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 (TH) 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: TH1, TH2, TH17, iTreg and TFH, which are modulated by cytokine milieus. TH1 and TH17 cells are known to be the primary effectors of inflammation. However, the pathogenic roles of either TH1 or TH17 in various inflammatory disorders remain unclear. For example, recent studies conflict with previously understood paradigm of TH17 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 TH cells, termed TH-GM, which represent a distinct helper T cell subset with unique developmental and functional characteristics. Identified herein is an inflammatory pathway mediated by TH-GM cells (TH-GM-mediated inflammatory pathway), which represents an independent inflammatory pathway apart from known non-TH-GM-mediated inflammatory pathways (e.g., TNF-α, IL-6, and IL-1b pathways of inflammation). The present disclosure provides methods and compositions for diagnosing inflammatory disorders that are TH-GM-mediated, and modulating TH-GM cell function for the treatment of inflammatory disorders mediated by the TH-GM pathway.


Accordingly, in one aspect, the present disclosure provides a method of diagnosing a TH-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 TH-GM-mediated inflammatory disorder.


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


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


In some aspects, the present disclosure provides a method of treating a TH-GM-mediated inflammatory disorder in a patient in need thereof, comprising administering to said patient an effective amount of a modulating agent that modulates TH-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 TH-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 TH-GM cell function, comprising contacting an isolated population of TH-GM cells, or an isolated population of CD4+ precursor cells, with a candidate agent, and determining a readout of TH-GM function in the presence or absence of the candidate agent, wherein a change in the readout of TH-GM function indicates that the candidate agent is a modulator of TH-GM function.


The present disclosure enables the identification or classification between inflammatory disorders that are either primarily TH-GM-mediated, or primarily non-TH-GM-mediated (e.g., mediated by TNF-α, IL-6, and/or IL-1β3), 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 TH-GM-mediated, or primarily non-TH-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 TH-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-ID 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 MOG35-55/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 MOG35-55/CFA (FIG. 1C, n=5 per group) or immunized twice with MOG35-55/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 2nd 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 TH1, TH17 or Treg 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 MOG35-55/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 MOG35-55/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 MOG35-55-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 MOG35-55/CFA-immunized Stat5+/+ and Stat5−/− mice (n=3 per group) before disease onset and challenged with MOG35-55 at various concentrations for 24 h. GM-CSF secretion was measured by ELISA (FIG. 8A). Golgiplug was added in the last 4 h of MOG35-55 (20 μg/ml) challenge and the frequencies of IL-17+ and GM-CSF+ cells among CD4+CD44hiT cells were measured (FIG. 8B). In FIGS. 8C and 8C, splenocytes were obtained from MOG35-55/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+CD44hi 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 MOG35-55/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 MOG35-55/CFA-immunized WT mice before disease onset and challenged with MOG35-55 (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+CD44hi 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 MOG35-55/CFA-immunized Stat5+/+ and Stat5−/− mice before disease onset and challenged with MOG35-55 (20 μg/ml) in the absence or presence of IL-7 for 48 h. Frequencies of GM-CSF+ and IL-17+ cells among CD4+CD44hi 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 CD62LhiCD44lo and CD62LloCD44hi T cells from MOG35-55/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 2nd immunization, as indicated by arrows. Data represent two independent experiments (FIG. 12A). Spinal cord sections were obtained from EAE mice at day 11 after 2nd 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 TH cells is distinct from TH17 and TH1. 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 TH-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 TH-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 TH-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, TH1 (IL-12+anti-IL-4), TH17 (TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and TH-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 TH-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 TH-GM cells. In FIGS. 20A and 20B, naïve CD4+ T cells were activated with anti-CD3 and anti-CD28 under TH1-(IL-12+anti-IL-4), TH17-(TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and TH-GM-(GM-CSF+ TH, 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 MOG35-55-specific TH subsets.



