Methods and compositions for modulating th-gm cell function

Abstract

Disclosed herein is a T helper ("T H - GM" cell) that is regulated by IL-7 / STAT5 and secretes GM-CSF / IL-3. Also disclosed are methods and compositions for modulating T H - GM function to treat, for example, inflammatory disorders. Also provided are methods for specifically identifying T H - GM-mediated (e.g., TNF-a mediated) inflammatory disorders that are different from and / or in addition to non-T H - GM-mediated (e.g., TNF-a mediated) inflammatory disorders. Also provided are diagnostic and prognostic methods for T H - GM-mediated inflammatory disorders (e.g., rheumatoid arthritis).

Description

[0001] Related applications

[0002] This application claims the benefit of Singapore Patent Application No. 10201406130P, filed on September 26, 2014. The entire teachings of the above application are incorporated herein by reference. Background Technology

[0003] Numerous studies have established current models of immunity and inflammation, as well as dysregulation in immune and inflammatory disorders. Currently, it is understood that CD4... + Auxiliary T(T) H T cells play a crucial role in the host's defense against various pathogens by coordinating adaptive and innate immune responses. When the T cell receptor (TCR) is activated by a homologous antigen, naïve CD4+... + T cells are directed to differentiate into at least five major subsets: T H 1. T H 2. T H 17. iT reg And T FH They are regulated by the cytokine environment. It is known that T... H 1 cell and T H 17 cells are the main effector cells in inflammation. However, T cells... H 1 or T H The pathogenic role of 17 in various inflammatory conditions remains unclear. For example, recent studies and previously known T... H The paradoxical nature of multiple sclerosis (MS) (Haak et al., 2009) makes identifying potential drug targets for MS treatment more challenging. Similarly, although rheumatoid arthritis (RA) is traditionally understood as a disease mediated by tumor necrosis factor-α (TNF-α), up to 40% of RA patients do not respond to TNF-α treatment.

[0004] Therefore, there remains a significant unmet need for effective treatments for autoimmune and inflammatory conditions such as MS and RA. Summary of the Invention

[0005] This disclosure relates in part to T cells that regulate the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) / IL-3 by interleukin-7 (IL-7) / signal transduction and activator of transcription 5 (STAT5). H The cells were identified as T cells. H -GM represents a distinct subset of helper T cells with unique developmental and functional characteristics. The subsets identified in this paper are composed of T cells... H -GM cell-mediated inflammatory pathway (TH -GM-mediated inflammatory pathway), which represents, in addition to known non-T... H Independent inflammatory pathways beyond GM-mediated inflammatory pathways (such as TNF-α, IL-6, and IL-1β inflammatory pathways). This disclosure provides for the diagnosis of T-mediated inflammatory pathways. H -GM-mediated inflammatory conditions and regulation of T H -GM cell function to treat T H Methods and compositions for GM pathway-mediated inflammatory conditions.

[0006] Therefore, in one aspect, this disclosure provides a method for diagnosing patients suffering from inflammatory conditions using T... H - A method for GM-mediated inflammatory conditions, the method comprising: a) contacting a sample collected from a patient with an inflammatory condition with a detection agent that detects peptide or nucleic acid levels of STAT5 (e.g., phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, or IL-3, or combinations thereof; and b) quantifying peptide or nucleic acid levels of STAT5 (e.g., phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, or IL-3, or combinations thereof, relative to a reference level, wherein an increase in the levels of STAT5 (e.g., phosphorylated STAT5 (Tyr694)), interleukin-7 (IL-7), GM-CSF, or interleukin-3 (IL-3), or combinations thereof, indicates that the patient has T inflammatory conditions. H -GM-mediated inflammatory conditions.

[0007] In another aspect, this disclosure provides isolated GM-CSF-secreting T helper cells (T cells). H -GM) group, wherein T H -GM cells differentiate from differentiation cluster 4 (CD4+) precursor cells in the presence of IL-7 and activated STAT5, and wherein the T H -GM cells express GM-CSF and IL-3.

[0008] In another aspect, this disclosure provides an adjustment of T H -GM function method, the method includes making the T H -GM or CD4+ precursor cells, or both, and regulatory T cells H - GM function regulator contact.

[0009] In some respects, this disclosure provides a treatment for patients in need using T H - A method for treating GM-mediated inflammatory conditions, the method comprising administering an effective amount of regulated T to the patient. H - A regulator of GM cell function.

[0010] In other respects, this disclosure provides a method for treating rheumatoid arthritis in patients who have shown limited response to antitumor necrosis factor-α (TNF-α) therapy, the method comprising administering to the patient an effective amount of TNF-α-modulated T2-α. H - A regulator of GM function.

[0011] In another aspect, this disclosure provides a method for treating STAT5-mediated inflammatory conditions in patients in need, the method comprising administering to the patient an effective amount of a STAT5 modulatory agent.

[0012] In another aspect, this disclosure provides a method for screening to identify T H A method for regulating GM cell function, the method comprising isolating T cells... H -Contact a population of GM cells or an isolated population of CD4+ precursor cells with the candidate agent, and determine T in the presence or absence of the candidate agent. H - Readings for the GM function, where T H A change in the -GM function reading indicates that the candidate drug is T. H - A regulator of GM function.

[0013] This disclosure enables the implementation primarily in T H -GM-mediated inflammatory conditions or primarily non-T-mediated conditions H -GM-mediated (e.g., TNF-α, IL-6, and / or IL-1β-mediated) inflammatory conditions, or a combination thereof, can be identified or classified. Therefore, using the methods described herein, it is possible to determine whether a patient with, for example, RA has a predominantly T-mediated inflammatory condition. H -GM-mediated RA, or primarily non-T H -GM-mediated RA, or both. This distinction allows for more targeted and customized approaches to treating inflammatory conditions such as RA, for which current treatments are only 40% effective. Furthermore, this disclosure provides methods and compositions for predicting the progression of inflammatory conditions to tailor treatment according to disease stages. This document also provides methods for treating inflammatory conditions, particularly those caused by T... H Compositions and methods for GM-mediated inflammatory conditions. Attached Figure Description

[0014] The above will become apparent from the following more detailed description of exemplary embodiments of the invention.

[0015] Figure 1A-1D This study describes how Stat5 conditional mutant mice are resistant to EAE. They received MOG... 35-55 / CFA's two immunizations Stat5 + / + Mice and Stat5 - / - Clinical EAE scores (Figure 1A) and morbidity in mice (Figure 1B). Data represent three independent experiments (Figure 1A) or pooled data from three experiments (Figure 1B, n=18 per group). Mice receiving MOG... 35-55 EAE mice immunized with CFA once (Figure 1C, n=5 per group) or treated with MOG 35-55 Clinical scores of EAE mice immunized twice with / LPS (Figure 1D). Data represent two independent experiments.

[0016] Figure 2A-2D The reduced neuroinflammation was depicted in Stat5 conditional mutant mice. Histology of spinal cord sections obtained from EAE mice on day 9 after the second immunization (Fig. 1A). The images shown represent two independent experiments with three mice in each group. Scale bar: 200 μm (top), 50 μm (bottom). CD4 in the spinal cord sections was detected by immunofluorescence. + Cells and CD11b + Cell staining (Fig. 1B). The images shown represent two independent experiments, with three mice in each group. Scale bar: 200 μm. CNS monocytes were analyzed by flow cytometry at the peak of the disease (Fig. 2C and 2D). The right panel shows the cell percentage (Fig. 2C, right side) or cell number (Fig. 2D, right side) pooled from the two experiments (n=9).

[0017] Figures 3A and 3B depict the resistance of Stat5-deficient mice to EAE and T... H 1 cell, T H 17 cells or T cells reg Cell-related. During the peak of the disease, CNS-invasive CD4 cells... + Flow cytometry analysis of IL-17 and IFN-γ expression on T cells (Figure 3A). Data represent three independent experiments. Stat5 was analyzed by flow cytometry at the peak of the disease. + / + EAE mice and Stat5 - / - CD4 in the CNS of EAE mice + CD25 in T cells + The percentage (Figure 3B).

[0018] Figures 4A-4C The CD4+ in the peripheral blood of conditional Stat5 mutant mice was described. + There was no defect in T cell production. On day 7 (Figure 4A) and day 21 (Figure 4B), cells were received from patients receiving MOG... 35-55 / CFA Immunization Stat5 + / + Mice and Stat5- / - Spleens were obtained from mice. CD4 counts were analyzed by flow cytometry. + T cells and CD8 + The proportion of T cells. Calculate CD4. + The absolute number of T cells (right figure). Data represent two independent experiments (Figure 4A) or a pooled sum of two independent experiments (Figure 4B). Stat5 was determined by intracellular cytokine staining. + / + EAE mice and Stat5 - / - Spleen CD4 in EAE mice + IL-17 and IFN-γ expression in T cells (Figure 4C). Data represent three independent experiments. *p<0.5, **p<0.005, ***p<0.0005.

[0019] Figures 5A-5D Describes the Stat5 defect type CD4 + T cells can infiltrate the CNS, but cannot induce effective neuroinflammation. Stat5 was measured. + / + EAE mice and Stat5 - / - Spleen CD4 in EAE mice + T cell expression of CCR6, CXCR3, and CD69. The data represent two independent experiments, each with three to five mice (Figure 5A). In the first MOG... 35-55 CNS infiltrative CD4 counts were analyzed on days 7, 9, and 21 following CFA immunization. + T cells (Figs. 5B-5D). Cell counts (Fig. 5D). Data represent two independent experiments, with three mice in each group. *p<0.5.

[0020] Figures 6A-6C Stat5 is shown - / - Mice's resistance to EAE is not due to CD4 in the absence of STAT5. + Caused by any defect in T cell survival. Received different numbers of Stat5. + / + and Stat5 - / - CD4 + Rag2 T-cell metastasis - / - CD4 in recipient mice + T cell infiltration (Figure 6A) and clinical score (Figure 6B). CD4+ in the CNS at day 21 after EAE induction (disease peak). + Clinical T cell scores and incidence (Figure 6C). *p<0.05, ***p<0.0005.

[0021] Figures 7A-7C Describes the Stat5 defect type CD4 +Intrinsic defects in T cell-induced encephalitis. (This was addressed by adoptively transferring 2 million MOGs.) 35-55 Specific Stat5 + / + or Stat5 - / - CD4 + T cells followed by Rag2 - / - Clinical EAE scores (Fig. 7A) and morbidity (Fig. 7B) in mice (n=5 per group). CNS infiltrative CD4 counts were measured at the peak of the disease. + IL-17 and IFN-γ expression in T cells (Figure 7C). Data represent two independent experiments. *p<0.05.

[0022] Figures 8A-8D Depicting the spleen Stat5 - / - CD4 + The induction of GM-CSF in T cells is weakened. Figures 8A-8D In the middle, before the onset of the disease, receiving MOG 35-55 / CFA Immunization Stat5 + / + Mice and Stat5 - / - Spleen cells were obtained from mice (n=3 per group) and treated with various concentrations of MOG. 35-55 The spleen cells were attacked for 24 hours. GM-CSF secretion was measured by ELISA (Figure 8A). In MOG 35-55 (20 μg / ml) Golgiplug was added within the last 4 hours of the attack and CD4 was measured. + CD44 hi IL-17 in T cells + and GM-CSF + Cell occurrence rate (Fig. 8B). In Fig. 8C and 8C, from MOG-received cells... 35-55 / CFA Immunization Stat5 - / - Mice, Stat3 - / - Spleen cells were obtained from mice or wild-type control mice and stimulated with PMA / ionomycin for 4 hours in the presence of Golgiplug. Splenic CD4 count was measured by intracellular cytokine staining. + CD44 hi IL-17 in T cells + and GM-CSF + Cell occurrence rate. *p<0.05, ***p<0.001.

[0023] Figures 9A-9C Depicting the infiltration of Stat5 in the CNS - / - CD4 + The induction of GM-CSF in T cells is reduced. Figure 9A shows the Stat5 level measured at the peak of the disease.+ / + Mice and Stat5 - / - CNS-infiltrating CD4 in mice + Expression of IL-17, IFN-γ, and GM-CSF in T cells. Calculation of IL-17. + Cells or IFN-γ + GM-CSF in cells + Percentage of cells (bottom right, Figure 9A). Rag2 at the peak of disease during adoptive transfer of EAE. - / - CNS-infiltrating CD4 in receptor mice + IL-17, IFN-γ, and GM-CSF expression in T cells (Figure 9B). No MOG was tested or received. 35-55 / CFA Immunization Stat5 + / + Mice and Stat5 - / - Temporal analysis of cytokine mRNA expression in the mouse CNS (n=3 per group at each time point). RT-PCR data were normalized relative to Rn18S, and expression in unexperimented mice was set as 1 (Figure 9C). Data represent two independent experiments. *p<0.05.

[0024] Figures 10A-10C This demonstrates that STAT5-mediated GM-CSF induction is independent of IL-23 or IL-1β signaling. In Figure 10A, purified CD4... + T cells were cultured with TGF-β and IL-6 for 3 days, followed by starvation for 6 hours. The cells were then treated with various cytokines for 30 minutes, and pSTAT3 and pSTAT5 were identified by Western blotting. STAT3 and STAT5 were further analyzed after dissociation. Figure 10B shows Stat5. + / + EAE mice and Stat5 - / - Splenic CD4 counts of EAE mice (n=3 per group) + mRNA expression of IL-23R and IL-1R1 in T cells. RT-PCR data were normalized relative to β-actin. In Figure 10C, data from patients receiving MOG prior to disease onset are shown. 35-55 Spleen cells were obtained from wild-type mice immunized with CFA and administered via MOG in the absence or presence of IL-2. 35-55 The spleen cells were challenged with 20 μg / ml for 48 hours. CD4 count was measured by flow cytometry. + CD44 hi GM-CSF in T cells + and IL-17 + Cell occurrence rate. *p<0.05.

[0025] Figure 11A-11CThe study depicted IL-7-induced STAT5 activation promoting autoreactive CD4. + Expression of GM-CSF in T cells. Pre-disease response from MOG administration. 35-55 / CFA Immunization Stat5 + / + Mice and Stat5 - / - Spleen cells were obtained from mice and treated with MOG in the absence or presence of IL-7. 35-55 The spleen cells were challenged with 20 μg / ml for 48 hours. CD4 count was measured by flow cytometry. + CD44 hi GM-CSF in T cells + and IL-17 + Cell prevalence (Fig. 11A). GM-CSF secretion was measured by ELISA (Fig. 11B). Data represent two independent experiments, with two to three mice in each group. Cells from mice receiving MOG were sorted. 35-55 Spleen CD62L of CFA-immunized mice hi CD44 lo and CD62L lE CD44 hi T cells. Cells were stimulated with anti-CD3 and anti-CD28 for 4 hours in the absence or presence of IL-7, and then the cells were collected for GM-CSF expression analysis by RT-PCR (Fig. 11C). *p<0.05.

[0026] Figures 12A-12F depict the neutralization and attenuation of GM-CSF expression by IL-7Rα and its improvement in EAE. Clinical scores of EAE mice (n=5) treated with anti-IL-7Rα or normal IgG every other day starting from day 5 post-second immunization, as indicated by arrows. Data represent two independent experiments (Figure 12A). Spinal cord sections were obtained from EAE mice on day 11 post-second immunization. Histological assessment of immune cell infiltration was performed. The images shown represent three individuals per group. Scale bar: 200 μm (top), 50 μm (Figure 12B, bottom). CD4 in the spleen of EAE mice. + and CD8 + The percentage of T cells. Data represent two independent experiments (Figure 12C). Figures 12D and 12E illustrate CD4+ in the CNS of EAE mice that received different treatments. + GM-CSF in T cells + IL-17 + and IFN-γ + Cell occurrence rate. mRNA expression of IFN-γ, IL-17 and GM-CSF in the CNS of EAE mice (Figure 12F). *p<0.05.

[0027] Figures 13A and 13B depict the T expression of GM-CSF. H Cell differentiation is different from T cells H 17 and T H 1. In the presence of various combinations of cytokines and neutralizing antibodies as shown, naïve CD4 was induced by plate-bound anti-CD3 and soluble anti-CD28. + T cells were sensitized. The expression of GM-CSF, IL-17, and IFN-γ was analyzed by intracellular staining (Fig. 13A) or RT-PCR (Fig. 13B).

