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PURPOSE The rho-associated coiled-coil–containing protein kinase-2 (ROCK2) signaling pathway regulates the Th17/regulatory T cells balance and controls profibrotic pathways. Selective ROCK2 inhibition with belumosudil (KD025) may offer a novel approach to the management of chronic graft-versus-host disease (cGVHD). PATIENTS AND METHODS A phase IIa, open-label, dose-finding study of belumosudil enrolled 54 patients with cGVHD who had received one to three prior lines of therapy (LOTs). The primary end point was overall response rate (ORR). RESULTS The median time from cGVHD diagnosis to enrollment was 20 months. Seventy-eight percent of patients had severe cGVHD, 50% had ≥ 4 organs involved, 73% had cGVHD refractory to their last LOT, and 50% had received ≥ 3 prior LOTs. With an overall median follow-up of 29 months, the ORR (95% CI) with belumosudil 200 mg once daily, 200 mg twice daily, and 400 mg once daily was 65% (38% to 86%), 69% (41% to 89%), and 62% (38% to 82%), respectively. Responses were clinically meaningful, with a median duration of response of 35 weeks, and were associated with quality-of-life improvements and corticosteroid (CS) dose reductions. CS treatment was discontinued in 19% of patients. The failure-free survival rate was 76% (62% to 85%) and 47% (33% to 60%) at 6 and 12 months, respectively. The 2-year overall survival rate was 82% (69% to 90%). Belumosudil was well-tolerated, with low rates of cytopenia. There were no unexpected adverse events and no apparent increased risk of infection, including cytomegalovirus infection and reactivation. CONCLUSION Belumosudil treatment resulted in a high ORR and overall survival rate and demonstrated quality-of-life improvements, CS dose reductions, and limited toxicity. Data from the study indicated that belumosudil may prove to be an effective therapy for patients with treatment-refractory cGVHD.

Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder characterized by chronic inflammation and fibrosis. Rho-associated coiled-coil-containing protein kinases (ROCKs) are key coordinators of tissue response to injury, regulating multiple functions, such as gene expression and cell migration, proliferation and survival. Relevant to cGVHD and autoimmunity, only the ROCK2 isoform drives a pro-inflammatory type 17 helper T (Th17) cell response. Moreover, ROCK2 inhibition shifts the Th17/regulatory T (Treg) cell balance toward Treg cells and restores immune homeostasis in animal models of autoimmunity and cGVHD. Furthermore, the selective inhibition of ROCK2 by belumosudil reduces fibrosis by downregulating both transforming growth factor-β signaling and profibrotic gene expression, thereby impeding the creation of focal adhesions. Consistent with its anti-inflammatory and antifibrotic activities, belumosudil has demonstrated efficacy in clinical studies, resulting in an overall response rate of >70% in patients with cGVHD who failed 2 to 5 prior lines of systemic therapy. In summary, selective ROCK2 inhibition has emerged as a promising novel therapeutic approach for treating cGVHD and other immunologic diseases with unique mechanisms of action, targeting both immune imbalance and detrimental fibrotic responses.

In vitro induced human regulatory T cells (iTregs) have demonstrated in vivo therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human iTregs generated in vitro from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4

