence
	endoplasmic				AI747699
	reticulum protein ///
	RIKEN cDNA
	1700030K09 gene
10404630	RIO kinase 1	10575184	WW domain	10536898	interferon
	(yeast)		containing E3		regulatory factor
			ubiquitin protein		5
			ligase 2
10518069	EF hand domain	10537712	glutathione S-	10505044	fukutin
	containing 2		transferase kappa
			1
10469672	glutamic acid	10511541	dpy-19-like 4 (C.	10605370	membrane
	decarboxylase 2		elegans)		protein,
					palmitoylated
10526941	RIKEN cDNA	10394816	predicted gene	10363669	DnaJ (Hsp40)
	D830046C22 gene		9282		homolog,
					subfamily C,
					member 12
10567448	dynein, axonemal,	10587503	SH3 domain	10496727	dimethylarginine
	heavy chain 3		binding glutamic		dimethylaminohydrolase
			acid-rich protein		1
			like 2
10437885	myosin, heavy	10411359	proteolipid	10587683	B-cell
	polypeptide 11,		protein 2		leukemia/lymphoma
	smooth muscle				2 related
					protein A1a ///
					B-cell
					leukemia/lymphoma
					2 related
					protein A1d ///
					B-cell
					leukemia/lymphoma
					2 related
					protein A1b ///
					B-cell
					leukemia/lymphoma
					2 related
					protein A1c
10600122	X-linked	10579939	ubiquitin specific	10458816	toll-like receptor
	lymphocyte-		peptidase 38 ///		adaptor molecule
	regulated 3B /// X-		predicted gene		2
	linked lymphocyte-		9725
	regulated 3C /// X-
	linked lymphocyte-
	regulated 3A
10587665	RIKEN cDNA	10370242	poly(rC) binding	10513008	Kruppel-like
	4930579C12 gene		protein 3		factor 4 (gut)
		10350753	glutamate-	10550906	plasminogen
			ammonia ligase		activator,
			(glutamine		urokinase
			synthetase)		receptor
		10456296	mucosa	10362674	U3A small
			associated		nuclear RNA
			lymphoid tissue
			lymphoma
			translocation gene
			1
		10380571	guanine	10473190	DnaJ (Hsp40)
			nucleotide binding		homolog,
			protein (G		subfamily C,
			protein), gamma		member 10
			transducing
			activity
			polypeptide 2 ///
			ABI gene family,
			member 3
		10369413	sphingosine	10477581	ribosomal
			phosphate lyase 1		protein L5
		10552276	ubiquitin-	10571774	aspartylglu-
			conjugating		cosaminidase
			enzyme E2H ///
			predicted gene
			2058
		10394532	ubiquitin-	10395356	anterior gradient
			conjugating		homolog 3
			enzyme E2F		(Xenopus laevis)
			(putative) ///
			ubiquitin-
			conjugating
			enzyme E2F
			(putative)
			pseudogene
		10556463	aryl hydrocarbon	10392440	solute carrier
			receptor nuclear		family 16
			translocator-like		(monocarboxylic
					acid
					transporters),
					member 6
		10471994	kinesin family	10352815	interferon
			member 5C		regulatory factor
					6
		10395328	sorting nexin 13
		10599348	glutamate
			receptor,
			ionotropic,
			AMPA3 (alpha 3)
		10601595	RIKEN cDNA
			3110007F17 gene
			/// predicted gene
			6604 ///
			predicted gene
			5167 ///
			predicted gene
			2411 ///
			predicted gene
			14957
		10372891	SLIT-ROBO Rho
			GTPase activating
			protein 1
		10355024	islet cell
			autoantigen 1-like
		10518147	podoplanin
		10473537	olfactory receptor
			1123
		10424411	tumor
			susceptibility gene
			101
		10439960	centrosomal
			protein 97
		10551852	CAP-GLY
			domain containing
			linker protein 3
		10599291	reproductive
			homeobox 4E ///
			reproductive
			homeobox 4G ///
			reproductive
			homeobox 4F ///
			reproductive
			homeobox 4A ///
			reproductive
			homeobox 4C ///
			reproductive
			homeobox 4B ///
			reproductive
			homeobox 4D
		10587315	glutathione S-
			transferase, alpha
			4
		10447167	metastasis
			associated 3
		10480288	nebulette
		10491300	SKI-like
		10596637	mitogen-activated
			protein kinase-