[0028] Figures 14A-14D The effects of IL-2 and IL-6 on naive T cells were demonstrated. H -The role of GM differentiation. Anti-CD3 alone or anti-CD3 plus anti-CD28 activates nascent CD4 after 72 hours. + Expression of GM-CSF and IFN-γ in T cells (Figure 14A). Figure 14B shows the initial CD4 cells sorted in the presence of neutralizing antibodies against IL-12 and IFN-γ, with or without the addition of IL-6. + T cells were stimulated with anti-CD3 and anti-CD28. GM-CSF was measured by intracellular staining. + and IL-17 + Cell occurrence rate (Fig. 14B). In Fig. 14C, cells from Stat3 will be analyzed under the conditions shown. + / + Mice and Stat3 - / - Initial CD4 in mice + T cell polarization for 72 hours. GM-CSF analysis. + and IL-17 + Cell occurrence rate. In Figure 14D, initial CD4 cells were observed in the presence of IL-2 or anti-IL-2. + T cells were activated using anti-CD3 and anti-CD28 agents. GM-CSF analysis was performed. + IL-17 + and IFN-γ + Cell occurrence rate.

[0029] Figures 15A-15F depict the IL-7-STAT5 signaling pathway that connects the T cells of naïve precursor cells. H -GM differentiation programmed. Initial CD4 was induced using plate-bound anti-CD3 and soluble anti-CD28 in the presence of various concentrations of IL-7 as shown. + T cells were sensitized. GM-CSF and IFN-γ expression were analyzed by intracellular staining (Fig. 15A) or ELISA (Fig. 15B). In Fig. 15C and 15D, Stat5 was sensitized in the presence of IL-7. + / + and Stat5 - / -Initial CD4 + T cells were activated for 3 days using anti-CD3 and anti-CD28 agents. The expression of GM-CSF, IL-17, and IFN-γ was analyzed by intracellular cytokine staining (Figure 15C). GM-CSF secretion was measured by ELISA (Figure 15D). From Stat5... - / - CD4 cells isolated from mice or control mice and stimulated by IL-7 + Immunoblots of pSTAT5 and STAT5 in T cells (Fig. 15E). Using Stat5 + / + and Stat5 - / - CD4 + T cells were subjected to ChIP assays using either normal IgG or STAT5-specific antibodies. The binding of the antibodies to the Csf2 promoter region was detected by RT-PCR (Figure 15F).

[0030] Figures 16A and 16B depict T H - Differentiation conditions for GM subsets. Initial CD4 in the presence of IL-7 and / or anti-IFN-γ as shown. + T cells were activated with anti-CD3 and anti-CD28 agents. The expression of GM-CSF, IL-17, and IFN-γ was analyzed (Figure 16A). Initial T cells... H 1 (IL-12 + anti-IL-4), T H 17 (TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and T H mRNA expression of T-bet and RORγt in GM cells (IL-7+ anti-IFN-γ) (Figure 16B). RT-PCR data were normalized relative to Gapdh, and expression in initial T cells was set to 1.

[0031] Figures 17A-17E The diagram illustrates IL-7 in initial CD4. + T cells induce STAT5 activation and GM-CSF expression, but IL-2 does not. Figures 17A-17C show the initial CD4 activation at the various time points indicated. + Flow cytometry of T cells or cells activated with anti-CD3 and anti-CD28 antibodies against CD25 and CD127 on their surface. Initial CD4+ stimulation with IL-2 or IL-7 for 30 minutes. + Activation of STAT5 in T cells (Fig. 17D). Fig. 17E shows naïve CD4 cells stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 or IL-7. + GM-CSF mRNA expression in T cells. RT-PCR data were normalized relative to β-actin, and expression in naïve T cells activated for 2 hours in the absence of cytokines was set as 1.

[0032] Figures 18A-18C It shows that both IL-2 and IL-7 can activate CD4. + STAT5 activation and GM-CSF expression are induced in T cells. As shown in Figures 18A and 18B, CD4+ activation is induced. + T cells were activated for 3 days with anti-CD3 and anti-CD28. After standing in fresh culture medium, cells were stimulated with IL-2 or IL-7 at various time points. pTyr-STAT5 and β-actin were detected by Western blotting (Fig. 18A). GM-CSF mRNA expression was measured by RT-PCR (Fig. 18B). RT-PCR data were normalized relative to β-actin, and expression in cells without cytokine stimulation was set to 1. ChIP assays were performed as shown in Fig. 18C using normal IgG or STAT5-specific antibodies. Antibody binding to the Csf2 promoter region was detected by RT-PCR.

[0033] Figure 19 The T-structure was characterized by microarray analysis. H -GM is a subset of surface molecules selectively expressed at high or low levels. These surface molecules, specific to each lineage, serve as biomarkers, characterizers, and potential targets for the 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 Th1, Th17, and ThGM shown in the illustrations is the same as the order shown for the bars in each chart.

[0034] Figure 20A-20D This shows that IL-3 preferentially occurs in T H -GM cells express this expression. In Figures 20A and 20B, it is found in T... H 1 (IL-12 + anti-IL-4), T H 17 (TGF-β+IL-6+anti-IFN-γ+anti-IL-4) and T H -GM(GM-CSF + T H Under polarization conditions of IL-7+ (anti-IFN-γ+ anti-IL-4), the initial CD4 + T cells were activated with anti-CD3 and anti-CD28. GM-CSF and IL-3 expression were analyzed by intracellular staining (Fig. 20A). mRNA expression of IL-3, EBI-3, PENK, or RANKL cytokines was measured by RT-PCR (Fig. 20B). The incidence of IL-3+ cells differentiated in the absence or presence of IL-7 was shown (Fig. 20C). GM-CSF and IL-3 expression in wild-type or STAT5-deficient TH cells producing GM-CSF was also shown (Fig. 20D).

[0035] Figure 21 Depicting adoption transfer 6×10 5 Various MOGs 35-55 Specific T H After subset Rag2 - / - Clinical EAE scores of mice (n = 3-6 mice per group).

[0036] Figures 22A-27E depict how inhibiting STAT5 activation suppresses in vitro T... H -GM cell differentiation. CD4... + T cells were pre-incubated for 1 hour with either a STAT5 inhibitor (Calbiochem) (Fig. 22A) or a JAK3 inhibitor (Fig. 22B) or a mediator (-) as indicated, followed by stimulation with IL-7 for 30 minutes. STAT5 activation (Tyr694 phosphorylation) was determined by Western blotting. CD4+ + T cells were pre-incubated for 1 hour with either a STAT5 inhibitor or a mediator (-) at the indicated concentration, followed by stimulation with IL-6 for 30 minutes. STAT3 activation (Tyr705 phosphorylation) was determined by Western blotting (Figure 22C). In Figure 22D, CD4+ was... + T cells were pre-incubated for 1 hour with either a STAT5 inhibitor or a mediator (-) at the indicated concentration, followed by stimulation with IFN-γ for 30 minutes. STAT1 activation (Tyr701 phosphorylation) was determined by Western blotting. In Figure 22E, naïve CD4 cells were... + T cells are isolated and under neutral or favorable conditions. H GM cells were activated for 3 days under conditions with different concentrations of STAT5 inhibitors. The expression of GM-CSF and IFN-γ was analyzed by intracellular cytokine staining and flow cytometry.

[0037] Figures 23A-23D The study depicts how targeting STAT5 activation with a chemical inhibitor improves EAE. (Figure 23A) Clinical EAE scores of wild-type control mice (n=5) or mice treated with a STAT5 inhibitor (Calbiochem). Arrows indicate treatment points. (Figure 23B) Histology of the spinal cord in EAE mice treated with different methods at day 18. (Figure 23C) CNS-infiltrating CD4+ at disease peak. + Intracellular staining and flow cytometry of T cells. (Fig. 23D) Whole CNS cells were collected for RNA extraction. GM-CSF mRNA expression was analyzed by RT-PCR. Data represent two independent experiments. *p<0.05.

[0038] Figures 24A-24E The T that produces GM-CSF is describedH Cellular involvement in human rheumatoid arthritis (RA). Plasma concentrations of GM-CSF and TNF-α in healthy controls (HC, n = 32) and RA (n = 47) were quantified by ELISA (Figure 24A). In Figures 24B and 24C, peripheral blood mononuclear cells (PBMCs) were collected from healthy controls (HC) and RA patients and stimulated with PMA / ionomycin in the presence of Golgiplug for 4 hours, followed by intracellular cytokine staining. CD44 is shown. + Representative flow cytometry data of GM-CSF, IFN-γ, and IL-17 in T cells (Figure 24B) and statistical values ​​for each group with n>=9 (Figure 24C). Figure 24D shows GM-CSF in peripheral blood of RA patients (n=18). + IFN-γ - T H The correlation between cell presence and plasma GM-CSF levels was investigated. CD4+ levels in synovial fluid derived from RA patients were analyzed using intracellular cytokine staining and flow cytometry. + Cytokine expression on T cells (Fig. 24E). Representative images of three cases are shown. *p<0.05, **p<0.01, ***p<0.001; ns, no significance.

[0039] Figures 25A-25E The distinguishable roles of GM-CSF and TNF-α in mouse AIA were depicted. Figure 25A shows knee swelling in wild-type mice 7 days after intra-articular injection of 100 μg mBSA in an AIA model, which received control IgG, GM-CSF-specific neutralizing antibody, and TNF-α-specific neutralizing antibody alone or in combination at the time points shown (arrows) (n = 5 per group). Figure 25B shows Stat5 levels 7 days after induced arthritis. + / + Mice and Stat5 - / - Knee swelling in mice (n=6 per group). Data represent more than three independent experiments. Representative images of joint sections stained with H&E (Fig. 25C) or Safranin-O / Fast Green (Fig. 25D) on day 7 after arthritis induction are shown in Fig. 25C. Scale bar: 500 μm (Fig. 25C upper and Fig. 25D) or 100 μm (Fig. 25C lower). Arrows in the upper image (Fig. 25C) indicate bone destruction. Stat5 quantification by ELISA is shown in Fig. 25E. + / + AIA mice and Stat5 - / - Serum concentrations of GM-CSF, IFN-γ, and TNF-α in AIA mice. Statistical values ​​are shown for each group n>=8. *p<0.05, **p<0.01, ***p<0.001.

[0040] Figures 26A-26D The study depicted the resistance of mice lacking Stat5 in T cells to CIA. (Figure 26A) Stat5 levels were observed on day 40 after immunization with collagen II / CFA in a CIA model. + / + Mice and Stat5 - / - Representative image of paw swelling in mice. (Fig. 26B) Stat5 within 40 days after collagen II / CFA immunization. + / + Mice and Stat5 - / - Clinical scores of mice (n=5 per group). Data represent two independent experiments. (Fig. 26C) Representative images of foot sections stained with H&E at day 40. (Fig. 26D) Serum concentrations of TNF-α quantified by ELISA (n=8 per group). *p<0.05, **p<0.01, ***p<0.001.

[0041] Figures 27A-27E Describes STAT5 defect type CD4 + T cells are defective in their arthrogenic potential. (Figures 27A and 27B) Stat5 on day 7 after induction of AIA + / + Mice and Stat5 - / - CD4 in the spleen (Fig. 27A) and inguinal lymph nodes (Fig. 27B) of mice + Representative flow cytometry of T cells. (Figures 27C and 27D) T cells from Stat5 on day 7 after induction of AIA. + / + Mice and Stat5 - / - Mice were used to collect synovial tissue, which was then dissociated into single cells. CD45 levels were calculated. + White blood cell count (Figure 27C). CD45 count was analyzed by flow cytometry. + CD4 in the grade + The percentage of T cells and the cell count were calculated (Fig. 27D). (Fig. 27E) Antigen-reactive CD4 cells that have undergone in vitro expansion. + Histological analysis of joint sections from wild-type unexperimented mice on day 7 following T-cell transfection and subsequent intra-articular injection of mBSA. Scale bar: 100 μm. Data represent two independent experiments. *p<0.05; ns, not significant.

[0042] Figures 28A-28G depict the T-phase of STAT5 regulation that produces GM-CSF. H Cells are crucial for AIA. On day 7 after arthritis induction, Stat5... + / + Mice and Stat5 - / -Spleen and synovial tissue were collected from mice. (Fig. 28A) Spleen fractions of wild-type AIA mice (n=3) were stimulated for 18 hours under the conditions shown. The level of GM-CSF in the supernatant was quantified by ELISA. (Figs. 28B-28D) Spleen CD4 levels were measured after 4 hours of restimulation with PMA / ionomycin in the presence of Golgiplug. + CD44 hi Effector T cells (Fig. 28B) or synovial infiltrating CD4 + Intracellular staining and flow cytometry of GM-CSF, IL-17, and IFN-γ in T cells (Fig. 28C and 28D). Representative images and statistics for each group of n=5 (Fig. 28B, right panel) or n=3 (Fig. 28D, right panel) are shown. Data represent two independent experiments. (Fig. 28E) Protein expression of several pro-inflammatory cytokines in synovial tissue was measured by ELISA. (Fig. 28F and 28G) Representative images of joint sections stained with H&E (Fig. 28F) or safranin-O / Fixed Green (Fig. 28G) on day 7 after intra-articular injection of mBSA alone into the right knee joint and intra-articular injection of mBSA supplemented with GM-CSF into the left knee joint. Scale bars: 500 μm, 50 μm, or 200 μm, as shown. Data represent two independent experiments. *p<0.05, **p<0.01, ***p<0.001; ns, no significant difference. "Spleen cells" represents the leftmost column in each group, "CD4 depleted". + "T cells in spleen cells" represents the middle column in each group, and "CD4" + "T cells" represent the rightmost bar in each group.

[0043] Figures 29A-29C The loss of STAT5 was described as causing antigen-specific CD4. + Impaired GM-CSF production by T cells was observed on day 7 after arthritis induction. + / + Mice and Stat5 - / - Spleen and inguinal lymph nodes were collected from mice and dissociated into single-cell suspensions. The cells were then stimulated with mBSA (20 μg / ml) for 24 hours (Figure 29A). Golgiplug was added during the last 4 hours of culture, followed by intracellular staining and flow cytometry. CD4 cells were shown. + CD44 hi Representative plots of GM-CSF, IL-17, and IFN-γ expression in effector T cells, representing two independent experiments. (Figs. 29B and 29C) Quantification of secreted cytokines in supernatant (n=3 per group) by ELISA. Data represent two independent experiments. *p<0.05; ns, no significance.

[0044] Figures 30A-30C The study depicted how loss of STAT5 reduced the expression of IL-6 and IL-1β in the synovial tissue of arthritic mice. Figures 30A-30C Stat5 levels were measured by qPCR and ELISA on day 5 or 7 after arthritis induction. + / + Mice and Stat5 - / - Expression of mRNA (Fig. 30A and 30C) and protein (Fig. 30B) of several pro-inflammatory cytokines in synovial tissue of mice (n>=3 per group). qPCR data were normalized relative to Rn18S.

[0045] Figures 31A-31D The study depicted how SAT5-induced GM-CSF expression mediates CD11b in inflamed synovial tissue. + Cell accumulation. (Figure 31A) Stat5 analysis by flow cytometry. + / + AIA mice and Stat5 - / - CD11b in the spleen of AIA mice + Cell occurrence rate. Statistical values ​​for each group n=3 are shown (right panel). (Figure 31B) Statistical values ​​from Stat5 on day 7 after arthritis induction. + / + Mice and Stat5 - / - Mice were used to collect synovial tissue, which was then dissociated into a single-cell suspension. CD45 levels were analyzed by flow cytometry. + CD11b in the grade + Percentage of bone marrow cells. Statistical values ​​for each group (n=5) are shown in the right figure. (Figure 31C) Synovial CD45 within 7 days after induced arthritis. + CD11b with graded gating + and CD4 + Representative flow cytometry of the cells. (Fig. 31D) Stat5 on day 7 after intra-articular injection of mBSA alone into the right knee joint and intra-articular injection of mBSA supplemented with GM-CSF into the left knee joint. + / + Mice and Stat5 - / - CD4 in mouse synovial tissue + CD11b + And B220 + Flow cytometry analysis of cell infiltration. Representative images are shown. All data shown represent two independent experiments. **p<0.01; ns, no significance.

[0046] Figures 32A-32D The study depicted GM-CSF-mediated neutrophil accumulation in arthritic mice. (Fig. 32A) Neutrophil accumulation in the synovial CD45 region within 7 days after arthritis induction. + CD11b +Flow cytometry analysis of gated Ly6C and Ly6G expression in fractionation. (Fig. 32B) Sorted Ly6C from synovial tissue of AIA mice. hi Ly6G - Cells and Ly6C lo Ly6G hi Giemsa staining of cells. Scale bar: 100 μm (left) or 20 μm (right). (Fig. 32C) Stat5 on day 7 after intra-articular injection of mBSA alone into the right knee joint and intra-articular injection of mBSA supplemented with GM-CSF into the left knee joint. + / + Mice and Stat5 - / - Ly6C in mouse synovial tissue hi Ly6G - and Ly6C lo Ly6G hi Population flow cytometry analysis. (Fig. 32D) Knee swelling in wild-type mice (n=5 per group) treated with Ly6G-specific neutralizing antibody (1A8) or IgG control within 3 days after intra-articular injection of mBSA in an AIA model. Arrows indicate the time points of antibody administration. *p<0.05.