Background & Aims Fibrosis, the primary cause of morbidity in chronic liver disease, is induced by pro-inflammatory cytokines, immune cell infiltrates, and tissue resident cells that drive excessive myofibroblast activation, collagen production, and tissue scarring. Rho-associated kinase 2 (ROCK2) regulates key pro-fibrotic pathways involved in both inflammatory reactions and altered extracellular matrix remodelling, implicating this pathway as a potential therapeutic target. Methods We used the thioacetamide-induced liver fibrosis model to examine the efficacy of administration of the selective ROCK2 inhibitor KD025 to prevent or treat liver fibrosis and its impact on immune composition and function. Results Prophylactic and therapeutic administration of KD025 effectively attenuated thioacetamide-induced liver fibrosis and promoted fibrotic regression. KD025 treatment inhibited liver macrophage tumour necrosis factor production and disrupted the macrophage niche within fibrotic septae. ROCK2 targeting in vitro directly regulated macrophage function through disruption of signal transducer and activator of transcription 3 (STAT3)/cofilin signalling pathways leading to the inhibition of pro-inflammatory cytokine production and macrophage migration. In vivo, KDO25 administration significantly reduced STAT3 phosphorylation and cofilin levels in the liver. Additionally, livers exhibited robust downregulation of immune cell infiltrates and diminished levels of retinoic acid receptor-related orphan receptor gamma (RORγt) and B-cell lymphoma 6 (Bcl6) transcription factors that correlated with a significant reduction in liver IL-17, splenic germinal centre numbers and serum IgG. Conclusions As IL-17 and IgG–Fc binding promote pathogenic macrophage differentiation, together our data demonstrate that ROCK2 inhibition prevents and reverses liver fibrosis through direct and indirect effects on macrophage function and highlight the therapeutic potential of ROCK2 inhibition in liver fibrosis. Lay summary By using a clinic-ready small-molecule inhibitor, we demonstrate that selective ROCK2 inhibition prevents and reverses hepatic fibrosis through its pleiotropic effects on pro-inflammatory immune cell function. We show that ROCK2 mediates increased IL-17 production, antibody production, and macrophage dysregulation, which together drive fibrogenesis in a model of chemical-induced liver fibrosis. Therefore, in this study, we not only highlight the therapeutic potential of ROCK2 targeting in chronic liver disease but also provide previously undocumented insights into our understanding of cellular and molecular pathways driving the liver fibrosis pathology., Graphical abstract, Highlights • ROCK2 inhibition with the small-molecule inhibitor KD025 prevents and reverses hepatoxin-induced liver fibrosis. • ROCK2 inhibition attenuates profibrogenic immune function. • KD025 exerts direct effects on liver macrophages resulting in decreased TNF secretion and impeded migration. • KD025 administration attenuates T cell IL-17 production and B-cell IgG production, which indirectly contributes to downregulation of profibrogenic macrophage function.

Rho-associated coiled-coil kinase (ROCK)2 targeting down-regulates autoimmune responses in animal models and patients, however the underlying molecular mechanism is still an enigma. We report that ROCK2 binds phosphorylated-STAT3 and its kinase activity controls the formation of ROCK2/STAT3/JAK2 complex and optimal STAT3 phosphorylation in human CD4+ T cells during T helper 17 (TH17)-skewing. Moreover, chromatin-immunoprecipitation sequencing (ChIP-seq) analysis revealed that, genome-wide, about 70% of ROCK2 and STAT3 peaks overlapped and co-localized to several key genes controlling TH17 and T follicular helper (TFH) cell functions. Specifically, the co-occupancy of ROCK2 and STAT3 on the Irf4 and Bcl6 genes was validated by ChIP-qPCR analysis. Furthermore, the binding of ROCK2 to both the Irf4 and Bcl6 promoters was attenuated by STAT3 silencing as well as by selective ROCK2 inhibitor. Thus, the present study demonstrated previously unidentified evidence that ROCK2-mediated signaling in the cytosol provides a positive feed-forward signal for nuclear ROCK2 to be recruited to the chromatin by STAT3 and potentially regulates TH17/TFH gene transcription.

Contains fulltext : 196332.pdf (Publisher’s version ) (Closed access) Reducing the activities of the pro-inflammatory cytokine IL-17 is an effective treatment strategy for several chronic autoimmune disorders. Rho-associated coiled-coil containing kinase 2 (ROCK2) is a member of the serine-threonine protein kinase family that regulates IL-17 secretion in T cells via signal transducer and activator of transcription 3 (STAT3)-dependent mechanism. We reported here that the selective ROCK2 inhibitor KD025 significantly reduced in vitro production of IL-17 in unfractionated human peripheral blood mononuclear cells (PBMCs) stimulated with the dectin-1 agonist Candida albicans. C. albicans induced IL-17 was reduced by 70% (p < 0.0001); a similar reduction (80%) was observed in PBMC stimulated with the Toll-like receptor 2 agonist Staphylococcus epidermidis (p < 0.0001). Treatment of PBMC with KD025 was not associated with a reduction in IL-1beta, IL-6 or IL-1alpha levels; in contrast, a 1.5 fold increase in the level of IL-1 receptor antagonist (IL-1Ra) was observed (p < 0.001). KD025 down-regulated C. albicans-induced Myosin Light Chain and STAT3, whereas STAT5 phosphorylation increased. Using anti-CD3/CD28 activation of the TCR, KD025 similarly suppressed IL-17 independent of a reduction in IL-1beta. Thus, ROCK2 directly regulates IL-17 secretion independent of endogenous IL-1 and IL-6 supporting development of selective ROCK2 inhibitors for treatment of IL-17-driven inflammatory diseases.