			activated protein
			kinase 3
		10518019	DNA-damage
			inducible protein
			2 /// regulatory
			solute carrier
			protein, family 1,
			member 1
		10384685	RIKEN cDNA
			1700093K21 gene
		10439483	Rho GTPase
			activating protein
			31
		10353844	neuralized
			homolog 3
			homolog
			(Drosophila)
		10459604	RIKEN cDNA
			4933403F05 gene
		10488892	transient receptor
			potential cation
			channel,
			subfamily C,
			member 4
			associated protein
		10542822	RAB15 effector
			protein
		10553354	neuron navigator
			2
		10425966	ataxin 10
		10360506	thymoma viral
			proto-oncogene 3
		10531610	RasGEF domain
			family, member
			1B
		10417787	guanine
			nucleotide binding
			protein (G
			protein), gamma 2
		10381588	granulin
		10437080	tetratricopeptide
			repeat domain 3
		10509560	ribosomal protein
			L38
		10466886	na
		10580457	NEDD4 binding
			protein 1
		10451061	runt related
			transcription
			factor 2
		10433953	yippee-like 1
			(Drosophila)
		10447461	stonin 1
		10501909	methyltransferase
			like 14 /// Sec24
			related gene
			family, member D
			(S. cerevisiae)
		10519693	sema domain,
			immunoglobulin
			domain (Ig), short
			basic domain,
			secreted,
			(semaphorin) 3D
		10385557	CCR4-NOT
			transcription
			complex, subunit
			6
		10413047	plasminogen
			activator,
			urokinase
		10406663	arylsulfatase B
		10430113	Rho GTPase
			activating protein
			39
		10475830	mitochondrial
			ribosomal protein
			S5
		10410892	RAS p21 protein
			activator 1
		10515994	stromal
			membrane-
			associated
			GTPase-activating
			protein 2
		10410099	CDC14 cell
			division cycle 14
			homolog B (S.
			cerevisiae)
		10428157	ring finger
			protein 19A
		10563643	tumor
			susceptibility gene
			101
		10412260	follistatin ///
			thyroid hormone
			receptor
			associated protein
			3
		10386539	similar to
			ubiquitin A-52
			residue ribosomal
			protein fusion
			product 1
		10415574	cyclin I
		10494978	protein tyrosine
			phosphatase, non-
			receptor type 22
			(lymphoid)
		10511416	thymocyte
			selection-
			associated high
			mobility group
			box
		10562500	dpy-19-like 3 (C.
			elegans)
		10568135	proline-rich
			transmembrane
			protein 2 ///
			RIKEN cDNA
			2900092E17 gene
		10514466	Jun oncogene
		10500847	membrane
			associated
			guanylate kinase,
			WW and PDZ
			domain containing
			3
		10549760	zinc finger
			protein 580
		10549377	RIKEN cDNA
			1700034J05 gene
		10430174	apolipoprotein
			L9a ///
			apolipoprotein
			L9b
		10474333	elongation
			protein 4 homolog
			(S. cerevisiae)
		10560791	predicted gene,
			EG381936 ///
			predicted gene
			6176
		10407159	ankyrin repeat
			domain 55
		10603659	mediator complex
			subunit 14
		10576854	cortexin 1
		10353775	BEN domain
			containing 6
		10573865	predicted gene
			3579
		10356886	solute carrier
			organic anion
			transporter family,
			member 4C1
		10507273	phosphatidylinositol
			3 kinase,
			regulatory
			subunit,
			polypeptide 3
			(p55)
		10424252	WDYHV motif
			containing 1
		10518735	splA/ryanodine
			receptor domain
			and SOCS box
			containing 1
		10562576	pleckstrin
			homology domain
			containing, family
			F (with FYVE
			domain) member
			1
		10375667	ring finger
			protein 130
		10528268	protein tyrosine
			phosphatase, non-
			receptor type 12
		10593205	REX2, RNA
			exonuclease 2
			homolog (S.
			cerevisiae)
		10576056	microtubule-
			associated protein
			1 light chain 3
			beta
		10547916	parathymosin
		10377689	gamma-
			aminobutyric acid
			receptor
			associated protein
		10602307	ovary testis