[0047] Figures 33A-33C The effects of GM-CSF on enhancing neutrophil migration and delaying apoptosis in vitro were depicted. (Fig. 33A) Percentage of neutrophils migrating in the presence or absence of GM-CSF as a chemiflutic agent at 3 hours after the start of the migration assay. (Fig. 33B) Microscopic image of CFSE-labeled neutrophils at the bottom of the lower chamber during the migration assay. (Fig. 33C) Sorted neutrophils were cultured in vitro for 24 hours in the presence or absence of GM-CSF (20 ng / ml). Neutrophils undergoing apoptosis were investigated by co-staining with annexin V and propidium iodide (PI). Representative flow cytometry of three replicates is shown. *p<0.05.

[0048] Figure 34A-34I The expression of pro-inflammatory cytokines in bone marrow cells and synovial fibroblasts mediated by GM-CSF was depicted. Synovial tissue was dissected from wild-type AIA mice and dissociated into single-cell suspensions. (Fig. 34A) Flow cytometry images depicting differential expression based on surface markers were used to classify the cells into different populations. (Fig. 34B) CD45 levels were measured by qPCR. + TCRβ + (abbreviated as TCRβ) + CD45 + TCRβ - CD11c - CD11b+ (CD11b + ) and CD45 + TCRβ - CD11c + (CD11c + mRNA expression of several pro-inflammatory cytokines in the population. qPCR data were normalized relative to GAPDH. (Figure 34C) Sorted Ly6C hi Ly6G - and Ly6C lo Ly6G hi Group (in CD11b) + mRNA expression of IL-6, IL-1β, and TNF-α in cells (gated). qPCR data were normalized relative to GAPDH. (Figs. 34D and 34E) mRNA expression of IL-6 and IL-1β in BMDM (Fig. 34D) and BMDC (Fig. 34E) after 1 hour of stimulation with 20 ng / ml GM-CSF. qPCR data were normalized relative to GAPDH. (Figs. 34F and 34G) BMDM (Fig. 34F) and BMDC (Fig. 34G) were stimulated with the indicated concentration of GM-CSF for 18 hours (n=3 per group). IL-6 secretion in the culture supernatant was quantified by ELISA. (Fig. 34H) BMDM was sensitized with LPS (100 μg / ml) in the presence of the indicated concentration of GM-CSF for 6 hours (n=3 per group), followed by stimulation with ATP (5 mM) for 30 minutes. IL-1β secretion in the culture supernatant was quantified by ELISA. (Figure 34I) Cells were cultured in DMEM medium supplemented with 10% FBS for more than 20 days, with more than 5 passages to obtain synovial fibroblasts. The synovial fibroblasts were stimulated with GM-CSF (20 ng / ml) for 1 hour and collected for RNA extraction. IL-1β mRNA expression was measured by qPCR. qPCR data were normalized relative to GAPDH. All data shown represent two independent experiments. *p<0.05, **p<0.01. Detailed Implementation

[0049] The exemplary embodiments of the present invention are described below.

[0050] This disclosure relates in part to what is referred to as "T" H Identification of GM-CSF-secreting T helper cells. As detailed in this article, IL-7 / STAT5 signaling transduction converts precursor CD4+ cells into T helper cells. H -GM differentiation is programmed, a process further regulated by IL-2 and IL-23 signal transduction. T H-GM cells are characterized by the production of, for example, GM-CSF and IL-3. T H -GM cells and known helper T cells H 1 and T H 17. They differ in aspects such as differentiation conditions, transcriptional regulation, and effector cytokine expression. For example, they mediate T... H 1 and T H 17. IL-12 / IFN-γ and TGF-β / IL-6 (e.g., promoting their development) potently inhibit naïve CD4. + Precursor cells develop into T H -GM, thus determining that T H -GM cells are transmitted through different T cells H 1 and T H The phylogenetic development of 17. Therefore, this disclosure provides a unique approach to mediating T. H 1 or T H The factor of 17 mediates T H -GM functions (e.g., its differentiation and pathogenicity) through different factor networks.

[0051] As shown in this article, with T H 1 cell and T H Compared to 17 cells, T H -GM cells preferentially induce EAE, indicating that T H -GM cells represent the main effector cells in the pathogenesis of autoimmune neuroinflammation in humans. Furthermore, blocking IL-7 signaling and / or inhibiting STAT5 function (e.g., eliminating expression or inhibiting STAT5 activity) alleviates the effects on T cells. H - Reduced GM-CSF production in GM cells is associated with autoimmune neuroinflammatory disease. Furthermore, blocking T... H -GM-CSF secreted by GM cells improves experimental arthritis in a non-TNF-α dependent manner, suggesting a method for treating patients with rheumatoid arthritis, for example, those unresponsive to TNF-α antagonists. Therefore, this disclosure enables the identification of GM-CSF secreted by T cells. H -GM pathway-mediated inflammatory conditions (e.g., RA) (e.g., those mediated by T) H -GM via, for example, GM-CSF and / or IL-3 or with T H -Pathogenicity caused by any factor related to the GM pathway) or by pathways such as TNF-α, IL-6 and / or IL-1β (i.e., non-T) H -GM-mediated pathways) mediated inflammatory conditions. For example, patients with RA may have predominantly T-mediated inflammatory conditions. H -GM-mediated or primarily non-T H-GM-mediated (e.g., TNF-α-mediated or IL-6-mediated) types of RA. This disclosure enables T H -GM-mediated inflammation and non-T H - GM-mediated classification of inflammation allows for more accurate diagnosis, prognosis, and treatment of individuals with inflammatory conditions such as RA or MS.

[0052] As shown in this paper, this disclosure identifies a subset of helper T cells (T cells). H -GM) provides the molecular basis for the directional development of this subset from initial precursor cells in vitro and in vivo, and confirms T H -GM cells are the main pathogenic cells in autoimmune diseases and inflammatory conditions such as MS and RA. Therefore, this article provides a method for diagnosing diseases mainly caused by T cells. H -GM cell-mediated inflammatory conditions, thereby enabling the identification of compositions and methods for RA patients who are unresponsive to TNF-α treatment (e.g., TNF-α inhibitor-based treatment); and for regulating T cells. H Compositions and methods for treating autoimmune and inflammatory conditions using GM-mediated functions. Regulation of T... H Methods for improving GM function include, for example, administering an effective dose of regulatory-mediated T. H Drugs that modulate the function of GM-mediated factors (e.g., IL-2 / IL-7 / STAT5 / GM-CSF / IL-3) networks (e.g., signal transduction, expression, or activity) to regulate T H - The functions of GM cells (e.g., development and pathogenicity). Specifically, this disclosure provides methods for identifying and diagnosing inflammatory conditions, such as multiple sclerosis (MS) and rheumatoid arthritis (RA), as being primarily caused by T cells. H -GM cell-mediated (i.e., T) H -GM pathway mediated) or mainly by non-T H Methods and combinations mediated by GM mechanisms (e.g., TNF-α, IL-6, and / or IL-1β pathways), or both. This article also provides methods and combinations for treating inflammatory conditions, particularly those caused by T... H Compositions and methods for GM-mediated inflammatory conditions.

[0053] Therefore, in one aspect, this disclosure provides a method for diagnosing patients suffering from inflammatory conditions using T... H -GM-mediated inflammatory conditions. In some embodiments, the method includes contacting a sample collected from a patient with an inflammatory condition with a detection agent that detects T. H -GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, at peptide or nucleic acid levels; and quantitative T H- The peptide or nucleic acid levels of GM-mediated factors (e.g., STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof), where T is relative to a reference level. H An increased level of GM-mediated factors (such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof) indicates that the patient has T. H -GM-mediated inflammatory conditions, thus diagnosing patients with inflammatory conditions caused by T H -GM-mediated inflammatory conditions.

[0054] As used in this article, "T" H -GM-mediated "inflammatory disease" refers to a subtype of inflammatory disease (such as a subtype of RA or MS) that is mediated by T-mediated regulation as described in this article. H -GM function pathway factors (“T”) H This is caused by any one or more physiological functions of the network of "GM-mediated factors". Such factors include, for example, GM-CSF, activated STAT5, IL-7, IL-2, and IL-3. In one specific embodiment, STAT5 is activated STAT5, wherein the tyrosine residue at position 694 is phosphorylated.

[0055] In some implementations, T is relative to the reference level. H The absence of increased levels of GM-mediated factors (such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof) indicates that the patient has non-T syndrome. H -GM-mediated inflammatory conditions.

[0056] In some embodiments, the method further includes if, relative to a reference level, T H If the levels of GM-mediated factors (such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof) do not increase, then administer TNF-α treatment as described in this article to the patient.

[0057] As used in this article, "non-T" H -GM-mediated inflammatory conditions refer to inflammatory conditions (e.g., RA or MS) primarily caused by factors such as TNF-α, IL-6, or IL-1β (and / or factors in the TNF-α, IL-6, or IL-1β pathways). Therefore, "T..." H -GM-mediated inflammatory conditions are primarily (or solely) caused by non-T... H -GM-mediated "inflammatory disease pathways (such as those associated with TNF-α, IL-6, or IL-1β) are caused by one or more different pathways."

[0058] However, as those skilled in the art will understand, T H -GM-mediated inflammatory conditions do not necessarily exclude the possibility that the inflammatory conditions may also be partly non-T-mediated. H -GM-mediated (e.g., mediated by factors in the TNF-α, IL-6, or IL-1β pathways, and / or the TNF-α, IL-6, or IL-1β pathways). Therefore, it is classified or diagnosed as "T". H -GM-mediated "and mainly / basically T" H -GM-mediated "synonyms" and classified as "non-T" H -GM-mediated "and "primarily / basically non-T" H -GM-mediated" is synonymous. For example, without being bound by any specific theory, inflammatory conditions in their early stages may be T... H -GM-mediated. As the inflammatory condition progresses to a late stage characterized by, for example, tissue damage, the inflammatory symptoms gradually become non-T... H -GM-mediated. In some implementations, T H -GM-mediated inflammatory conditions are defined by clinical criteria as having a significant impact on T... H - GM function regulation is responsive in certain conditions; non-T H -GM-mediated inflammatory conditions are those that, as determined by clinical criteria, respond to treatments such as TNF-α, IL-6, or IL-1β. In some implementations, the inflammatory condition can respond to T... H -GM function was regulated and responded to TNF-α, IL-6 and / or IL-1β treatment.

[0059] In some embodiments, the sample may be, for example, peripheral blood, cerebrospinal fluid, synovial fluid, or synovium, or a combination thereof.

[0060] In some embodiments, the inflammatory condition is an autoimmune condition. In some embodiments, the inflammatory condition may be caused by T... H - Any GM cell-mediated inflammatory condition, including but 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 rheumatica, psoriasis, psoriatic arthritis, Raynaud's syndrome / phenomenon, reactive arthritis (Reiter syndrome), sarcoidosis, scleroderma, and Hughlen syndrome. (syndrome), ulcerative colitis, uveitis, or vasculitis.

[0061] As used herein, "detector" refers to, for example, an antibody, peptide, small molecule, or nucleic acid that binds to a target polypeptide or nucleic acid (e.g., STAT5 (e.g., phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, or IL-3) and enables the quantification of the target polypeptide or nucleic acid. The detector may be detectably labeled or quantifiable by other means known in the art.

[0062] In some embodiments, the detection agent is an antibody that binds to a peptide of STAT5, IL-7, GM-CSF, or IL-3. In one embodiment, the antibody is an antibody that binds to activated STAT5 (e.g., phosphorylated STAT5) as described herein. The antibodies against STAT5 (e.g., phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, or IL-3 used in the methods of this invention are known in the art and commercially available (e.g., STAT5Ab: C-17 from Santa Cruz Biotech; phosphorylated STAT5 (Tyr694)Ab: #9351 or #9359 from CellSignaling; IL-7Ab: clone BVD10-40F6 from BD Pharmingen; IL-7RAb: clone SB / 14 from BD Pharmingen; GM-CSF Ab: clone MP1-22E9 from BD Pharmingen; IL-3Ab: clone MP2-8F8 from BD Pharmingen).

[0063] In other embodiments, the detection agent is a nucleic acid that binds to nucleic acids of STAT5, IL-7, GM-CSF, and / or IL-3. Nucleic acid molecules encoding sequences of, for example, STAT5, IL-7, GM-CSF, and / or IL-3, or fragments thereof or oligonucleotides thereof, that hybridize under high stringency to polypeptide sequences of, for example, STAT5, IL-7, GM-CSF, and / or IL-3 can be used as probes to monitor the expression levels of nucleic acids of STAT5, IL-7, GM-CSF, and / or IL-3 in samples used in the diagnostic methods of this disclosure. Methods for quantifying nucleic acid levels are conventional and available in the art.

[0064] In some embodiments, the method further includes contacting the sample with a detection agent that detects polypeptide or nucleic acid levels of one or more genes (and gene products) listed in Table 1. As described herein, Table 1 lists genes related to T...H 1 cell or T H Compared to 17 cells, in T H -Genes differentially expressed in GM cells and in T H -Genes that are differentially expressed on the surface of GM cells.

[0065] In one specific implementation, the method further includes contacting the sample with a detection agent that detects peptide or nucleic acid levels of basic helical-loop-helical family member e40 (BHLHe40), chemokine (CC motif) receptor 4 (CCR4), and / or CCR6.

[0066] Standard methods can be used to quantify peptide levels in any sample. These methods include, for example, ELISA, Western blotting, immunohistochemistry, fluorescence-activated cell sorting (FACS) using antibodies against peptides, and quantitative enzyme immunoassay techniques, which are known in the art. These methods are conventional and readily available in the art. Similarly, methods for quantifying nucleic acid levels (e.g., mRNA) are known in the art.

[0067] In the diagnostic method disclosed herein, an increase in the levels of STAT5 (e.g., activated phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, and / or IL-3 relative to a reference level indicates that the patient has T. H -GM-mediated inflammatory conditions.

[0068] In some implementations, an increase of 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 has T. H -GM-mediated inflammatory conditions. In one specific implementation, 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 has T. H -GM-mediated inflammatory conditions.

[0069] In some implementations, a lack of increase of at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 150% in STAT5 (e.g., activated phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, and / or IL-3 levels relative to a reference level indicates that the patient has non-T-cell inflammatory disease. H -GM-mediated inflammatory conditions.

[0070] In some implementations, equivalent (or unchanged) levels of STAT5 (e.g., activated phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, and / or IL-3 relative to a reference level indicate that the patient has non-T-cell inflammatory disease. H -GM-mediated symptoms. As used herein, a level “comparable” to a reference level means a level that remains unchanged, or a change relative to a reference level that is not statistically significant according to clinical criteria. In some embodiments, a comparable level (or a level that remains unchanged) may include a level that does not increase by at least 40%, at least 50%, at least 60%, or at least 70% relative to a reference level, because, for example, it may not indicate a clinically significant change. In some embodiments, relative to a reference level, T H Decreased levels of GM-mediated factors (such as STAT5 (e.g., activated phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, and / or IL-3) can also indicate that the patient has non-T-cell leukemia. H -GM-mediated symptoms.

[0071] In some embodiments, the reference level is a level used for comparison purposes and may be obtained from, for example, samples previously taken from the same patient; normal healthy subjects; samples from subjects without autoimmune diseases or inflammatory conditions; subjects diagnosed as having a predisposition to autoimmune diseases but not yet exhibiting symptoms; patients already receiving treatment for autoimmune diseases; or samples of purified reference peptides or nucleic acid molecules (e.g., STAT5) of this disclosure at known normal concentrations. "Reference standard or reference level" means a value or value derived from a reference sample, or a value or range accepted in the art as an indicator of health (e.g., an individual without inflammatory conditions). A normal reference standard or reference level may also be a value or value derived from a normal subject without autoimmune diseases. In one embodiment, the reference sample, reference standard, or reference level is matched to the subject's sample by at least one of the following criteria: age, weight, body mass index (BMI), disease stage, and overall health status. A standard curve of the levels of purified DNA, RNA, or mRNA within a normal reference range may also be used as a reference. A standard curve of the levels of purified proteins within a normal reference range may also be used as a reference.