FIGS. 22A-27E depict inhibition of STAT5 activation suppresses TH-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 TH-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 TH 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 TH 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 TH 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+CD44hi 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+CD44hi 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 Ly6ChiLy6G and Ly6CloLy6Ghi cells from synovial tissues of AIA mice. Scale bar, 100 μm (left) or 20 μm (right). (FIG. 32C) Flow cytometric analysis of Ly6ChiLy6G and Ly6CloLy6Ghi 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+ TCRβCD11cCD11b+ (CD11b+) and CD45+ TCRβCD11c+ (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 Ly6ChiLy6G and Ly6CloLy6Ghi 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 “TH-GM”. As detailed herein, IL-7/STAT5 signaling programs the differentiation of precursor CD4+ cells to TH-GM, a process which is further modulated by IL-2 and IL-23 signaling. TH-GM cells are characterized by, e.g., GM-CSF and IL-3 production. TH-GM cells are distinct from the known helper T cells TH1 and TH17, 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) TH1 and TH17, respectively, potently suppress the development of TH-GM from naïve CD4+ precursor cells, establishing that TH-GM cells develop via a lineage distinct from TH1 and TH17. Thus, the present disclosure provides a distinct network of factors, unique from factors known to mediate TH1 or TH17, that mediate TH-GM function (e.g., its differentiation and pathogenicity).


As shown herein, TH-GM cells preferentially induce EAE as compared with TH1 and TH17 cells, indicating that TH-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 TH-GM cells. Further, blockade of TH-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 TH-GM pathway (e.g., a disorder that results from TH-GM pathogenicity through the action of, e.g., GM-CSF and/or IL-3, or any factor associated with the TH-GM pathway), or an inflammatory disorder that is mediated by, e.g., TNF-α, IL-6, and/or IL-1β pathways (i.e., non-TH-GM-mediated pathway). For example, a patient who has, e.g., RA may be afflicted with a type of RA that is primarily TH-GM-mediated, or primarily non-TH-GM-mediated (e.g., TNF-α-mediated or IL-6 mediated). The present disclosure enables the classification between TH-GM-mediated and non-TH-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 TH-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 TH-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 TH-GM function to treat autoimmune and inflammatory disorders. The methods of modulating TH-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 TH-GM function in an effective amount to modulate the function (e.g., development and pathogenicity) of TH-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 TH-GM cells (i.e., TH-GM pathway mediated) or by non-TH-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 TH-GM-mediated.


Accordingly, in one aspect, the present disclosure provides a method of diagnosing a TH-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 TH-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 TH-GM-mediating factor (e.g., STAT5, IL-7, GM-CSF or IL-3, or a combination thereof), wherein an increased level of a TH-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 TH-GM-mediated inflammatory disorder, thereby diagnosing a TH-GM-mediated inflammatory disorder in a patient suffering from an inflammatory disorder.


As used herein, a “TH-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 TH-GM function (a “TH-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 TH-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-TH-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 TH-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-TH-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 “TH-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-TH-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 TH-GM-mediated inflammatory disorder does not necessarily exclude the possibility that the inflammatory disorder could also be partially non-TH-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 “TH-GM-mediated” is synonymous with “primarily/predominantly TH-GM-mediated”, and a classification as “non-TH-GM-mediated” is synonymous with “primarily/predominantly non-TH-GM-mediated.” For example, without wishing to be bound by any particular theory, an inflammatory disorder in its early stage may be TH-GM-mediated. As the inflammatory condition advances to a late stage characterized by, e.g., tissue damage, the inflammatory disorder becomes progressively non-TH-GM-mediated. In some embodiments, a TH-GM-mediated inflammatory disorder is a condition that is responsive to modulation of TH-GM function, as determined by clinical standards; a non-TH-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 TH-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 TH-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 TH-GM cells as well as genes that are differentially expressed on the surface of TH-GM cells, as compared to TH1 or TH17 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 TH-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 TH-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 TH-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-TH-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-TH-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 TH-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-TH-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 TH-GM-mediated inflammatory disorder does not have a non-TH-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 TH-GM-mediated inflammatory disorder responds to modulation of TH-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 TH-GM-mediated inflammatory disorder does not exclude the possibility that the inflammatory disorder is also partially (though not primarily) contributed by a non-TH-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 TH-GM cell function to the patient diagnosed or classified as having a TH-GM-mediated inflammatory disorder. As described herein, in some embodiments, the modulating agent inhibits TH-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-TH-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 TH-GM-mediated inflammatory disorder or a non-TH-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 TH-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 TH-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 TH-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 TH-GM-mediated inflammatory disorder; or a comparable level of the TH-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-TH-GM-mediated inflammatory disorder, thereby classifying the patient suffering from an inflammatory disorder as a TH-GM-mediated inflammatory disorder or a non-TH-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-TH-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 TH-GM-mediated inflammatory disorder and the patient can be treated with an agent that modulates TH-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-TH-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 TH-GM-mediated and non-TH-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 TH-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-TH-GM-mediated. However, in this scenario, the patient may receive a combined treatment with an agent that modulates TH-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 TH-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 TH-GM cells. During, for example, the late stages of an inflammatory disorder, TH-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 TH-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-TH-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 TH-GM-mediating factor, as described herein, and one or more non-TH-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 TH-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 TH-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 TH-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 TH-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 (TH-GM). In one embodiment, the TH-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 TH-GM cells express GM-CSF and IL-3.