			transcribed ///
			predicted gene
			15085 ///
			predicted gene
			15127 ///
			predicted gene,
			OTTMUSG00000019001 ///
			leucine zipper
			protein 4 ///
			predicted gene
			15097 ///
			predicted gene
			15091 ///
			predicted gene
			10439 ///
			predicted gene
			15128
		10426835	DIP2 disco-
			interacting protein
			2 homolog B
			(Drosophila)
		10439798	DAZ interacting
			protein 3, zinc
			finger
		10375614	glutamine
			fructose-6-
			phosphate
			transaminase 2
		10361882	NHS-like 1
		10419274	glia maturation
			factor, beta
		10424781	glutamate
			receptor,
			ionotropic, N-
			methyl D-
			aspartate-
			associated protein
			1 (glutamate
			binding)
		10546960	na
		10514713	WD repeat
			domain 78
		10394954	grainyhead-like 1
			(Drosophila)
		10437205	Purkinje cell
			protein 4
		10464251	attractin like 1
		10496251	3-
			hydroxybutyrate
			dehydrogenase,
			type 2
		10396383	solute carrier
			family 38,
			member 6
		10585794	cytochrome P450,
			family 11,
			subfamily a,
			polypeptide 1
		10385719	Sec24 related
			gene family,
			member A (S.
			cerevisiae)
		10407358	polyadenylate
			binding protein-
			interacting protein
			1
		10498775	golgi integral
			membrane protein
			4
		10584435	von Willebrand
			factor A domain
			containing 5A
		10466304	deltex 4 homolog
			(Drosophila)
		10598292	forkhead box P3
			/// RIKEN cDNA
			4930524L23 gene
			/// coiled-coil
			domain containing
			22
		10472440	Tax1 (human T-
			cell leukemia
			virus type I)
			binding protein 3
		10398455	protein
			phosphatase 2,
			regulatory subunit
			B (B56), gamma
			isoform
		10493076	SH2 domain
			protein 2A
		10409152	RIKEN cDNA
			1110007C09 gene
		10542880	RIKEN cDNA
			4833442J19 gene
		10378523	Smg-6 homolog,
			nonsense
			mediated mRNA
			decay factor (C.
			elegans)
		10531560	anthrax toxin
			receptor 2
		10467319	retinol binding
			protein 4, plasma
		10395978	predicted gene
			527
		10471715	mitochondrial
			ribosome
			recycling factor
		10511755	WW domain
			containing E3
			ubiquitin protein
			ligase 1
		10353754	zinc finger
			protein 451
		10477572	chromatin
			modifying protein
			4B
		10359161	sterol O-
			acyltransferase 1
		10462035	lactate
			dehydrogenase B
		10543319	family with
			sequence
			similarity 3,
			member C
		10579052	predicted gene
			10033
		10475532	sulfide quinone
			reductase-like
			(yeast)
		10428857	metastasis
			suppressor 1
		10475144	calpain 3 ///
			glucosidase,
			alpha; neutral C
		10396645	zinc finger and
			BTB domain
			containing 1
		10428302	Kruppel-like
			factor 10
		10577882	heparan-alpha-
			glucosaminide N-
			acetyltransferase
		10548069	dual-specificity
			tyrosine-(Y)-
			phosphorylation
			regulated kinase 4
		10436053	developmental
			pluripotency
			associated 2
		10401564	RIKEN cDNA
			1110018G07 gene
		10471535	family with
			sequence
			similarity 129,
			member B
		10349404	mannoside
			acetylglucosaminyltransferase
			5
		10520173	amiloride-
			sensitive cation
			channel 3
		10508860	solute carrier
			family 9
			(sodium/hydrogen
			exchanger),
			member 1
		10374500	vacuolar protein
			sorting 54 (yeast)
		10387723	RIKEN cDNA
			2810408A11 gene
		10488020	thioredoxin-
			related
			transmembrane
			protein 4
		10411126	junction-
			mediating and
			regulatory protein
		10345706	DNA segment,
			Chr 1, Brigham &
			Women's Genetics
			0212 expressed
		10364375	cystatin B
		10480379	mitochondrial
			ribosomal protein
			S5
		10521243	G protein-
			coupled receptor
			kinase 4
		10497920	ankyrin repeat
			domain 50