[0072] In some implementation schemes, individuals already diagnosed or classified as having T H -GM-mediated inflammatory conditions: Patients with inflammatory conditions do not have non-T... H -GM-mediated inflammatory conditions (i.e., inflammatory conditions without TNF-α, IL-6, or IL-1β-mediated inflammation). In other words, being diagnosed with T... H - Patients with GM-mediated inflammatory conditions have a negative effect on T H -GM function regulation (e.g., inhibition of STAT5, IL-7, GM-CSF, and / or IL-3) responds, but does not respond to TNF-α treatment (or shows a limited response), as determined by clinical criteria. However, as described in this article, T H -GM-mediated inflammatory conditions do not preclude the possibility that the inflammatory conditions are also partially (though not primarily) caused by non-T... H Caused by GM-mediated pathways (e.g., TNF-α, IL-6, IL-1β).

[0073] In some embodiments, the method of this disclosure further includes administering medication to a person diagnosed or classified as having T... H - Patients with GM-mediated inflammatory conditions should be given an effective dose of modulated T. H - A regulator of GM cell function. As described herein, in some embodiments, the regulator inhibits T... H -GM function.

[0074] In some embodiments, the method of this disclosure further includes administering medication to individuals diagnosed or classified as having non-T-cell diabetes. H - Patients with GM-mediated inflammatory conditions should be treated with effective doses of, for example, TNF-α, IL-6, or IL-1β, as described in this article.

[0075] In some respects, this disclosure also provides a method for classifying patients with inflammatory conditions as having T... H -GM-mediated inflammatory conditions or non-T H -GM-mediated inflammatory conditions. In some embodiments, the method includes contacting a sample collected from a patient with an inflammatory condition with a detection agent that detects T. H -GM-mediated factors, such as STAT5 (e.g., phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, or IL-3, or combinations thereof, at peptide or nucleic acid levels. In some aspects, the method further includes quantifying T H -GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, peptide or nucleic acid levels, where T is relative to a reference level. H Elevated levels of GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, indicate that the patient has T-cell hypothalamus. H -GM-mediated inflammatory conditions; or T relative to reference levels. H - Equivalent levels of GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, indicate that the patient has non-T-cell hypothalamic disease. H -GM-mediated inflammatory conditions, thus classifying patients with inflammatory conditions as T H -GM-mediated inflammatory conditions or non-T H -GM-mediated inflammatory conditions.

[0076] In other aspects of this disclosure, the methods disclosed herein may further include measuring non-T... H -GM-mediated inflammatory conditions involve peptide or nucleic acid levels of factors. Such factors include, for example, TNF-α, IL-6, and IL-1β.

[0077] For example, in some aspects, this disclosure provides a method for determining a treatment regimen for a patient with an inflammatory condition. For illustration, the method includes quantifying peptide or nucleic acid levels, such as activated STAT5 or GM-CSF, in samples collected from the patient with the inflammatory condition, and quantifying peptide or nucleic acid levels, such as TNF-α, in samples collected from the patient. At least four scenarios can be considered.

[0078] In the first case, if the level of activated STAT5 or GM-CSF increases (e.g., by at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 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 T... H -GM-mediated inflammatory conditions and can be treated by regulating T H - GM-functional drugs were used to treat the patients, as described in this article.

[0079] In the second scenario, if the level of activated STAT5 or GM-CSF is comparable to the first reference level and the TNF-α level is increased relative to the second reference level (e.g., by at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 150%), then the patient is classified as having non-T-cell TNF-α syndrome. H -GM-mediated inflammatory conditions and can be treated with, for example, TNF-α therapy.

[0080] In the third scenario, if the level of activated STAT5 or GM-CSF increases relative to the first reference level, and the level of TNF-α also increases relative to the second reference level, and these increases are equivalent within clinical and / or statistical criteria (e.g., both GM-CSF and TNF-α increase by at least 50% relative to their respective reference levels), then the patient is classified as having T-cell activating factor (T-cell activating factor) to an equivalent degree. H -GM-mediated and non-T H -GM-mediated (e.g., TNF-α-mediated) inflammatory conditions. In such cases, an effective amount of T-regulatory agents can be used. H The patient was treated with a drug that enhances GM function and an effective dose of, for example, TNF-α. As shown in this article, the combination of these two drugs can have a synergistic effect.

[0081] In the fourth scenario, if the levels of activated STAT5 or GM-CSF increase relative to the first reference level, and the levels of TNF-α also increase relative to the second reference level, but one increases more than the other, then the inflammatory condition is primarily mediated by the pathway showing the greater increase. For example, if GM-CSF increases by 40% relative to the reference level and TNF-α increases by 90% relative to the reference level, then the inflammatory condition is primarily non-T... H -GM-mediated. However, in this case, the patient can receive treatment with T-modulated... HCombination therapy with medications that enhance GM function and TNF-α therapy (e.g., anti-TNF-α therapy) is effective because GM-CSF levels are increased by, for example, at least 40% relative to the reference level.

[0082] In some implementations, the first reference level and the second reference level are obtained from the same reference sample.

[0083] In one related aspect, this disclosure also provides a method for tailoring treatment to a patient with inflammatory conditions based on the progression of the patient's inflammatory condition. In the illustrative examples above, the first case (T) H Elevated levels of GM-mediated factors, such as STAT5 or GM-CSF, but with TNF-α levels comparable to reference levels, may indicate that the patient is in the early stages of an inflammatory condition. Without being bound by any specific theory, during, for example, the early stages of an inflammatory condition, naive T cells are stimulated by antigens and programmed by IL-7 / STAT5 to differentiate into T cells that produce GM-CSF / IL-3. H -GM cells. During, for example, the late stages of inflammatory diseases, T... H -GM cytokines (e.g., IL-3 and GM-CSF) gradually stimulate more inflammatory cells, such as macrophages and neutrophils, thereby causing the production of, for example, TNF-α, IL-6, and IL-1β, leading to widespread inflammation. Therefore, in the illustrative example above, the second scenario (levels of activated STAT5 or GM-CSF comparable to the first reference level and increased TNF-α levels) could indicate that the patient is in a late stage of an inflammatory condition characterized by, for example, tissue damage. Therefore, this disclosure enables the application of one or more T... H -GM-mediated factors (e.g., STAT5 (e.g., activated phosphorylated STAT5 (Tyr694)), IL-7, GM-CSF, and / or IL-3) and one or more non-T H The quantifiable levels of GM-mediated factors (e.g., TNF-α, IL-6, IL-1β) can be used to predict patient prognosis, thereby tailoring treatment based on disease progression. Therefore, as those skilled in the art will appreciate, treatment can be tailored based on one or more T cells as described herein. H -GM-mediated factors, and one or more non-T factors H - Monitoring the levels of GM-mediated factors (such as TNF-α, IL-6, and IL-1β) in patients with inflammatory conditions is crucial for monitoring disease progression and ensuring effective and tailored treatment.

[0084] In a related aspect, this disclosure also provides a method for predicting the progression of inflammatory conditions in patients in need. In some embodiments, the method includes a) quantifying T in a first sample collected from a patient with inflammatory conditions. H - GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, peptide or nucleic acid levels; and b) quantifying peptide or nucleic acid levels, such as TNF-α, IL-6, or IL-1β, or combinations thereof, in a second sample collected from the patient, wherein i) relative to a first reference level, T H - Increased levels of GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, and unchanged levels of TNF-α, IL-6, or IL-1β, or combinations thereof, relative to a second reference level, indicate that the patient is in an early stage of an inflammatory condition, as described herein; or ii) increased levels of T, γ, IL-α, IL-6, or IL-1β, or combinations thereof, relative to a first reference level. H - The levels of GM-mediated factors, such as STAT5, IL-7, GM-CSF, or IL-3, or combinations thereof, remain unchanged, while the levels of TNF-α, IL-6, or IL-1β, or combinations thereof, increase relative to a second reference level, indicating that the patient is in a late stage of an inflammatory condition, as described herein. In some embodiments, the method further includes administering an effective amount of modulated T... H - GM-functional agents and / or, for example, TNF-α therapy, as described herein.

[0085] In some implementations, the first sample and the second sample are identical.

[0086] In various respects, this disclosure also provides an isolated GM-CSF-secreting T helper cell (T cell). H -GM) group. In one implementation, the T H -GM cells differentiate from precursor cells (e.g., CD4+ cells) in the presence of signal transducer and activator of transcription 5 (STAT5) and / or IL-7, and wherein the T cells are... H -GM cells express GM-CSF and IL-3.

[0087] In some implementations, the T H -GM cells differentiate from precursor cells (e.g., CD4+ cells) in the presence of agents that inhibit IL-12, IFN-γ, TGF-β, and / or IL-6. Similarly, precursor cells (e.g., CD4+ precursor cells) differentiate into T cells. H - GM cell differentiation is inhibited by IL-12, IFN-γ, TFG-β and / or IL-6.

[0088] In some implementations, TH -GM cells are differentiated from precursor cells in vitro under artificial conditions, but the T cells mentioned therein... H -GM cells retain the physiological characteristics described in this article.

[0089] In some implementations, the T H -GM cells are further characterized by the overexpression of one or more genes listed in Table 1. For example, T H -GM cells are further characterized by overexpression of, for example, members of the basic helical-loop-helical family e40 (BHLHe40), proenkephalinogen (PENK), IL-2, serine (or cysteine) peptidase inhibitor clade B member 6b (serine protease inhibitor b6b), neuron spike protein 1 (Nrn1), stearoyl-CoA desaturase 1 (Scd1), or phosphotriesterase-associated C1q-like protein 3 (Pter), or combinations thereof.

[0090] In some implementations, the T H -GM cells are further characterized by low expression of one or more genes listed in Table 1. For example, the T... H -GM cells are further characterized by low expression of lymphocyte antigen 6 complex locus A (Ly6a); CD27; or lymphocyte selectin (Sell).

[0091] As described in this article, identification mediates T H - GM function (e.g., its differentiation and pathogenicity) differs from known mediators of T H 1 or T H The 17-factor network enables targeted regulation of T H -GM function to treat T H -GM-mediated conditions, such as those caused by abnormal T cells H -Symptoms caused by GM function. Therefore, in some aspects, this disclosure provides a method for regulating T H -GM function method, the method includes making the T H -GM or differentiation cluster 4 (CD4+) precursor cells or both of these and regulatory T H - GM function modulator contact. In one embodiment, the modulator is contacted with the T in vitro or in vivo. H -Contact with GM cells or CD4+ precursor cells.

[0092] As used in this article, "T" H "GM function" refers to T H -GM cells' orientation, development, maintenance, survival, proliferation, or activity, or combinations thereof. Therefore, regulation (e.g., enhancing or inhibiting) T cells... H-Medications that regulate T-GM function are drugs that regulate T. H -GM cells T H -A drug that directs, promotes, sustains, proliferates, or activates GM, or a combination thereof. For example, T... H - The GM function can be adjusted by modifying the following aspects: from CD4 + Precursor T cell targeting; already targeted to T H CD4+ in the GM developmental pathway + Precursor cell development; T H -Maintenance of the GM phenotype; in the effect T H - Survival or proliferation of GM cells during development; and / or effector T cells. H - GM cell activity (e.g., regulating the function of secretory factors such as GM-CSF or IL-3). For example, regulating T... H -GM functions include, but are not limited to, adjusting: T H - Number of GM cells; T H - GM cell survival; T H - GM cell proliferation; and / or T H -GM cell activity. T H -GM cell activity, as described herein, includes cytokines, chemokines, growth factors, enzymes, and T cells. H -Activities induced by other factors secreted by GM cells, and by T H -Activities induced by direct contact with GM cells.

[0093] As used in this article, the T helper cell subset "T" H "-GM" refers to cells that are similar to T cells. H 1 cell and T H 17 cells, differentiated from precursor CD4+ precursor cells, but via pathways different from and unique to T cells. H 1 or T H 17. Factors (T) of the known subset of factors for cell subtype orientation and development H -GM-mediated factors) subset mediate pathways for orientation and development, as described herein. In some implementations, T H -GM cells produce a different and unique set of genes (see, for example, Table 1) and via mediators T H 1 or T H The known pathogenic factors of the 17 cell subtypes involve different mechanisms and pathways that produce pathogenicity. For example, T... H -GM cells are directed and develop through the function of IL-7 / STAT5 (its regulator), and exert pathogenicity through GM-CSF / IL-3 (its effector).

[0094] In some respects, this disclosure provides a treatment for patients in need using T H - A method for treating GM-mediated inflammatory conditions, the method comprising administering an effective amount of regulated T to the patient. H - A regulator of GM cell function. In some embodiments, the patient has previously been diagnosed with T cells as described herein. H -GM-mediated inflammatory conditions.

[0095] In some aspects, this disclosure also provides a method for treating rheumatoid arthritis in patients who have shown a limited response to TNF-α treatment, the method comprising administering to the patient an effective amount of TNF-α-modulated T-α. H - A regulator of GM function.

[0096] As used herein, “limited response” means no response or a negligible response such that the patient was not treated by the treatment, as determined by clinical criteria.

[0097] "Treatment" or "treating" means treating, preventing, and protecting against, and particularly refers to administering a medicine or performing a medical procedure to a patient to prevent (prevent) the occurrence of or reduction of the degree or likelihood of a condition or event in which the patient is distressed. It also refers to reducing the severity of one or more symptoms associated with a disease or condition. In this application, it can refer to improving one or more of the following: pain, swelling, redness, or inflammation associated with an inflammatory condition or autoimmune disease. As used herein and as is fully understood in the art, "treatment" is a method for obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, reducing or improving one or more symptoms, reducing the severity of the disease, stabilizing the disease state (e.g., preventing it from worsening), delaying or slowing disease progression, and / or improving or alleviating the disease state. "Treatment" can also mean prolonged survival compared to the expected survival without treatment.

[0098] The "effective amount" of a drug is the amount sufficient to achieve a beneficial or desired outcome, including clinical results. The "effective amount" depends on the context in which it is applied. In the context of administering a composition that modulates an autoimmune response, as a T... H The effective amount of a modulator of GM function is an amount sufficient to achieve such regulation compared to the response obtained without drug administration. An effective amount can confer disease remission, such as suppressing and / or inhibiting T cells as defined herein. H-The immediate, short-term, or long-term benefits of GM function. An effective amount may be administered in one or more doses. As used herein, "effective amount" is intended to mean an amount sufficient to reduce one or more clinically significant symptoms in a patient by at least 10%, at least 25%, at least 50%, at least 75%, or sufficient to improve one or more clinically significant symptoms in a patient.

[0099] In some implementations, the regulator inhibits T H -GM functions, for example, to reduce inflammation. The inhibitory effect conferred by modulators (inhibitors) does not necessarily imply a specific biological mechanism of action. In fact, the terms "antagonist" or "inhibitor," as used herein, encompass those mediating T... H Factors contributing to GM function (e.g., IL-7, IL-7 receptor, STAT5, GM-CSF, IL-3, IL-2, IL-2 receptor, PENK, RANKL, JAK1 / 3, or in T...) H -All possible pharmacological, physiological, and biochemical interactions, whether direct or indirect, with respect to any of the differentially expressed genes in GM cells (e.g., those listed in Tables 1 and 2), and including interactions with T at the protein and / or nucleic acid levels, or via other mechanisms. H - Interactions of GM function factors (or its active fragments).

[0100] In some implementations, suppressing T H -Regulators of GM function include those that block mediating T H Antibodies, peptides (e.g., those that bind to and inhibit soluble receptors such as IL-7), small molecules, nucleic acids (e.g., antisense, small interfering RNA molecules, short hairpin RNA, microRNA), or proteins (e.g., cytokines), or combinations thereof, that function (e.g., express and / or are active) in the GM-functioning factor. Methods for designing, generating, and using such inhibitors are known and available in the art.

[0101] As used herein, “binding” may be used interchangeably with “specific binding”, meaning a peptide that recognizes and binds to a sample that naturally includes a peptide of the present disclosure, such as a biological sample containing a peptide of the present disclosure, but substantially does not recognize and bind to other molecules in the sample (e.g., soluble receptors) or antibodies. In one embodiment, an antibody specifically binds to an activated STAT5 peptide, but does not bind to non-STAT5 peptides.

[0102] As used in this article, “antibody” refers to a complete antibody or an antigen-binding fragment of an antibody, including complete antibodies or antigen-binding fragments that have been modified or engineered or are used as human antibodies.