In some embodiments, the TH-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 TH-GM cell is inhibited by IL-12, IFN-γ, TFG-β, and/or IL-6.


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


In some embodiments, the TH-GM cells are further characterized by an overexpression of one or more genes listed in Table 1. For example, the TH-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 TH-GM cells are further characterized by an underexpression of one or more genes listed in Table 1. For example, the TH-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 TH1 or TH17) that mediate TH-GM function (e.g., its differentiation and pathogenicity) enables targeted modulation of TH-GM function to treat TH-GM-mediated disorders, e.g., disorders that result from aberrant TH-GM function. Thus, in some aspects, the present disclosure provides a method of modulating TH-GM function, comprising contacting the TH-GM, or cluster of differentiation 4 (CD4+) precursor cells, or both, with a modulating agent that modulates TH-GM function. In one embodiment, the modulating agent is contacted with the TH-GM cells or CD4+ precursor cells in vitro or in vivo.


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


As used herein, a T helper subset cell “TH-GM” refers to a cell that, similar to TH1 and TH17 cells, differentiates from precursor CD4+ precursor cells, but which commits and develops through a pathway that is mediated by a subset of factors (the TH-GM-mediating factors) that is distinct and unique from the known subset of factors that commit and develop TH1 or TH17 cell subtypes, as described herein. In some embodiments, a TH-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 TH1 or TH17 cell subtypes. For example, a TH-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 TH-GM-mediated inflammatory disorder in a patient in need thereof, comprising administering to said patient an effective amount of a modulating agent that modulates TH-GM cell function. In certain embodiments, the patient is previously diagnosed as having a TH-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 TH-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 TH-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 TH-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 TH-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 TH-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 TH-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 TH-GM function at the protein and/or nucleic acid level, or through another mechanism.


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


In some embodiments, the modulating agent that inhibits TH-GM function can be a small molecule that inhibits, directly or indirectly, one or more of the factors that mediate TH-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 TH-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 TH-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 TH-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-TH-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 TH-GM function. Accordingly, an agent that modulates TH-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 TH-GM function and a TNF-α therapy will depend on the nature of the therapeutic agent(s). In addition, an agent that modulates TH-GM function and a TNF-α therapy may or may not be administered on similar dosing schedules. For example, the agent that modulates TH-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 TH-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 TH-GM cells as well as the identification of genes differentially produced by TH-GM cells relative to TH1 or TH17 enables the use of TH-GM cells to identify novel therapeutics for modulating TH-GM function, thereby enabling new therapeutics for treating TH-GM-mediated disorders (e.g., inflammatory disorders). Thus, in further aspects, the present disclosure provides a method of screening to identify a modulator of TH-GM cell function, comprising contacting an isolated population of TH-GM cells, or an isolated population of CD4+ precursor cells, with a candidate agent, and measuring a readout of TH-GM function in the presence or absence of the candidate agent, wherein a change in the readout of TH-GM function indicates that the candidate agent is a modulator of TH-GM function.