		10593723	acyl-CoA
			synthetase
			bubblegum family
			member 1
		10375634	mitogen-activated
			protein kinase 9
		10384555	aftiphilin
		10468113	Kv channel-
			interacting protein
			2
		10423363	progressive
			ankylosis
		10538150	transmembrane
			protein 176 A
		10396485	synaptic nuclear
			envelope 2
		10401007	protein
			phosphatase 2,
			regulatory subunit
			B (B56), epsilon
			isoform
		10419151	eosinophil-
			associated,
			ribonuclease A
			family, member 1
		10390768	SWI/SNF related,
			matrix associated,
			actin dependent
			regulator of
			chromatin,
			subfamily e,
			member 1
		10478145	protein
			phosphatase 1,
			regulatory
			(inhibitor) subunit
			16B
		10433057	calcium binding
			and coiled coil
			domain 1
		10545921	MAX
			dimerization
			protein 1
		10392449	WD repeat
			domain,
			phosphoinositide
			interacting 1
		10545608	sema domain,
			immunoglobulin
			domain (Ig), TM
			domain, and short
			cytoplasmic
			domain
		10567219	ADP-ribosylation
			factor-like 6
			interacting protein
			1
		10471201	c-abl oncogene 1,
			receptor tyrosine
			kinase
		10505841	predicted gene
			13271 ///
			predicted gene
			13290 ///
			predicted gene
			13277 ///
			predicted gene
			13276
		10414360	lectin, galactose
			binding, soluble 3
		10403258	guanosine
			diphosphate
			(GDP)
			dissociation
			inhibitor 2
		10476759	Ras and Rab
			interactor 2
		10430866	cytochrome P450,
			family 2,
			subfamily d,
			polypeptide 10
		10432619	POU domain,
			class 6,
			transcription
			factor 1
		10521972	protocadherin 7
		10350646	ER degradation
			enhancer,
			mannosidase
			alpha-like 3
		10440993	regulator of
			calcineurin 1
		10505008	solute carrier
			family 44,
			member 1
		10566670	olfactory receptor
			478
		10356172	phosphotyrosine
			interaction domain
			containing 1
		10418506	stabilin 1
		10419429	olfactory receptor
			723 /// olfactory
			receptor 724
		10581434	dipeptidase 2
		10401365	zinc finger,
			FYVE domain
			containing 1
		10591188	olfactory receptor
			843
		10565846	signal peptidase
			complex subunit 2
			homolog (S.
			cerevisiae)
		10467258	myoferlin
		10548547	predicted gene
			6600
		10523012	deoxycytidine
			kinase
		10348547	ubiquitin-
			conjugating
			enzyme E2F
			(putative)
		10483667	corepressor
			interacting with
			RBPJ, 1
		10584071	PR domain
			containing 10
		10585249	protein
			phosphatase 2
			(formerly 2A),
			regulatory subunit
			A (PR 65), beta
			isoform
		10546137	ankyrin repeat
			and BTB (POZ)
			domain containing
			1
		10484720	olfactory receptor
			1166
		10571415	vacuolar protein
			sorting 37A
			(yeast)
		10595189	solute carrier
			family 17
			(anion/sugar
			transporter),
			member 5
		10584426	olfactory receptor
			910
		10585986	myosin IXa
		10401753	VPS33B
			interacting
			protein, apical-
			basolateral
			polarity regulator
		10349793	dual
			serine/threonine
			and tyrosine
			protein kinase
		10527528	SWI/SNF related,
			matrix associated,
			actin dependent
			regulator of
			chromatin,
			subfamily e,
			member 1
		10485767	olfactory receptor
			1277
		10557459	mitogen-activated
			protein kinase 3
		10471486	endoglin
		10420846	frizzled homolog
			3 (Drosophila)
		10405849	olfactory receptor
			466
		10568691	RIKEN cDNA
			A130023I24 gene
		10351111	dynamin 3,
			opposite strand ///
			microRNA214 ///
			microRNA 199a-2
		10540785	RIKEN cDNA
			6720456B07 gene
		10540923	makorin, ring
			finger protein, 2
		10413416	interleukin 17
			receptor D
		10386636	ubiquitin specific
			peptidase 22
		10383799	transcobalamin 2