[0103] In one specific implementation, the antibody binding mediates TH -GM function of any one or more factors and inhibits their function. For example, the antibody binds to IL-7, IL-7 receptor (IL-7R), IL-2, IL-2 receptor (IL-2R), STAT5, or Jenners kinase 1 / 3 (JAK1 / 3), or combinations thereof and inhibits their function. In other embodiments, the antibody binds to GM-CSF (or its receptor), IL-3, PENK, or RANKL, or combinations thereof and inhibits their function. In some embodiments, the antibody binds to the genes listed in Table 1 and inhibits their function. In some embodiments, the antibody binds to and inhibits a protein or any functional fragment thereof. Methods for designing, generating, and using suitable antibodies are known and available to those skilled in the art. Examples of antibodies suitable for use in this disclosure include, for example, daclizumab, basiliximab, mavrilimumab, MOR103, KB003, namilumab, and MOR Ab-022.

[0104] The terms “protein” and “peptide” are used interchangeably and can include full-length peptides or functional fragments thereof (e.g., degradation products, alternative splicing isoforms of peptides, enzyme cleavage products of peptides), peptides bound to substrates or ligands, or free (unbound) forms of peptides. The term “functional fragment” refers to a portion of a full-length protein that retains some or all of the activities (e.g., biological activities, such as the ability to bind homologous ligands or receptors) of the full-length peptide.

[0105] In some implementations, suppressing T H -Regulators of GM function can directly or indirectly inhibit T-mediated... H -GM function is derived from one or more specific biological proteins (e.g., cytokines) that contribute to the function of GM. Such cytokines include, for example, IL-12, IFN-γ, TGF-β, and IL-6.

[0106] In some implementations, suppressing T H -Regulators of GM function can directly or indirectly inhibit T-mediated... H -One or more small molecules that are the functional components of GM. As used herein, "small molecule" refers to an organic compound that is biologically active and not a polymer, or a compound that is complexed with an inorganic compound (e.g., a metal). Small molecules generally have a molecular weight of less than about 3 kilodaltons. Known examples of small molecules include CAS 285986-31-4 (Calbiochem), pimozide, and tofacitinib.

[0107] In other embodiments, the modulator enhances T in conditions such as viral infections, fungal infections, bacterial infections, cancer, and / or related conditions. H -GM functionality. In one implementation, enhance T H Modulators of GM function include, for example, CD28 activators; initial (precursor) CD4 + IL-7 and / or IL-2 on T cells; STAT5 activators; or T H - Effector factors of GM cells (e.g., GM-CSF, IL-3).

[0108] In another aspect, this disclosure provides a method for treating STAT5-mediated inflammatory conditions in patients in need, the method comprising administering to the patient an effective amount of a STAT5 modulatory agent.

[0109] As used herein, "STAT5-mediated" inflammatory conditions refer to an inflammatory condition caused by abnormal STAT5 function (abnormal enhancement or inhibition) and responding to regulation of STAT5 function, as determined by clinical criteria. In some embodiments, the STAT5 is an activated STAT5 (e.g., phosphorylated STAT5 (Tyr694)).

[0110] In some embodiments, the inflammatory condition is an autoimmune condition. In some embodiments, the inflammatory condition can be any inflammatory condition 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 rheumatica, psoriasis, psoriatic arthritis, Raynaud's syndrome / phenomenon, reactive arthritis (Reiter's syndrome), sarcoidosis, scleroderma, Hughlen's syndrome, ulcerative colitis, uveitis, or vasculitis.

[0111] In some implementations, the term "patient" refers to a mammal, preferably a human, but may also include animals requiring veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), livestock (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0112] 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.

[0113] In some embodiments, the method of this disclosure further includes administering TNF-α treatment to a patient. In some embodiments, the patient is identified as having non-T... H - TNF-α treatment is administered to patients with GM-mediated inflammatory conditions. As described herein, in some embodiments, TNF-α treatment is administered if a quantitative level of TNF-α increases relative to a reference level by, for example, at least 40%.

[0114] Examples of TNF-α treatment include both TNF-α inhibitor-based and non-TNF-α inhibitor-based therapies. Specifically, TNF-α inhibitor-based therapies include etanercept, adalimumab, infliximab, golimumab, and pegylated certolizumab pegol. Examples of non-TNF-α inhibitor-based therapies include corticosteroids (e.g., prednisone), nonsteroidal anti-inflammatory drugs (e.g., methotrexate), and JAK inhibitors (e.g., tofacitinib). Other examples of non-TNF-α inhibitor-based therapies include anakinra, abatacept, rituximab, and tocilizumab.

[0115] The effective amount can be adjusted by T. H TNF-α therapy can be administered before, simultaneously with, or after drugs that regulate T-GM function. H -GM-regulating agents and TNF-α therapy can be administered together in a single dose, or in separate doses, such as simultaneously or sequentially, or both. Administration of T-regulating agents... H The duration of interaction between GM-positive drugs and TNF-α treatment will depend on the nature of one or more of the drugs. Furthermore, regulation of T... H - GM-regulating agents and TNF-α therapy may or may not be administered according to similar dosing regimens. For example, regulating T... H - GM-regulating agents and TNF-α treatments may have different half-lives and / or act on different time scales to modulate T H - GM-functioning agents are administered at a greater frequency than TNF-α treatment, or vice versa. The number of days between treatment administrations can be appropriately determined by those skilled in the art, based on the safety and pharmacokinetics of each drug.

[0116] Identification T H -GM cells and identification relative to TH 1 or T H 17 by T H -Genes resulting from GM cell differences enable the use of T H -GM cells to identify T cells used to regulate T H -Novel therapeutic agents with GM function, thereby enabling the treatment of T H Novel therapeutics for GM-mediated conditions (such as inflammatory conditions) have become possible. Therefore, in another aspect, this disclosure provides a method for screening to identify T... H A method for regulating GM cell function, the method comprising isolating T cells... H - A population of GM cells, or an isolated population of CD4+ precursor cells, is contacted with the candidate drug, and T is measured in the presence or absence of the candidate drug. H - Readings for the GM function, where T H A change in the -GM function reading indicates that the candidate drug is T. H - A regulator of GM function.

[0117] The candidate drugs used in this article refer to those that can regulate T-mediated H -GM function factors (e.g., expression and / or activity) regulate T H -GM function agents. Such candidates include, for example, antibodies, peptides, small molecules, nucleic acids (e.g., antisense, small interfering RNA molecules), or proteins (e.g., cytokines), or combinations thereof. Candidate agents can be engineered to target T-mediated T as described herein. H -Any of the factors that contribute to GM function (at the protein and / or nucleic acid levels), including the genes listed in Table 1 (e.g., those in T... H -GM cells preferentially upregulated genes, in T H -GM genes preferentially overexpressed / underexpressed on the surface of cells.

[0118] As used in this article, "reading" refers to T, which can be measured or quantified. H -Any change (or no change) in GM function. For example, candidate drugs can be evaluated against, for example, T. H -The role of GM-CSF secretion in GM cells, or its effect on T H -The role of GM cell abundance (via T cells) H -The role of GM cells in directed / development / proliferation, as described herein. The measurements used to determine such readings are known and available in the art, and are illustrated herein by example.

[0119] In some implementations, the change in the presence of the candidate agent is a decrease in the measured reading, indicating a reduction in the effect of T. H-Inhibition of GM function (e.g., reduced production of GM-CSF or IL-3, or T...) H -Reduction in GM cell abundance), thereby identifying the candidate drug as a T H -An inhibitor of GM function.

[0120] In some implementations, the change in the presence of the candidate agent is an increase in the measured reading, indicating that T H - Enhanced GM function (e.g., increased production of GM-CSF or IL-3, or T H -Increased GM cell abundance), thereby identifying the candidate drug as a T H -A enhancer for GM function.

[0121] In some implementations, the readings may be those listed in Tables 1 and 2 at T H -GM cells preferentially upregulate or downregulate any one or more genes. Therefore, downregulation in T H -The candidate drug for the gene preferentially upregulated in GM cells is T. H -Inhibitors of GM function. Similarly, upregulation in T H -The candidate drug for the gene preferentially downregulated in GM cells is T. H -A enhancer for GM function.

[0122] In some respects, if precursor CD4+ cells are used, then in T... H - Screening methods can be performed under GM polarization conditions, as described herein. For example, these methods can be performed in the presence of a combination of IL-7 / STAT5, TCR activation, CD28 co-stimulation, and blocking of IFN-γ and IL-4.

[0123] Unless otherwise stated, the definitions of the terms used herein apply to all aspects and embodiments of this disclosure.

[0124] Implementation of this disclosure includes the use of conventional techniques in molecular biology (e.g., recombinant DNA), microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, such as those described in, for example, the following literature: *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989) and *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook and Russell, 2001), collectively and individually referred to herein as “Sambrook”; *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.M. Calos, 1987); and *Current Protocols*. *In Molecular Biology* (edited by F.M. Susubel et al., 1987, including supplements up to 2001); *PCR: The Polymerase Chain Reaction* (edited by Mullis et al., 1994); *Current Protocols in Immunology* (edited by J.E. Coligan et al., 1991); *The Immunoassay Handbook* (edited by D. Wild, Stockton Press, NY, 1994); *Bioconjugate Techniques* (edited by Greg T. Hermanson, Academic Press, 1996); *Methods of Immunological Analysis* (edited by R. Masseyeff, W.H. Albert, and N.A.).Staines (ed.), Weinheim: VCH Verlags gesellschaft mbH, 1993; Harlow and Lane (1988), Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York; and Harlow and Lane (1999), Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (collectively and individually referred to as "Harlow and Lane" in this article); Beaucage et al. (eds.), Current Protocols in Nucleic Acid Chemistry (John Wiley & Sons, New York, 2000); and Agrawal (ed.), Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Humana Press, New Jersey, 1993).

[0125] illustration

[0126] method

[0127] mice

[0128] Stat5 f / f The mice were provided by L. Hennighausen (National Institute of Diabetes and Digestive and Kidney Diseases). Stat3 was produced as described. f / f mice 2 Cd4-Cre transgenic mice were purchased from Taconic Farms. Rag2 - / -Mice were obtained from Jean-Pierre Abastado (Singapore Immunology Network). All mice had a C57BL / 6 genetic background and were housed under specific pathogen-free conditions at the National University of Singapore (NUS). All experiments were conducted using mice aged 6–8 weeks and were approved by the NUS Institutional Animal Care and Use Committee.

[0129] Patients and controls

[0130] Blood samples (n=47) and synovial fluid samples (n=3) were collected from RA patients who visited the Department of Rheumatology and Immunology at Affiliated Drum Tower Hospital, Nanjing University Medical School. All patients met the American College of Rheumatology (ACR) classification criteria for RA. Age- and sex-matched healthy controls (n=32) were obtained from the Medical Examination Center at Affiliated Drum Tower Hospital. The study protocol was approved by the Ethics Committee of Affiliated Drum Tower Hospital, Nanjing University Medical School.

[0131] In vitro T cell differentiation

[0132] CD4 was obtained from the spleen and lymph nodes via positive selection and magnetic separation (Miltenyi Biotech). + T cells, followed by purification of initial CD4 cells sorted using FACS Aria. + T cell population (CD4) + CD25 - CD62L hi CD44 lo ). Initial CD4 + T cells were stimulated for 3–4 days with plate-bound antiCD3 (3 μg / ml; BD Pharmingen) and antiCD28 (1 μg / ml; BD Pharmingen) in the presence of different combinations of neutralizing antibodies and cytokines: for neutral conditions, no cytokines or neutralizing antibodies were added; for T cells… H1. Conditions: IL-12 (10 ng / ml) and anti-IL-4 (10 μg / ml, BD Pharmaceuticals); for T H 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 alternative T H 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 cells were stimulated with plate-bound anti-CD3 (2 μg / ml) and soluble anti-D28 (1 μg / ml) in addition to IL-7 and / or anti-IFN-γ (10 μg / ml) as shown. + T cells. 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 non-essential amino acids, and 5 μM β-mercaptoethanol. After polarization for 3–4 days, cells were washed and restimulated for 4–5 hours in the presence of Golgiplug with phorbol 12-myristate 13-acetate (PMA) and iomycin, followed by fixation and intracellular staining using the Cytofix / Cytoperm kit from BD Pharmaceuticals. Foxp3 staining was performed using a kit from eBioscience. Cells were acquired on LSR II (BD Biosciences) and analyzed using FlowJo software (Tree Star).

[0133] EAE-induced

[0134] The EAE induction procedure has been modified from previous reports. 3 For active EAE induction, on days 0 and 7, 300 μg of MOG was administered in 100 μl CFA containing 5 mg / ml heat-inactivated Mycobacterium tuberculosis strain H37Ra (Difco) at two sites on the posterior ventral side. 35-55 Mice were immunized. Pertussis toxin (List Bio Labs) was administered intraperitoneally at a dose of 500 ng per mouse on days 1 and 8. For a single MOG... 35-55 / CFA immunization was performed using a similar procedure only on days 0 and 1. In alternative active EAE induction, LPS (600 μg / ml in IFA, O111:B4 from Sigma) was used as adjuvant. For Rag2 - / - Active EAE induced in mice, metastasis originates from Stat5 f / f Or Cd4-Cre; Stat5 f / f mouse CD4 + T cells, followed by MOG as described above. 35-55 / CFA immunization. Clinical symptoms were scored as follows: 0 points: no clinical signs; 1 point: loss of tail tone; 2 points: gait waddling; 3 points: hind limb paralysis; 4 points: hind limb and forelimb paralysis; 5 points: death. IL-7Rα neutralizing antibody (SB / 14, BD Pharmingen) and an isotype control were administered intraperitoneally at 200 μg per mouse every other day. For analysis of CNS infiltrating cells, both spinal cord and brain were collected from perfused mice, minced, and monocytes were isolated by gradient centrifugation using Percoll (GE Healthcare).

[0135] For using Stat5 + / + or Stat5 - / - CD4 + Passive EAE induction of T cells was performed. Spleen cells and lymphocytes were collected 10–14 days post-immunization and passed through a 70 μm cell filter (BD Falcon). Cells were then in vitro with MOG in the presence of IL-23 (5 ng / ml) and IL-1β (2 ng / ml). 35-55 (20 μg / ml) were cultured together for 3 days. After collection, CD4... + T cells were purified to a purity >90% through positive selection. CD4+ + T cells (2 million in sterile PBS) were injected intraperitoneally into Rag2. - / - In mice, pertussis toxin was administered the following day. Mice were monitored daily for EAE signs as described above. For the transfer of various T... H EAE induction for subsets follows a similar procedure as described above. Different subset bias conditions are as follows: non-biased, only MOG. 35-55 ;T H 1: MOG 35-55 Added IL-12 (10 ng / ml) and anti-IL-4 (5 μg / ml); T H 17: MOG 35-55In addition to TGF-β (3 ng / ml), IL-6 (10 ng / ml), anti-IFN-γ (5 μg / ml), and anti-IL-4 (5 μg / ml); T cells expressing GM-CSF H MOG 35-55 In addition, IL-7 (2 ng / ml) and anti-IFN-γ (5 μg / ml) were added. 6 × 10⁶ cells were transferred to each recipient mouse. 5 CD4 + T cells.

[0136] Antigen-induced arthritis (AIA)

[0137] In short, on day 0, mice were subcutaneously immunized at two sites on the posterior ventral side with 100 μg of methylated bovine serum albumin (mBSA, Sigma-Aldrich) in 100 μl of complete Freund's adjuvant (CFA) containing 5 mg / ml heat-inactivated Mycobacterium tuberculosis strain H37Ra (Difco). On day 1, pertussis toxin (List Bio Labs) was administered intraperitoneally at a dose of 250 ng per mouse. Arthritis was induced on day 7 post-immunization by intra-articular injection of 100 μg of mBSA (in 10 μl saline) into the right posterior knee joint. The same volume of saline was injected into the left posterior knee joint as a control. Joint swelling was recorded by measuring the difference in diameter between the right and left knee joints using calipers over 7 days after arthritis induction. To assess the effect of GM-CSF administration, AIA was induced by intra-articular injection of mBSA alone in the right knee or intra-articular injection of mBSA supplemented with 100 ng GM-CSF (ImmunoTools) in the left knee. To assess the effect of blocking GM-CSF and / or TNF-α, neutralizing antibodies specific to GM-CSF (MP1-22E9, BD Pharmingen) and / or TNF-α (MP6-XT3, BDPharmingen) were administered intraperitoneally to mice at the indicated times (100 μg of each antibody per mouse).

[0138] For AIA induction via adoptive transfer, spleen cells and inguinal LN cells were isolated from mice immunized with mBSA / CFA on day 7 and cultured in vitro for 3 days with mBSA (10 μg / ml) in the presence of IL-7 (2 ng / ml). After collection, CD4+ cells were... + T cells were purified to a purity >90% using positive selection (Medtronic Biotechnology Co., Ltd.). Then, CD4+ was... +T cells (1 million in sterile PBS) were transferred into wild-type unexperimented mice, and mBSA was injected into the joints the next day.