As used herein, a candidate agent refers to an agent that may modulate TH-GM function by modulating the function (e.g., expression and/or activity) of a factor that mediates TH-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 TH-GM function, as described herein, including the genes listed in Table 1 (e.g., genes preferentially upregulated in TH-GM cells, genes preferentially overexpressed/underexpressed on the surface of TH-GM cells).


As used herein, “readout” refers to any change (or lack of change) in TH-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 TH-GM cells, or its effect on the abundance of TH-GM cells (through an effect on the commitment/development/proliferation of TH-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 TH-GM function (e.g., decrease in GM-CSF or IL-3 production, or decrease in the abundance of TH-GM cells), thereby identifying the candidate agent as an inhibitor of TH-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 TH-GM function (e.g., increase in GM-CSF or IL-3 production, or increase in the abundance of TH-GM cells), thereby identifying the candidate agent as an enhancer of TH-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 TH-GM cells. Thus, a candidate agent that downregulates a gene that is preferentially upregulated in a TH-GM cell is a inhibitor of TH-GM function. Similarly, a candidate agent that upregulates a gene that is preferentially downregulated in a TH-GM cell is an enhancer of TH-GM function.


In certain aspects, the method of screening, if performed with precursor CD4+ cells, is performed under TH-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


Stat5f/f mice were provided by L. Hennighausen (National Institute of Diabetes and Digestive and Kidney Diseases). Stat3f/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+CD25CD62LhiCD44lo) 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 TH 1 conditions, IL-12 (10 ng/ml), and anti-IL-4 (10 μg/ml, BD Pharmingen); for TH 17 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 TH 17 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 MOG35-55 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 MOG35-55/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 Stat5f/f or Cd4-Cre; Stat5f/f mice were transferred, followed by MOG35-55/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 MOG35-55 (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 TH subsets, similar procedures was performed as described above. Different subsets skewing conditions were as follows: Non-skewed, MOG35-55 only; TH1: MOG35-55 plus IL-12 (10 ng/ml) and anti-IL-4 (5 μg/ml); TH17: MOG35-55 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 TH: MOG35-55 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 g 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 t (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 Grinwald-Giemsa Staining


Monocytes/macrophages (Ly6ChiLy6G) and neutrophils (Ly6CloLy6Ghi) 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 Stat5f/f or Cd4-Cre; Stat5f/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 TH17 differentiation by restraining IL-17 production (Laurence et al., 2007; Yang et al., 2011). However, the function of STAT5 in TH17-mediated pathogenesis is not well understood. To explore this question, EAE was induced in Cd4-Cre; Stat5f/f (Stat5−/−) mice, where Stat5 was specifically deleted in T cell compartment, and in littermate controls by immunizing the mice with MOG35-55/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 TH17 generation. Similar results were observed when a single MOG35-55/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 TH1 and TH17 cell development. Nevertheless, decreased CD4+CD25+ Treg population in the CNS of Stat5−/− mice was detected (FIG. 3B), indicating the resistance to EAE in Stat5−/− mice was unlikely due to altered Treg cells.


Example 2. Resistance to EAE in Stat5-Mutant Mice is Due to an Intrinsic Defect of Antigen Specific CD4+ T Cells Independent of TH1 and TH17 Generation