REFERENCES

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While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A method of treating an inflammatory disorder in a patient, comprising administering an effective amount of a modulating agent that inhibits Th-GM function, wherein the patient exhibits a limited response or no response to a TNF-α inhibitor therapy, wherein the inflammatory disorder is mediated by granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting T-helper (Th-GM) cellswherein the modulating agent is a signal transducer and activator of transcription 5 (STAT5) inhibitor, andwherein the inflammatory disorder is rheumatoid arthritis.

2. The method of claim 1, wherein the STAT5 inhibitor is an antibody or a small molecule.

3. The method of claim 1, wherein the modulating agent is pimozide or CAS 285986-31-4.

4. The method of claim 1, wherein the patient has an increased expression of STAT5, IL-7, GM-CSF or IL-3 relative to a reference level.

5. The method of claim 1, wherein the TH-GM cells are differentiated from precursor CD4+ cells in the presence of activated STAT5 and IL-7.

6. The method of claim 5, wherein the TH-GM cell expresses GM-CSF and IL-3.

7. The method of claim 5, wherein the TH-GM cells overexpress one or more of basic helix-loop-helix family, member e40 (BHLHe40), preproenkephalin (PENK), IL-2, serine or cysteine peptidase inhibitor, clade B member 6b (Serpinb6b), neuritin 1 (Nrn1), stearoyl-Coenzyme A desaturase 1 (Scdl), and phosphotriesterase related Clq-like 3 (Pter).

8. The method of claim 5, wherein the TH-GM cells underexpress one or more of lymphocyte antigen 6 complex, locus A (Ly6a); CD27; or selectin, lymphocyte (Sell).

9. The method of claim 1, further comprises contacting a sample collected from the patient who exhibits a limited response or no response to a TNF-α therapy with a detecting agent that detects a polypeptide or nucleic acid level of STAT5, IL-7, GM-CSF or IL-3, or a combination thereof; andquantifying the polypeptide or nucleic acid level of STAT5, IL-7, GM-CSF or IL-3, or a combination thereof, wherein an increased level of STAT5, IL-7, GM-CSF or IL-3, or a combination thereof relative to a reference level indicates that the patient suffers from the TH-GM-mediated inflammatory disorder.

10. The method of claim 9, further comprising contacting the sample with a detecting agent that detects a polypeptide or nucleic acid level of at least one gene selected from the group consisting of basic helix-loop-helix family member e40 (BHLHE40), Chemokine (C-C Motif) Receptor 4 (CCR4), and Chemokine (C-C Motif) Receptor 6 (CCR6).

11. A method of treating an inflammatory disorder in a patient, comprising administering an effective amount of a modulating agent that inhibits Th-GM function, wherein the patient exhibits a limited response or no response to a TNF-α inhibitor therapy,wherein the modulating agent is pimozide, andwherein the inflammatory disorder is rheumatoid arthritis.