[0139] Collagen-induced arthritis (CIA)

[0140] CIA was induced using a procedure similar to that described above for AIA, achieved by immunizing mice with chicken collagen II / CFA emulsion (purchased from Chondrex) followed by pertussis toxin injection. Mice were monitored and scored for arthritis: 0 points: normal; 1 point: mild swelling of the ankle or carpal joint or significant swelling limited to a single toe; 2 points: moderate swelling of the entire paw; 3 points: severe swelling of the entire paw with joint stiffness. The scores for each mouse's limbs were summed.

[0141] Histological analysis

[0142] For paraffin-embedded tissue, the spinal cord was fixed in 4% PFA. Knee or foot joints were removed, fixed in 10% formalin, and decalcified in 5% formic acid, followed by 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 / Fixed Green to assess cartilage consumption. For frozen tissue, the spinal cord was embedded in OCT (Tissue-Tek) and rapidly 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 a fluorescently conjugated secondary antibody (Invitrogen). For AIA experiments, knee joints were fixed in 10% formalin for 5 days, followed by decalcification in 5% formic acid for 5 days. Slices (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 assess cartilage destruction.

[0143] Cell sorting and May Grünwald-Gymsa staining

[0144] CD45 cells were isolated from spleen or synovial single-cell suspensions using FACS Aria. + CD11b + Upper-gated monocytes / macrophages (Ly6C) hi Ly6G - ) and neutrophils (Ly6C lo Ly6G hi The sorted cells were centrifuged and smeared on a glass slide, and then stained with McGraw-Grenfell and Giemsa dyes according to standard procedures.

[0145] Real-time PCR

[0146] Total RNA was extracted from cells using the RNeasy kit (Qiagen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using Superscript reverse transcriptase (Ingenium). Gene expression was measured using a SYBR qPCR kit (KAPA) on a 7500 real-time PCR system (Applied Biosystems). Actin b, Gapdh, or Rn18S were used as internal controls. Primer sequences are available upon request.

[0147] ELISA

[0148] The levels of TNF-α, IL-6, IL-1β, IFN-γ, GM-CSF, and IL-2 were measured using the Ready-SET-Go ELISA kit (eBioscience), and the level of IL-17 was measured using the DuoSet ELISA kit (R&D Systems) according to the manufacturer's instructions.

[0149] Chromatin Immunoprecipitation Assay

[0150] From Stat5 f / f Or Cd4-Cre; Stat5 f / f CD4 isolated from mice + T cells were activated for 3 days using plate-bound anti-CD3 and anti-CD28. Cells were stimulated for 45 minutes with IL-7 (20 ng / ml) or IL-2 (25 ng / ml). Cross-linking was performed by adding 1% final concentration of formaldehyde for 10 minutes, followed by glycine quenching. Cell lysates were disrupted by sonication and pre-cleaned with Protein G Dynabeads magnetic beads, followed by overnight precipitation at 4°C with anti-STAT5 antibody (Santa Cruz) or normal rabbit IgG (Santa Cruz). After washing and elution, cross-linking reversal was performed by incubation at 65°C for 8 hours as previously described. 5 The eluted DNA was purified and analyzed by RT-PCR using primers specific to the Csf2 promoter.

[0151] Statistical methods

[0152] Statistical significance was determined using GraphPad Prism 6.01 via Student's t-test. A p-value < 0.05 was considered significant. The p-values ​​for clinical scores were determined using two-way multivariate analysis of variance (ANOVA) for multiple comparisons. Unless otherwise stated, data are presented as mean and the standard error of the mean (mean ± SEM).

[0153] Example 1: Stat5 conditional knockout mice are resistant to EAE

[0154] STAT5 negatively regulates T by limiting IL-17 production. H 17 differentiation (Laurence et al., 2007; Yang et al., 2011). However, STAT5 in T H The function of Cd4-Cre;Stat5 in the pathogenesis of 17 is not fully understood. To investigate this issue, [further research is needed]. f / f (Stat5 - / - Mice (where Stat5 was specifically absent in T cell compartments) and littermate controls were tested using MOG on days 0 and 7. 35-55 / CFA was used to immunize mice to induce EAE. The development of paralysis was assessed by assigning clinical scores daily. Surprisingly, paralysis was observed in Stat5. - / - The incidence and severity of clinical disease were reduced in mice (Figures 1A and 1B), a result consistent with STAT5-based T... H The expected antagonistic effect in the production of 17 is the opposite. When a single MOG is performed... 35-55 Similar results were observed when EAE was induced by CFA immunization or by using LPS instead of CFA as an adjuvant (Figures 1C and 1D). (Compared to Stat5) - / - Consistent with the reduction of EAE disease in mice, Stat5 was observed. - / - A significant reduction in immune cell infiltration was observed in the spinal cord of mice (Figure 2A). Furthermore, in Stat5... - / - In mice, various immune cell populations, including CD4 + Cells, CD8 + Cells, B220 + Cells and CD11b + Cell infiltration was reduced (Figs. 2B-D, data not shown). However, in Stat5... + / + Mice and Stat5 - / - Among mice, CD4 in the CNS + IL-17 in T cells + and IFN-γ + The cell occurrence rate was comparable (Figure 3A), indicating that Stat5 - / - Mouse resistance to EAE and T H 1 and T H 17. Cell development was not involved. However, Stat5 was detected. - / - CD4 in the CNS of mice + CD25 + T regThe population decreased (Figure 3B), which indicates that Stat5 - / - The resistance of mice to EAE is unlikely to be due to T. reg The cells undergo changes.

[0155] Example 2: The resistance of Stat5 mutant mice to EAE is attributed to antigen-specific CD4. + Intrinsic defects of T cells and their relationship with T H 1 and T H 17. Irrelevant

[0156] Stat5 missing (Cd4-cre; Stat5) f / - Mice have been reported to exhibit peripheral lymphopenia, accompanied by CD4+. + and CD8 + Both of these T cell types are reduced (Yao et al., 2006). However, another study showed a deficiency of Stat5 (Cd4-cre; Stat5...). f / f It does not affect peripheral CD4. + The proportion of T cells (Burchill et al., 2007). In the experimental setting, peripheral CD4 was not detected due to Stat5 deficiency during EAE production. + The changes in the absolute number of T cells (Figures 4A and 4B) indicate that Stat5 - / - The resistance to EAE in mice was not caused by a decrease in peripheral lymphocytes. Furthermore, in Stat5... - / - CD4 in the spleen of mice + IL-17 was detected in T cells. + and IFN-γ + The increased presence of cells (Figure 4C) further supports the idea that Stat5... - / - Mice's resistance to EAE may be related to T H 1 and T H 17. This is irrelevant. To verify STAT5 in T... H 1 and T H 17. Functions in production, through T H 1 and T H Activation of initial CD4 under 17 polarization conditions + T cells were used for in vitro differentiation. Consistent with previous reports, STAT5 mediates the effect of IL-2 on T cells. H Inhibition of T17 differentiation (data not shown). Interestingly, no effect of IL-7, which also transduces signals via STAT5, on T was observed. H 17 differentiation has a significant effect (data not shown). However, when STAT5 is missing, a difference was observed in T... H IFN-γ under polarization conditions+ The number of cells decreased slightly (data not shown).

[0157] To confirm Stat5 - / - Is the resistance of mice to EAE caused by CD4? + T cell-mediated Rag2 - / - Mice using Stat5 + / + or Stat5 - / - CD4 + T cell regeneration, subsequently inducing EAE. We found that mice receiving Stat5 cells showed significantly higher efficacy compared to mice receiving wild-type cells. - / - CD4 + Rag2 T cells - / - The mice developed resistance to the disease (data not shown), which confirms the Stat5... - / - CD4 + T cells are impaired in their ability to promote EAE production.

[0158] Subsequently, the study investigated whether the lack of encephalitis-inducing effects was due to Stat5. - / - CD4 + This is caused by defects in T cell migration to the CNS. The chemokine receptor CCR6 has been confirmed to be essential for T cell migration. H 17 cells are essential for entering the CNS via the choroid plexus (Reboldi et al., 2009). Therefore, studying Stat5... - / - and Stat5 + / + CD4 + CCR6 expression was observed in both T cells and Stat5. + / + In comparison, Stat5 - / - CD4 in the spleen of mice + CCR6 + Cell proliferation (Fig. 5A). The expression of CXCR3 and CD69 was also investigated, showing increased expression on CD4 in the absence of STAT5. + Increased expression of these two molecules in T cells (Figure 5A). These results indicate that Stat5 - / - CD4 + T cells can infiltrate the CNS. Furthermore, Stat5 was observed (on days 7 and 9) during EAE induction. + / + Mice and Stat5 - / - CD4 present in the CNS of mice + The number of T cells was comparable (Fig. 5B). However, Stat5 - / - CD4 in the CNS of mice + T cells decreased significantly during the course of the disease (day 21) (Figures 5C and 5D). In summary, these results confirm Stat5... - / -CD4 + T cells can infiltrate the CNS, but cannot induce effective inflammation in the CNS during EAE.

[0159] To further rule out the possibility that the resistance of Stat5-deficient mice to EAE is due to autoreactive CD4 in the CNS. + The likelihood caused by any potential defects in T cell survival will increase the number of Stat5 cells compared to wild-type cells. - / - CD4 + T cells translocate to Rag2 - / - In mice, ensure that a sufficient number of autoreactive CD4 cells are present in the CNS during EAE production. + T cells. As shown in Figures 6A and 6B, although CD4 in the CNS was present in both groups of mice... + The number of T cells was similar, but they were in response to Stat5-deficient CD4. + Reduced disease severity was still observed in mice with T cells. Furthermore, a number of Stat5-deficient mice were observed to have similar numbers of CD4+ in their CNS to wild-type mice at the peak of EAE disease. + T cells, however, were relatively resistant to EAE compared to wild-type mice (Fig. 6C), further suggesting that the resistance of Stat5-deficient mice to EAE disease is unlikely to be due to CD4+ in the CNS. + T cell survival is impaired.

[0160] To further investigate these observations and Stat5 - / - CD4 + The causal relationship between intrinsic damage to T cells and MOG 35-55 Specific Stat5 + / + and Stat5 - / - CD4 + T cells translocate to Rag2 - / - In mice without further immunization to test whether these cells could mediate EAE production. As shown in Figures 7A and 7B, Stat5 was administered one week after transfer. + / + CD4 + Mice receiving T cells spontaneously developed EAE disease. Conversely, mice receiving Stat5... - / - CD4 + Mice with T-cell antibodies showed significantly reduced disease severity and incidence. Between the two groups, Rag2... - / - CD4 in the CNS of mice + IL-17 in T cells + and IFN-γ +The cell occurrence rate was comparable (Figure 7C), which further indicates that Stat5 - / - CD4 + Intrinsic defects in the encephalogenic effect of T cells and T H 1 and T H 17 is irrelevant. This is to exclude CD8. + T cells in Stat5 - / - The possible role of Rag2 in the resistance to EAE observed in mice. - / - Mouse using MOG 35-55 Specific Stat5 + / + or Stat5 - / - CD4 + T cells and an equal number of Stat5 cells + / + CD8 + T cell regeneration. Stat5 - / - CD4 + T cells and Stat5 + / + CD8 + T cell metastasis still failed to induce EAE (data not shown). In summary, these data confirm Stat5 - / - CD4 + T cells have an inherent deficiency in their encephalogenic effects. All patents, published applications, and relevant teachings cited herein are incorporated herein by reference in their entirety.

[0161] Example 3: Stat5 - / - CD4 + Reduced expression of GM-CSF in T cells

[0162] To test whether GM-CSF production is impaired due to Stat5 deficiency, MOG 35-55 Specific Stat5 + / + and Stat5 - / - CD4 + Its expression in T cells will be studied. This will be derived from the acceptance of MOG. 35-55 / CFA Immunization Stat5 + / + Mice and Stat5 - / - Mouse spleen cells were treated with various concentrations of MOG 35-55 The attack lasted 24 hours to study GM-CSF secretion. Observations were made in Stat5. + / + In cells, GM-CSF is produced via MOG 35-55 The dose-dependent manner increased (Figure 8A). Conversely, under all conditions, in Stat5... - / -In cells, GM-CSF production was severely reduced. To further verify this, mouse-derived spleen cells were stimulated with PMA / ionomycin in the presence of GolgiPlug during EAE production to perform intracellular staining for GM-CSF and IL-17. Although in Stat5... - / - IL-17 expression is enhanced in cells, but not in CD4 in the absence of STAT5. + CD44 hi GM-CSF was observed in cells + IL-17 - Cells and GM-CSF + IL-17 + The proportion of cells was significantly reduced (Figure 8B). Furthermore, in Stat5... - / - In the spleen of mice, MOG 35-55 Specific GM-CSF + The incidence of T cells was also significantly reduced (Figure 8C). In summary, these results indicate that STAT5 is a precursor to autoreactive CD4+. + GM-CSF expression is required in T cells. However, STAT3 is a key component of T cells. H One important transcription factor in the differentiation of 17 is required for GM-CSF expression (Figure 8D).

[0163] Subsequently, the induction of GM-CSF in the CNS during EAE production was investigated. Despite CNS infiltrative CD4... + The production of IL-17 and IFN-γ by T cells was not impaired due to Stat5 deficiency, but Stat5 was detected compared to control mice. - / - CD4 in the CNS of mice + GM-CSF + The incidence of these cells was reduced (Figure 9A). Further analysis showed that CD4... + IL-17 + In cells, CD4 + IFN-γ + GM-CSF in cells + The percentage was reduced (Figure 9A). Similarly, compared with control mice, those receiving MOG... 35-55 Specific Stat5 - / - CD4 + Rag2 T-cell metastasis - / - Mice also showed CD4 in the CNS + GM-CSF + The incidence of T cells decreased (Fig. 9B). On day 8 post-EAE induction, when Stat5... - / - Mice and Stat5 + / + A significant amount of CD4 was detected in mice.+ During T cell infiltration (Figs. 5C and 5D), Stat5 - / - GM-CSF mRNA expression in the CNS of mice was significantly lower than that in Stat5. + / + Mice (Fig. 9C). Meanwhile, in Stat5... - / - Mice and Stat5 + / + No significant differences in IL-17 and IFN-γ expression were detected among mice (Fig. 9C). In the later stage (day 14, Fig. 9C), Stat5... - / - Impaired cytokine-induced (IL-17 and IFN-γ) activity in the mouse CNS can be caused by Stat5. - / - CD4 + The inability of T cells to maintain neuroinflammation is illustrated by (Figs. 5C and 5D). Interestingly, GM-CSF induction precedes IL-23 induction in the CNS (Fig. 9C), suggesting that IL-23 may not be required for GM-CSF expression during the induction phase of EAE. In summary, these results indicate self-reactive CD4+. + The expression of GM-CSF in T cells is regulated by STAT5, and impaired GM-CSF production in the absence of STAT5 makes the mice resistant to EAE.

[0164] Example 4: IL-7-STAT5 signal transduction induces autoreactive CD4 + GM-CSF expression in T cells promotes neuroinflammation.

[0165] Subsequently, the mechanism by which STAT5 regulates GM-CSF expression was investigated. As demonstrated in this disclosure, neither IL-23 nor IL-1β appears to be a potent STAT5 stimulant (Figure 10A). Furthermore, in STAT5... - / - CD4 + In T cells, IL-1R1 expression remained unchanged, while IL-23Rα expression increased (Fig. 10B). These data suggest that the ability of STAT5 to induce GM-CSF expression may be independent of IL-23 and IL-1β signaling. Conversely, IL-2 and IL-7 potently activate STAT5 by inducing tyrosine phosphorylation (Fig. 10A). Therefore, further investigation is needed into the roles of these two cytokines in GM-CSF induction in autoreactive T cells. Using MOG... 35-55 Alone or MOG 35-55 The attack, in addition to IL-2, originates from accepting MOG. 35-55 Spleen cells from immunized wild-type mice. CD4 count was analyzed by intracellular cytokine staining. + T cells produce GM-CSF and IL-17. As shown in Figure 10C, IL-2 inhibits GM-CSF production. +The presence of T cells showed little effect. Conversely, IL-7 significantly promoted IL-17. - CD4 + CD44 hi T cells and IL-17 + CD4 + CD44 hi GM-CSF expression in both T cells (Fig. 11A). Furthermore, IL-7 functions in a STAT5-dependent manner, as Stat5 deficiency eliminates its effect on GM-CSF expression, as assessed by intracellular cytokine staining and ELISA (Fig. 11A lower panel and Fig. 11B).