Stat5 deletion (Cd4-cre; Stat5f/−) 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; Stat5f/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 TH1 and TH17 generation. To validate the function of STAT5 in TH1 and TH17 generation, the in vitro differentiation was performed by activating naïve CD4+ T cells under TH1- and TH17-polarizing conditions. In agreement with previous reports, that STAT5 mediated the suppressive effect of IL-2 on TH17 differentiation (data not shown). Interestingly, IL-7, which also signals through STAT5, was not observed to have a demonstrable effect on TH17 differentiation (data not shown). Nevertheless, a slight decrease of IFN-γ+ cells under TH1-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 TH17 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 showedincreased 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, MOG35-55-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 TH1 and TH17. To exclude the possible role of CD8+ T cells in the resistance to EAE observed in Stat5−/− mice, Rag2−/− mice were reconstituted with MOG35-55-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 MOG35-55-specific Stat5+/+ and Stat5−/− CD4+ T cells. Splenocytes derived from MOG35-55/CFA-immunized Stat5+/+ and Stat5−/− mice were challenged with various concentrations of MOG35-55 for 24 h, to examine the secretion of GM-CSF. GM-CSF production was observed to increase in a MOG35-55 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+CD44hi cells in the absence of STAT5 (FIG. 8B). Moreover, the frequency of MOG35-55-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 TH17 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 MOG35-55-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 MOG35-55-immunized wild-type mice were challenged with MOG35-55 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+CD44hi 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 CD62LhiCD44loT cells and CD62LloCD44hi T cells, suggesting IL-7 may directly act on CD4+ T cells to regulate GM-CSF expression. Thus, CD62LhiCD44lo and CD62LloCD44hi 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, CD62LloCD44hi T cells potently expressed GM-CSF, while CD62LhiCD44lo T cells expressed 30-fold lower GM-CSF amounts. STAT5 deletion resulted in reduced basal GM-CSF production in CD62LloCD44hi 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 TH Cells are Distinct from TH17 and TH1

Since both TH17 and TH1 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 TH1- or TH17-polarizing conditions. It was observed that anti-CD3 together with anti-CD28 induced the expression of GM-CSF (FIG. 14A). However, while TH1 differentiation conditions promoted IFN-γ expression and TH17 conditions promoted IL-17 expression as expected, both TH1 and TH17 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 TH17 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 TH17 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 TH17 and TH1.


Example 6. IL-7-STAT5 Programs GM-CSF-Expressing TH 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 TH cells versus TH1 and TH17 cells) indicates a distinct TH cell subset that is regulated by IL-7-STAT5 signaling. This finding was further explored by examining GM-CSF-expressing TH 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 TH 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 TH 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 TH was examined and observed that the expression of RORγt or T-bet in GM-CSF-expressing TH was significantly lower than those in TH17 or TH1 cells, respectively (FIG. 16B), confirming that the GM-CSF-expressing TH cells are distinct from TH1 and TH17 cells. Together, these data suggest that IL-7-STAT5 signaling direct the differentiation of a novel GM-CSF-expressing helper T cell subset, termed TH-GM.


Next, it was determined whether IL-2 signaling could influence TH-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 TH-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 TH-GM

To demonstrate TH-GM as distinct from known T cell subsets (e.g., TH1 and TH17), a whole transcriptome analysis was performed by microarray to validate its specificity compared with known T cell subsets, in particular TH17 cells. Naïve CD4+ T cells were differentiated into TH1, TH17 and TH-GM. Microarray analysis was performed to examine their gene expression profiles. Whole transcriptome clustering indicates TH-GM cells as representing a novel subset distinct from TH1 or TH17 cells. T cell lineage-specific gene expression is shown in Table 1. A list of 202 genes preferentially expressed in TH1 cells were identified, compared with naïve, TH17 or TH-GM cells (fold change >1.7), among which IFN-γ and T-bet are on the top of the list (Table 1). Similarly, TH17-feature genes, such as IL-17, IL-17F, RORγt and RORα, were identified in the list including 411 genes specific to TH17 cells (Table 1). The TH-GM cell-specific gene list (“Genes preferentially upregulated in TH-GM”—the TH-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 TH-GM subset, and another set of surface molecules which were selectively expressed at low level in TH-GM subset compared with other subsets were identified (FIG. 19 and Table 1). These molecules (also TH-GM signature genes) can be used for further characterization by surface markers to identify the TH-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 TH-GM cells are potent IL-3 producers as compared with TH1 and TH17 cells (FIGS. 20A, 20C and 20D). In addition, several other cytokines, including EBI-3, PENL and RANKL were found preferentially expressed in TH-GM cells (FIG. 20B), indicating diverse biological functions of TH-GM cells.