[0166] IL-7Rα in CD62L hi CD44 lo T cells and CD62L lo CD44 hi T cells express both of these substances, indicating that IL-7 can directly act on CD4. + T cells regulate GM-CSF expression. Therefore, during EAE production, T cells from Stat5... - / - CD62L was sorted from mice and littermate controls. hi CD44 lo T cells and CD62L lo CD44 hi T cells are then activated in the presence or absence of IL-7. As shown in Figure 11C, CD62L... lo CD44 hi T cells strongly express GM-CSF, while CD62L hi CD44 lo T cells express 1 / 30 of the GM-CSF level. STAT5 deficiency leads to CD62L... lo CD44 hi Reduced basal GM-CSF production in T cells. As expected, IL-7 promotes CD4 in a STAT5-dependent manner. + Expression of GM-CSF in these two subsets of T cells (Figure 11C).

[0167] To study the self-reactive CD4 +The role of IL-7-induced GM-CSF expression in EAE production was investigated in mice treated with an IL-7Rα-specific antibody (clone SB / 14) during EAE production. This treatment significantly reduced disease severity, accompanied by a reduction in CNS inflammation (Figs. 12A and 12B). Consistent with previous reports (Lee et al., 2012), this neutralizing antibody did not exhibit T-cell depletion activity (Fig. 12C). Notably, blocking IL-7 signaling resulted in the reduction of CNS-infiltrating CD4+. + Reduced GM-CSF expression in T cells (Figures 12D-12F). In summary, the findings of this invention demonstrate that IL-7 induces autoreactive CD4+. + STAT5-dependent GM-CSF expression in T cells promotes neuroinflammation.

[0168] Example 5: T expressing GM-CSF H Cells are different from T cells H 17 and T H 1

[0169] Because of T H 17 and T H Both of these can generate GM-CSF, thus determining whether the phenotype of IL-7 stimulation is associated with either of these subsets. To further understand CD4+ expressing GM-CSF... + Cell characteristics, in T H 1 or T H 17. Plate-bound anti-CD3 and soluble anti-CD28 stimulation of primordial CD4 under polarization conditions. + T cells. Anti-CD3 and anti-CD28 induced GM-CSF expression was observed (Fig. 14A). However, although as expected, T cells... H 1. Differentiation conditions promote IFN-γ expression and T H Condition 17 promotes IL-17 expression, but T H 1. Differentiation conditions and T H 17. Differentiation conditions: Both IL-12 and IFN-γ significantly inhibited GM-CSF production (Fig. 13A and 13B). Conversely, neutralization of IL-12 and IFN-γ promoted the production of GM-CSF-expressing cells (Fig. 13A), consistent with previous reports (Codarri et al., 2011). IL-23 and IL-1β did not increase naïve CD4+. + T cells differentiate into cells expressing GM-CSF (Figure 13A), which is related to the discovery of naïve CD4. + T cells do not express their receptors. TGF-β inhibits GM-CSF expression (El-Behi et al., 2011). IL-6 acts as a receptor for T cells. H17. A necessary cytokine for differentiation has a profound inhibitory effect on GM-CSF expression (Figure 14B), suggesting that STAT3 may be a negative regulator. To address this issue, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. - / - Purified initial CD4 in mice + T cells are used for cell differentiation. Surprisingly, in the absence of STAT3, T cells... H Cells polarized under 17 conditions expressed GM-CSF (Fig. 14C). Interestingly, even in the absence of exogenous IL-6, STAT3 still had a moderate inhibitory effect on the differentiation of cells expressing GM-CSF (Fig. 14C). Furthermore, RORγt and T-bet have been reported to inhibit CD4+ differentiation. + GM-CSF expression in T cells is unnecessary (El-Behi et al., 2011). Therefore, the data in this invention support the expression of GM-CSF in CD4 cells. + T cells, through a different process than T cells H 17 and T H A model of phylogenetic development for 1.

[0170] Example 6: IL-7-STAT5 will express GM-CSF T H Cell differentiation programming

[0171] The discoveries of the invention disclosed herein (including, for example, in vivo Stat5) - / - CD4 + Reduced GM-CSF expression in T cells; naïve CD4 + Induction of GM-CSF expression in T cells mediated by IL-7 / STAT5; and T cells expressing GM-CSF H Cells and T H 1 cell and T H (Compared to 17 cells, they exhibited different characteristics), indicating a distinct T cell type regulated by IL-7-STAT5 signaling. H Cell subsets. Activation of naïve CD4 cells with anti-CD3 and anti-CD28 in the presence of varying concentrations of IL-7. + T cells were used to study T cells expressing GM-CSF in vitro. H Cell differentiation was used to further investigate this finding. As shown in Figures 15A and 15B, IL-7 strongly promotes the production of GM-CSF-expressing cells and GM-CSF secretion. Furthermore, STAT5 mediates the IL-7-induced T cell differentiation of GM-CSF-expressing cells. HThe production of GM-CSF is not promoted without STAT5 (Fig. 15C and 15D). Further studies showed that IL-7-induced STAT5 activation directly binds to the promoter region of the Csf2 gene (Fig. 15E and 15F).

[0172] In expressing GM-CSF, T H During differentiation, a small proportion of cells expressing IFN-γ were generated (Fig. 15A). Therefore, the effect of blocking IFN-γ on the production of GM-CSF-expressing cells was tested, showing that the combination of IL-7 and IFN-γ neutralization induced GM-CSF production. + The highest occurrence rate of cells, in which very little IL-17 was detected. + Cells or IFN-γ + Cells (Fig. 16A). Furthermore, T cells expressing GM-CSF were investigated. H The neutron subset restricts the expression of transcription factors and observed that in T cells expressing GM-CSF... H In the study, the expression of RORγt or T-bet was significantly lower than that of T. H 17 cells or T cells H One cell (Fig. 16B) confirmed that T cells expressing GM-CSF were present. H Cells are different from T cells H 1 cell and T H 17 cells. In summary, these data indicate that IL-7-STAT5 signaling is guided by what is known as T... H -Novel expression of GM-CSF promotes the differentiation of a subset of helper T cells.

[0173] Subsequently, it was determined whether IL-2 signal transduction could affect T. H -GM from initial CD4 + T cell differentiation. Adding IL-2 or an antibody against IL-2 only affects GM-CSF. + The presence of cells had little effect (Figure 14D), indicating that IL-2 has a limited effect on T cells. H -GM has a very small role in differentiation. Unlike IL-7Rα, the high-affinity IL-2 receptor IL-2Rα plays a role in initial CD4 differentiation. + It is not expressed in T cells, but TCR activation gradually induces its expression (Figures 17A-17C). Therefore, the minimal role of IL-2 is at least partly due to the initial CD4+. + T cells do not respond to IL-2 stimulation. To support this view, IL-7 is present in the initial CD4+. +STAT5 activation and upregulation of GM-CSF mRNA expression are induced in T cells, but not with IL-2 (Figs. 17D and 17E). To further confirm this idea, activated CD4 cells were stimulated with either IL-2 or IL-7. + T cells showed that both cytokines induced STAT5 activation, Csf2 promoter binding, and GM-CSF mRNA upregulation. Figures 18A-18C It is noteworthy that, compared to IL-7, IL-2 induces prolonged STAT5 activation (Figure 18A).

[0174] Example 7: T H - Different gene expression profiles of GM

[0175] To confirm T H -GM differs from a known subset of T cells (e.g., T1). H 1 and T H 17) Whole transcriptome analysis was performed using microarrays to validate its compatibility with known subsets of T cells, particularly T cells. H Specificity compared to 17 cells. This resulted in the initial CD4... + T cells differentiate into T cells. H 1. T H 17 and T H -GM. Microarray analysis was performed to study their gene expression profiles. Whole transcriptome clustering showed T H -GM cells represent a type of cell different from T cells. H 1 cell or T H A novel subset of 17 cells. T cell lineage-specific gene expression is shown in Table 1. Compared with naïve cells, T... H 17 cells or T cells H Compared to GM cells, T cells were identified H A list of 202 preferentially expressed genes in cells (fold change > 1.7) is provided, with IFN-γ and T-bet at the top of the list (Table 1). Similarly, T-bet was identified in this list. H The list includes 17 characteristic genes, such as IL-17, IL-17F, RORγt, and RORα, and includes those that affect T. H 17 cells possess 411 cell-specific genes (Table 1). T H -GM cell-specific gene list ("T") H -Genes preferentially upregulated in GM" - T H The -GM characteristic genes) contain 210 genes, including the gene encoding GM-CSF, which is the top gene in this list (Table 1). Compared to other subsets, it was identified that in T... H -A set of surface molecules in the GM subset that are selectively expressed at high levels, and in T H- Another group of surface molecules in the GM subset are selectively expressed at low levels. Figure 19 (and Table 1). These molecules (also for T) H -GM characteristic genes) can be used to further characterize T cells through surface markers to identify T cells. H -GM subset. Several other genes of interest were also identified, including those encoding cytokines and transcription factors, particularly IL-3. Various helper T cells were differentiated in vitro, and T cell differentiation was confirmed. H -GM cells and T H 1 cell and T H Compared to 17 cells, these were potent IL-3-producing cells (Figs. 20A, 20C, and 20D). Furthermore, several other cytokines, including EBI-3, PENL, and RANKL, were found in T cells. H -GM cells preferentially express it (Figure 20B), which indicates that T H -GM cells perform a wide variety of biological functions.

[0176] Example 8: T H GM cells are the main pathogenic group.

[0177] To test the expression of GM-CSF, T H subset (T) H The hypothesis that MOGs are the primary encephalitis-inducing effector cells is supported by the theory that MOGs are primarily responsible for encephalitis. 35-55 Specific CD4 + Different subsets of T cells were adopted into Rag2 cells. - / - In mice, EAE was induced. Figure 21 As shown, with T H 17 subsets and T H Compared to a subset, T represents the GM-CSF. H Cells can preferentially induce EAE.

[0178] Example 9: Inhibition of STAT5 activity by chemical inhibitors weakened T H -GM-CSF expression and improvement of EAE

[0179] This study investigated potential therapeutic approaches for autoimmune neuroinflammation by disrupting STAT5 activation through chemical inhibitors. Phosphorylation of key tyrosine residues in the SH2 domain is crucial for STAT5 activation and function. Commercially available STAT5 inhibitors (CAS285986-31-4, Calbiochem) have been reported to selectively disrupt STAT5 tyrosine phosphorylation and DNA binding (Muller et al., 2008). First, this inhibitor was tested on CD4... +The inhibitory effect of IL-7 on STAT5 activation in T cells was investigated. At a concentration of 50 μM, the inhibitor exhibited approximately 50% inhibition, which increased further with increasing concentration (Figure 22A). STAT5 inhibitors have low affinity and therefore require high concentrations to completely block STAT5 activation, while JAK3 inhibitors showed potent inhibition even at low concentrations (Figure 22B). The specificity of the STAT5 inhibitor was then tested by investigating its effect on the activation of STAT3 and STAT1. As shown in Figures 22C and 22D, this STAT5 inhibitor showed minimal inhibition of STAT3 and STAT1 activation at relatively lower concentrations (50 μM or 100 μM).

[0180] The study investigated the effect of STAT5 on T. H -The effect of GM differentiation. As shown, STAT5 inhibitors inhibit T in a dose-dependent manner. H -GM differentiation (Figure 22E). T was observed during STAT5 inhibitor treatment. H 1. Differentiation decreased (data not shown), but T H Differentiation of 17 was not inhibited by STAT5 inhibitors (data not shown).

[0181] To investigate the therapeutic effect of targeting STAT5 activation in acute exacerbation of epidural anemia (EAE), commercially available STAT5 inhibitors were administered intraperitoneally to wild-type mice every other day after disease onset. The development of paralysis was assessed using daily assigned clinical scores. Inhibition of STAT5 reduced the severity of EAE, which was associated with decreased immune cell infiltration in the CNS (Figures 23A and 23B). Conversely, although JAK3 inhibitors potently blocked STAT5 activation (Figure 22B), they showed detrimental effects on EAE (Figure 23B). Notably, STAT5 inhibitors induced CNS-infiltrating CD4+. + Reduced GM-CSF production in T cells (Figures 23C and 23D). This study suggests that targeting STAT5 with chemical inhibitors could be a therapeutic intervention for MS.

[0182] Example 10: T that generates GM-CSF H Cells are related to human RA

[0183] Plasma concentrations of GM-CSF and TNF-α in peripheral blood of RA patients were investigated compared with sex / age-matched healthy controls (HC), and both cytokines were found to be elevated in RA (Figure 24A). Quantification of T cells that produce IFN-γ, IL-17, or GM-CSF in RA and HC was also performed. H In vitro cell occurrence rate. T cells producing IFN-γ and / or GM-CSF were detected in all samples. HHigh incidence of cells, but T cells producing IL-17 were observed. H Low cell occurrence rate (<1%) (Figure 24B). GM-CSF produced alone (GM-CSF) + IFN-γ - ) of T H The cells represented a fairly large population from both RA and HC (Fig. 24B). More importantly, this population was significantly more prevalent in the peripheral blood of RA than in HC (Fig. 24C). Conversely, between RA and HC, T cells that dually produce GM-CSF / IFN-γ... H Cells and T cells that produce IFN-γ alone H No significant differences were observed in the number of cells present (Fig. 24C). Therefore, in RA, T cells that produce GM-CSF alone in peripheral blood are... H The selective increase in the occurrence rate of cells indicates that T H Cellular secretion of GM-CSF is functionally associated with rheumatoid arthritis (RA). Furthermore, plasma GM-CSF concentration in RA was observed to be correlated with the concentration of T cells that produce GM-CSF alone. H Significant correlation between cell occurrence rates (Figure 24D).

[0184] To further evaluate the T that generates GM-CSF H The association between cells and rheumatoid arthritis (RA) was investigated by isolating monocytes from the synovial fluid of RA patients and analyzing their abundance. Compared to peripheral blood, T cells producing GM-CSF were observed in the synovial fluid. H A significant increase in cell incidence was observed, but the majority of these cells co-expressed IFN-γ (Fig. 24E). Similarly, T... H 1 and T H 17 The occurrence of both of these substances also increases in synovial fluid, especially with T H Compared to 1, T H 17 is still a relatively small group (Fig. 24E).

[0185] Example 11: GM-CSF mediates experimental arthritis in a non-TNF-α dependent manner

[0186] The elevated plasma levels of GM-CSF and TNF-α in RA compared to HC suggest therapeutic approaches targeting these two cytokines. In an antigen-induced arthritis (AIA) model, the efficacy of blocking GM-CSF and TNF-α was tested by treating arthritic mice. This model is a T-cell-driven RA model that is readily induced in the C57BL / 6 strain with rapid and synchronized disease onset, thus facilitating the exploration of RA pathogenesis. Blockage of either GM-CSF or TNF-α alone attenuated AIA production (Fig. 25A). Interestingly, the combination of GM-CSF-specific neutralizing antibodies and TNF-α-specific neutralizing antibodies showed superior efficacy in controlling arthritis production compared to either treatment alone (Fig. 25A). That is, targeting GM-CSF may have beneficial efficacy in treating arthritis in a manner independent of TNF-α activity. To further investigate the distinguishable roles of GM-CSF and TNF-α in mediating arthritis production, a mouse strain with conditional Stat5 deficiency in T cells (Cd4-Cre; Stat5) was used. f / f or simply Stat5 - / - AIA was induced in these stat5 knockout mice. These stat5 knockout mice were resistant to arthritis, as exemplified by less joint swelling, less infiltration of immune cells in the synovium, and reduced joint destruction (Figs. 25B-25D), even with significantly increased serum TNF-α and IFN-γ levels (Fig. 25E). Conversely, serum GM-CSF levels were significantly reduced in knockout mice (Fig. 25E), which may be the reason for resistance to arthritis, as further supported by results described below. Consistent results were also observed in collagen-induced arthritis (CIA) models. Figures 26A-26D In summary, these findings indicate that GM-CSF is an important pathogenic mediator in RA and also suggest the prospect of developing anti-GM-CSF drugs to treat RA patients who are unresponsive to anti-TNFα drugs, thereby impacting the T cells that produce GM-CSF. H Cellular markers are a novel biomarker for the diagnosis of rheumatoid arthritis (RA).

[0187] Example 12: Self-reactive T H Synovial inflammation is mediated by STAT5-regulated GM-CSF secretion.

[0188] Based on the association between GM-CSF and arthritis (RA), this study investigated GM-CSF-producing cells and the fundamental regulatory mechanism of GM-CSF expression in arthritis-prone mice. Spleen cells were collected from wild-type AIA mice and divided into three fractions: spleen cells, CD4-depleted cells, and... + T cells, spleen cells, and CD4 +T cells; and each fraction was stimulated with the same cell number under all conditions. Splenic cells produced low but detectable GM-CSF levels without stimulation, which increased significantly upon stimulation with PMA / ionomycin or mBSA antigen (Fig. 28A). CD4 depletion was observed under all conditions. + T cells in spleen cells almost completely eliminate GM-CSF production (Fig. 28A). Conversely, under all conditions, CD4+ production was significantly reduced compared to spleen cells. + T cells produced significantly elevated levels of GM-CSF (Figure 28A). These results strongly support CD4... + T cells are the primary GM-CSF producers, at least in the spleen of arthritic mice, which is partly related to the plasma GM-CSF concentration observed in RA and the proportion of T cells that produce GM-CSF alone. H The correlation of cell occurrence rate was consistent (Figure 24D). Therefore, the study of T H The functional significance of GM-CSF secreted by cells in the development of arthritis. Given that T cell-specific Csf2 knockout mice are unavailable and STAT5 is a key component of T cells... H GM-CSF is a key regulator of cellular expression, so conditional Stat5 knockout mice were used, which showed reduced GM-CSF levels and resistance to arthritis, as described above.

[0189] Consistent with previous studies (Burchill et al., 2007), CD4 was observed in peripheral lymphoid tissue and inflamed synovial tissue of STAT5-deficient mice on day 7 after AIA induction, compared with wild-type mice. + The similar prevalence of T cells (Figures 27A-27D) suggests that loss of STAT5 does not appear to reduce the prevalence of CD4+ in the peripheral blood. + T cell production and impaired infiltration in synovial tissue. To determine whether STAT5 is CD4... + The arthrogenic potential of T cells, which is essential for T cells, will be derived from Stat5. + / + AIA mice and Stat5 - / - Antigen-reactive CD4 cells amplified in vitro from AIA mice + T cells were transfected into wild-type, unexperimented mice, followed by intra-articular injection of mBSA. Stat5 was then administered. + / + CD4 + Mice receiving T cells showed abundant immune cell infiltration in the synovial tissue on day 7 after AIA induction (Fig. 27E). Conversely, mice receiving Stat5... - / - CD4 + Mice with T cells showed a significant reduction in synovial infiltration (Fig. 27E). Therefore, STAT5-deficient CD4 +T cells are defective in their potential to induce arthritis.

[0190] Multiple pieces of evidence support the important role of T cells in rheumatoid arthritis (RA). However, the pathogenic mechanisms of T cells remain not fully understood. Although T cells play a significant role in human RA... H 1 is synovial wettability CD4 + The majority of T cells are found in the arthritis animal population (Berner et al., 2000; Yamada et al., 2008), but in animal models of arthritis, defective IFN-γ signaling leads to increased disease susceptibility (Guedez et al., 2001; Irmler et al., 2007; Manoury-Schwartz et al., 1997; Vermeire et al., 1997). Conversely, T cells... H 17 cells have been shown to be crucial in animal models of arthritis (Pernis, 2009), but T... H The dominance of 17 cells in both peripheral blood and synovial compartments is limited in human RA (Yamada et al., 2008 and Figures 1B and 1E). As demonstrated in this paper, STAT5 regulates the production of GM-CSF by T cells. H Cellular enhancement can lead to the development of arthritis.

[0191] To verify the regulatory role of STAT5 in GM-CSF production, spleen cells derived from AIA mice were stimulated in vitro with PMA / ionomycin plus Golgiplug, followed by intracellular cytokine staining and flow cytometry. As expected, STAT5... - / - In mice, CD4 + CD44 hi The incidence of cells producing GM-CSF alone was significantly reduced in the population (Figs. 28, 34B). Notably, between the two groups, the incidence of cells producing IL-17 alone (T... H 17) or cells that produce IFN-γ alone (T cells) H No significant difference was observed in the occurrence rate of Stat5 (Figure 28B). Further studies combining mBSA restimulation and intracellular cytokine staining showed that Stat5... - / -The incidence of mBSA-specific GM-CSF-producing effector T cells in the spleen of mice was significantly lower than in controls (Fig. 29A). Furthermore, in vitro restimulation of splenocytes and inguinal lymph nodes (LNs) derived from AIA mice with mBSA to measure cytokine concentrations in the culture supernatant revealed a significant reduction in GM-CSF in the presence of STAT5 deficiency, although the levels of IL-17 and IFN-γ were comparable between the two groups (Figs. 29B and 29C). In summary, these results suggest that loss of STAT5 may specifically inhibit GM-CSF-producing effector Th cells in experimental arthritis, but not T cells. H 17 cells or T cells H 1 cell.

[0192] To investigate T cells that produce GM-CSF in synovial inflammation H Cellular involvement and STAT5 regulation of them, from dissected synovial tissue of AIA mice and studies T H Cellular cytokine production. Although GM-CSF has multiple cellular sources (Cornish et al., 2009), CD4... + T H Cells are the main GM-CSF-producing cells in the synovial tissue of AIA mice (Fig. 28C), consistent with observations in the spleen (Fig. 28A). Furthermore, in Stat5... - / - Detected in mice compared to Stat5 + / + Mice had significantly lower levels of synovial T cells that produced GM-CSF. H Percentage of cells (Fig. 28D). On the other hand, T between the two groups H 1 cell and T H 17 cells showed similar percentages for both (Figure 28C). It was expected that, compared to the control, Stat5... - / - The level of GM-CSF in the synovial compartments of mice was decreased. To address this issue, inflamed synovial tissue was collected from AIA mice for RNA and protein extraction to investigate cytokine levels via qPCR and ELISA. Specifically, on days 5 or 7 following arthritis induction, GM-CSF levels were compared with Stat5. + / + Compared to mice, in Stat5 - / -Lower levels of synovial GM-CSF were detected in mice, but not of IFN-γ or IL-17 (Figs. 28E and 30A-30C). Furthermore, two important pro-inflammatory cytokines, IL-6 and IL-1β, were persistently and significantly reduced in STAT5-deficient mice (Figs. 28E and 30A-30C), suggesting reduced synovial inflammation. Notably, TNF-α production was reduced on day 7, not day 5, in STAT5-deficient mice (Figs. 28E and 30A-C). In summary, these results indicate that pro-inflammatory T cells... H Cellular GM-CSF expression regulated by STAT5 is crucial for inducing synovial inflammation.

[0193] To determine T H Cellular GM-CSF production regulated by STAT5 plays a crucial role in mediating synovitis and arthritis. GM-CSF mixed with mBSA was administered via intra-articular injection into the left knee joint of mice immunized with mBSA / CFA, while mBSA was injected alone into the right knee joint. Injection of mBSA alone was sufficient to mediate STAT5-mediated GM-CSF production. + / + Abundant immune cell infiltration was induced in the synovial compartments of mice, but not in Stat5. - / - This was achieved in mice (Fig. 28F). Administration of GM-CSF along with mBSA effectively restored Stat5. - / - Synovial inflammation in mice (Fig. 34F). Consistently, safranin-O / fast green staining revealed inflammation in Stat5 mice. - / - In mice, severe cartilage consumption occurred after GM-CSF / mBSA injection, but not after mBSA injection alone (Fig. 28G). These results therefore support the view that arthrogenic T cells... H Cellular production of GM-CSF regulated by STAT5 is essential for mediating the pathogenesis of arthritis.

[0194] Example 13: GM-CSF derived from Th cells mediates the accumulation of neutrophils in synovial tissue

[0195] The mechanisms by which GM-CSF-producing Th cells induce synovial inflammation and drive arthritis were investigated. Bone marrow lineage-derived cells, including neutrophils, dendritic cells (DCs), and macrophages, express the GM-CSF receptor and are common GM-CSF targets (Hamilton, 2008). Importantly, these cells invade synovial compartments in RA patients and mouse models of arthritis and promote synovitis (McInnes and Schett, 2011). The infiltration of bone marrow lineage-derived cells into the synovial compartments of AIA mice was investigated. CD11b +Bone marrow cells represent the majority (approximately 70%) of synovial infiltrating leukocytes (Figure 31B). Although STAT5 deficiency does not affect CD4... + T H Cell infiltration changed, but when studied on day 7 after arthritis induction, it was different from Stat5. + / + Compared to mice, in Stat5 - / - CD11b in mouse synovium + Cell infiltration was significantly reduced (Figure 31B). This reduction is unlikely to be due to hematopoietic defects, as similar CD11b was detected in the spleens of both groups. + Cell occurrence rate (Figure 31A). Furthermore, over a 7-day period, CD11b... + Cells proliferate in the synovial tissue of wild-type mice, but not in STAT5-deficient mice (Fig. 31C). Notably, synovial CD11b cells are present in STAT5-deficient mice. + Selective ablation of cell accumulation can be partially restored by local application of GM-CSF during arthritis induced episodes (Fig. 31D). In summary, these results suggest that the accumulation of bone marrow cells in synovial compartments can be specifically inhibited by T-cell-specific STAT5 deficiency and the resulting GM-CSF insufficiency.

[0196] Subsequently, CD11b was analyzed. + Different cell populations, including dendritic cells (DCs), macrophages, and neutrophils. Recently reported to be characterized as CD11c. int CD11b hi Ly6C + / hi MHCII hi Mononuclear leukocyte-derived dendritic cells (MoDCs) are involved in the mBSA / IL-1β arthritis model (Campbell et al., 2011). In our AIA model, MoDCs were identified in low abundance in the spleen and synovial tissue (data not shown). Furthermore, in Stat5... + / + Mice and Stat5 - / - Comparable rates of MoDC occurrence were detected in both peripheral lymphoid tissue and synovial tissue in mice (data not shown). These results are consistent with previous studies that confirmed the unnecessary role of GM-CSF in MoDC differentiation (Greter et al., 2012).

[0197] Neutrophils possess significant cytotoxic potential and promote the initiation and progression of rheumatoid arthritis (RA) in multiple ways (Wright et al., 2014). RA disease activity and joint destruction have been shown to be directly associated with neutrophil influx into the joints (Wright et al., 2014). Based on differential expression of Ly6C and Ly6G, CD11b...+ Bone marrow cells can be divided into Ly6C lo Ly6G hi Population (neutrophils) and Ly6C hi Ly6G - Population (monocytes / macrophages). This study shows that in wild-type mice, Ly6C over a 7-day timeframe... lo Ly6G hi The population persists in the synovial tissue and represents synovial CD11b on day 7 after AIA induction. + The dominant cell population, which was persistently and significantly reduced in STAT5-deficient mice (Fig. 32A). Synovial infiltrative Ly6C was confirmed using Giemsa staining. lo Ly6G hi The population consisted of neutrophils, which exhibited typical polymorphic nuclear features with a ring nucleus (Fig. 32B). In contrast, synovial infiltrating Ly6C... hi Ly6G - The population exhibited a mononuclear morphology and was likely composed of mononuclear leukocytes / macrophages (Fig. 32B). Importantly, intra-articular administration of GM-CSF during arthritis induction effectively restored the accumulation of neutrophils in the synovial compartments of STAT5-deficient mice (Fig. 32C), suggesting a T-derived neutrophil origin. H GM-CSF in cells plays a crucial role in mediating the accumulation of neutrophils in inflamed joints.

[0198] Neutrophils are recruited during inflammation, where complex interactions between neutrophils and vascular endothelial cells guide neutrophil adhesion and migration from the circulation to inflamed tissue (Kolaczkowska and Kubes, 2013). In in vitro migration assays, neutrophil adhesion and migration across the endothelial cell monolayer were significantly enhanced by GM-CSF as a chemisorbent (Figs. 33A and 33B), suggesting that GM-CSF can mediate neutrophil recruitment to inflamed joints in AIA. Effective neutrophil apoptosis is crucial for inflammation resolution. However, in synovitis, neutrophil apoptosis is delayed, leading to prolonged survival and persistent inflammation (Wright et al., 2014). Therefore, the effect of GM-CSF on neutrophil survival was tested, and a significant effect of GM-CSF in delaying neutrophil apoptosis was found (Fig. 33C). In summary, these results indicate that GM-CSF can mediate neutrophil recruitment and maintain neutrophil survival within synovial compartments and promote persistent synovitis. To determine the crucial role of neutrophils in AIA, neutrophils were depleted in vivo using a neutralizing antibody (1A8) specific for Ly6G. Administration of the neutralizing antibody significantly improved joint swelling in AIA (Figure 32D). Therefore, neutrophils derived from T... H GM-CSF-mediated neutrophil accumulation in cells is important for AIA production.

[0199] Example 14: GM-CSF enhances the production of pro-inflammatory cytokines in bone marrow cells and synovial fibroblasts.

[0200] Cytokines are important mediators in the interaction between innate and adaptive immunity. As shown in this article, several pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) associated with the pathogenesis of rheumatoid arthritis (Choy and Panayi, 2001) were significantly reduced in the synovial tissue of STAT5-deficient AIA mice (Figs. 28E and 30A-30C). To understand the underlying mechanisms of the observed cytokine reduction, synovial cells were segregated into different populations based on differential expression of surface markers to investigate the cellular origin of these pro-inflammatory cytokines (Fig. 34A). CD45 was assessed by RT-PCR. + TCRβ + population (T cells), CD45 + TCRβ - CD11c - CD11b + Population (mainly monocytes / macrophages and neutrophils) and CD45 + TCRβ - CD11c +Cytokine mRNA expression levels in the population (dendritic cells). Similar to IL-17 (as a control), GM-CSF was primarily produced by synovial T cells (Fig. 34B), which further enhanced the T cells that produced GM-CSF. H The importance of cells. Conversely, IL-6 and IL-1β are primarily produced by bone marrow cells, such as CD11b. + Groups and CD11c + Population (Fig. 34B). TNF-α was expressed in all three populations, with relatively low abundance in T cells (Fig. 34B). Based on CD11b as described above + Differential expression of Ly6C and Ly6G in the population, further analysis of Ly6C lo Ly6G hi Population (neutrophils) and Ly6C hi Ly6G - The population (monocytes / macrophages) showed that monocytes / macrophages are likely the primary IL-6 producers, while neutrophils appear to be better producers of IL-1β and TNF-α (Fig. 34C). These results, along with the findings mentioned above (Figs. 28E and 30A-30C), suggest that T... H The link between GM-CSF secreted by cells and the expression of pro-inflammatory cytokines in bone marrow cells in synovitis.

[0201] To test the regulatory role of GM-CSF in the expression of IL-6 and IL-1β, bone marrow-derived macrophages (BMDM) and bone marrow-derived dendritic cells (BMDC) were cultured and stimulated with GM-CSF. Indeed, GM-CSF stimulation rapidly upregulated the mRNA expression of both IL-6 and IL-1β within 1 hour (Figs. 34D and 34E). Furthermore, GM-CSF significantly increased IL-6 secretion from BMDM in a dose-dependent manner (Fig. 34F), and even at low doses, it increased IL-6 secretion from BMDC (Fig. 34G). To induce the secretion of mature IL-1β, BMDM were sensitized with LPS for 6 hours, during which different concentrations of GM-CSF were added, followed by ATP stimulation. The addition of GM-CSF significantly enhanced the secretion of IL-1β into the culture supernatant, as measured by ELISA (Fig. 34H). Synovial fibroblasts, as active cells in synovial inflammation (Muller-Ladner et al., 2007), also showed increased IL-1β mRNA expression upon GM-CSF stimulation (Fig. 34I). No induction of TNF-α expression by GM-CSF was observed in BMDM, BMDC, or synovial fibroblasts (data not shown). Given the functional importance of IL-6 and IL-1β in arthritis development (Choy and Panayi, 2001), T...H GM-CSF secreted by cells may also mediate synovial inflammation by inducing the expression of IL-6 and IL-1β in bone marrow cells and synovial fibroblasts.

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[0323] While the invention has been specifically shown and described with reference to exemplary embodiments thereof, those skilled in the art will understand that various changes in form and detail may be made therein without departing from the scope of the invention as covered by the appended claims.

Claims

1. Regulation of granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted T helper cells (T cells). H Use of a modulator of GM-CSF function in the preparation of a medicament for treating patients with rheumatoid arthritis associated with a non-tumor necrosis factor α (TNF-α)-dependent inflammatory pathway, wherein the modulator is an inhibitor of signal transduction and transcription activator 5 (STAT5), wherein the modulator is an antibody or a small molecule compound wherein GM-CSF is present in the peripheral blood of the patient. + IFN-γ - T H The level of selective elevation in cells.

2. The use as described in claim 1, wherein the regulator inhibits T H -GM function.

3. The use as described in claim 1, wherein the regulator is pimozide.

4. The use as described in claim 1, wherein the regulator is a compound with CAS number 285986-31-4.

5. The use as described in claim 1, wherein the modulator is an antibody that binds to STAT5.

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