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

To test the hypothesis that GM-CSF-expressing TH subset (TH-GM) was the primary encephalitogenic effector cells, adoptive transfer of different subsets of MOG35-55-specific CD4+ T cells was performed into Rag2−/− mice for EAE induction. As shown in FIG. 21, GM-CSF-expressing TH cells were preferentially able to induce EAE compared with TH17 and TH1 subsets.


Example 9. The Suppression of STAT5 Activity by Chemical Inhibitor Attenuates GM-CSF Expression by TH-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 TH-GM differentiation was examined. As shown, STAT5 inhibitor suppressed TH-GM differentiation in a dosage-dependent manner (FIG. 22E). Reduced TH1 differentiation upon STAT5 inhibitor treatment was observed (data not shown), but TH17 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 TH 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 TH cells were quantified in RA and HC. High frequencies of IFN-γ- and/or GM-CSF-producing TH cells were detected in all samples, but observed low frequency (<1%) of IL-17-producing TH cells (FIG. 24B). GM-CSF-single-producing (GM-CSF+IFN-γ) TH 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 TH cells showed any significant difference in their frequencies between RA and HC (FIG. 24C). Therefore, the frequency of GM-CSF-single-producing TH cells in peripheral blood is selectively elevated in RA, suggesting a functional association of TH-cell-secreted GM-CSF with RA. Moreover, a significant correlation between plasma GM-CSF concentration and GM-CSF-single-producing TH cell frequency was observed in RA (FIG. 24D).


To further evaluate the association of GM-CSF-producing TH 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 TH cell frequency was observed in synovial fluid compared with peripheral blood, but most of these cells co-expressed IFN-γ (FIG. 24E). Similarly, both TH1 and TH17 frequencies were also increased in synovial fluid, with TH17 remaining to be a minor population compared with TH1 (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; Stat5f/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 TH cells as a new biomarker for RA diagnosis.


Example 12. STAT5-Regulated GM-CSF Secretion by Autoreactive TH 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 TH cell frequency in RA (FIG. 24D). Thus, the functional significance of TH-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 TH 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 TH1 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, TH17 cells are proven crucial in animal models of arthritis (Pernis, 2009), but predominance of TH17 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 TH 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+CD44hi population was significantly decreased in Stat5−/− mice (FIG. 2834B). Notably, no significant differences were observed in frequencies of IL-17-single-producing (TH17) or IFN-γ-single-producing (TH1) 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 TH17 or TH1 cells in experimental arthritis.


To investigate the involvement of GM-CSF-producing TH cells and their regulation by STAT5 in synovial inflammation, synovial tissues were dissected from AIA mice and examined cytokine production by TH cells. In spite of multiple cellular sources of GM-CSF (Cornish et al., 2009), CD4+ TH 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 TH cells was detected in Stat5−/− mice than Stat5+/+ mice (FIG. 28D). On the other hand, both TH1 and TH17 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 TH cells is crucial for evoking synovial inflammation.


To determine the critical role of STAT5-regulated GM-CSF production by TH 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 TH 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+ TH 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 CD11cintCD11bhiLy6C+/hiMHCIIhi, 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 Ly6CloLy6Ghi population (neutrophils) and Ly6ChiLy6G population (monocytes/macrophages). The present study shows that Ly6CloLy6Ghi 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 Ly6CloLy6Ghi population were neutrophils, which displayed typical polymorphonuclear characteristics with ring-shaped nuclei (FIG. 32B). In contrast, synovial-infiltrating Ly6ChiLy6G 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 TH-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 TH-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+ TCRβCD11c 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 TH 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, Ly6CloLy6Ghi population (neutrophils) and Ly6ChiLy6G 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 TH-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), TH-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









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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 diagnosing and treating a granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting T-helper cell (TH-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, IL-7, GM-CSF or IL-3, or a combination thereof;b) quantifying 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 a TH-GM-mediated inflammatory disorder,thereby diagnosing a TH-GM-mediated inflammatory disorder in the patient suffering from an inflammatory disorder; andc) treating the granulocyte macrophage colony-stimulating factor (GM-CSF)-secreting T-helper cell (TH-GM)-mediated inflammatory disorder in a patient by administering to said patient an effective amount of a modulating agent that modulates TH-GM function.
  • 2. The method of claim 1, wherein the detecting agent is an antibody that binds to the polypeptide of STAT5, activated STAT5, IL-7, GM-CSF or IL-3; or that binds to the nucleic acid of STAT5, IL-7, GM-CSF or IL-3.
  • 3. (canceled)
  • 4. (canceled)
  • 5. The method of claim 1, further comprising contacting the sample with a detecting agent that detects a polypeptide or nucleic acid level of at least one gene.
  • 6. The method of claim 5, wherein the at least one gene is basic helix-loop-helix family member e40 (BHLHE40), Chemokine (C-C Motif) Receptor 4 (CCR4), or Chemokine (C-C Motif) Receptor 6 (CCR6).
  • 7. The method of claim 1, wherein the sample is peripheral blood, cerebrospinal fluid, synovial fluid, or synovial membrane, or a combination thereof.
  • 8. The method of claim 1, wherein the reference level is obtained from a healthy human.
  • 9. The method of claim 1, wherein the increased level of STAT5, IL-7, GM-CSF or IL-3, or a combination thereof is at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% relative to the reference level.
  • 10. (canceled)
  • 11. An isolated population of GM-CSF-secreting T-helper cells (TH-GM), wherein the TH-GM cells are differentiated from precursor CD4+ cells in the presence of activated signal transducer and activator of transcription 5 (STAT5) and IL-7, and wherein the TH-GM cells express GM-CSF and IL-3.
  • 12. The isolated cells of claim 11, wherein the TH-GM cells are further characterized by an overexpression of one or more genes, or wherein the TH-GM cells are further characterized by an underexpression of one or more genes.
  • 13. (canceled)
  • 14. The isolated cells of claim 12, 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 (Scd1), or phosphotriesterase related C1q-like 3 (Pter).
  • 15. The isolated cells of claim 12, wherein the TH-GM cells underexpress one or more of lymphocyte antigen 6 complex, locus A (Ly6a); CD27; or selectin, lymphocyte (Sell).
  • 16. (canceled)
  • 17. (canceled)
  • 18. The method of claim 1, wherein the patient is previously diagnosed as having a TH-GM-mediated inflammatory disorder.
  • 19. A method of claim 1, wherein the inflammatory disorder is rheumatoid arthritis or multiple sclerosis and wherein the patient exhibits limited response to tumor necrosis factor alpha (TNF-α) therapy.
  • 20. The method of claim 19, wherein the TNF-α therapy is TNF-α inhibitor-based therapy or non-TNF-α inhibitor-based therapy, or both.
  • 21. The method of claim 1, wherein the modulating agent inhibits TH-GM function.
  • 22. The method of claim 1, wherein the modulating agent is an antibody, a polypeptide, a small molecule, a nucleic acid, or a cytokine, or a combination thereof.
  • 23. The method of claim 22, wherein the modulating agent inhibits the function of STAT5, activated STAT5, interleukin-7 (IL-7), IL-7 receptor, IL-2, or IL-2 receptor, GM-CSF, IL-3, preproenkephalin (PENK), or receptor activator of nuclear factor kappa-β ligand (RANKL), janus kinase 1/3 (JAK1/3), or a combination thereof.
  • 24. (canceled)
  • 25. The method of claim 22, wherein the cytokine is IL-12, interferon-γ (IFNγ), transforming growth factor-β (TGF-β), or IL-6, or a combination thereof.
  • 26. The method of claim 22, wherein the modulating agent inhibits the function of a gene or a gene product.
  • 27-29. (canceled)
  • 30. A method of treating a signal transducer and activator of transcription 5 (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.
  • 31.-33. (canceled)
Priority Claims (1)
Number Date Country Kind
1020140613OP Sep 2014 SG national
PCT Information
Filing Document Filing Date Country Kind
PCT/SG2015/050344 9/25/2015 WO 00