Introduction cGVHD is characterized by an imbalance between effector and regulatory arms of the immune system that results in over production of IL-17 and IL-21. A reduction in the regulatory T (Treg) cells limits the ability of the immune system to re-calibrate this pro-inflammatory environment. KD025 is an oral Rho kinase 2 (ROCK2) selective inhibitor. In vitro data demonstrate KD025 modulates immune homeostasis by shifting the Th17/Treg balance towards Treg. Methods 3 cohorts (C1: 200 mg QD, C2: 200 mg BID, and C3: 400 mg QD) of patients (pts) with cGVHD after 1-3 prior lines of systemic therapy were treated in 28-Day cycles until disease progression. The primary endpoint is overall response rate (ORR) (partial/complete response) per 2014 NIH criteria. Additional endpoints include duration of response (DOR), corticosteroid (CS) dose reductions, Lee Symptom Scale (LSS) score and PD. Blood samples were collected at Cycle 1 Day 1 (C1D1), C2D1, C4D1 and C6D25, and shipped at ambient temperature. PBMC were isolated and kept frozen until analysis. Intracellular expression of IL-17A and FOXP3 was determined by flow cytometry. Results Data as of 6-June-2018 for C1 and C2 are included; C3 data will be available 4Q18. 17 and 16 pts were enrolled in C1 and C2 with median age 52 years, time from cGVHD diagnosis to KD025 treatment of 19 months, and 3 prior regimens. Median duration of treatment was37 (C1) and 33 (C2) weeks. KD025 demonstrated ORR of 65% in C1 and 63% in C2. Responses were rapid (76% at first assessment at 8 weeks) and durable (≥20 weeks) in 82% (C1) and 50% (C2) of responders. Median CS dose was reduced 44% (C1) and 26% (C2). 76% and 56% of pts achieved CS dose reductions. 5/33 (15%) pts discontinued CS. 65% (C1) and 50% (C2) achieved a meaningful improvement (≥7 point reduction) in the LSS score. Common AEs were increased LFTs 42%, URI 33%, anemia 27%, nausea 24%, diarrhea 24% and fatigue 21%. Grade 3+ LFT increases were reported for 6 (18%) pts. 2 pts discontinued treatment due to AEs possibly related to KD025 (headache, diarrhea). SAEs were reported for 11/33 pts, none considered related to KD025. There was no apparent increased risk of infection. Exploratory PD analyses revealed an early increase in the percentage of CD4+ Treg cells by C2D1 with a simultaneous decrease in Th17 cells. The percentage of CD4+ Tregs continued to increase, and the Th17 cells continued to decrease through C6D25. Conclusion KD025 achieved responses with little toxicity. Responses are clinically meaningful with durability, reductions in CS doses and improvement in LSS score. PD data indicate KD025 may restore the Th17/Treg balance.

Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.

Targeted inhibition of Rho-associated kinase (ROCK)2 downregulates the proinflammatory T cell response while increasing the regulatory arm of the immune response in animals models of autoimmunity and Th17-skewing human cell culture in vitro. In this study, we report that oral administration of a selective ROCK2 inhibitor, KD025, reduces psoriasis area and severity index scores by 50% from baseline in 46% of patients with psoriasis vulgaris, and it decreases epidermal thickness as well as T cell infiltration in the skin. We observed significant reductions of IL-17 and IL-23, but not IL-6 and TNF-α, whereas IL-10 levels were increased in peripheral blood of clinical responders after 12 wk of treatment with KD025. Collectively, these data demonstrate that an orally available selective ROCK2 inhibitor downregulates the Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via a defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.

Yin-Yang T Cell Signaling Immune responses are kept in check by CD4 + regulatory T cells (T reg ) that suppress other immune cells, including CD4 + effector T cells (T eff ). T reg and T eff cells have many signaling components in common, yet triggering through their T cell receptors (TCRs) leads to very different outcomes. Zanin-Zhorov et al. (p. 372 , published online 25 March) compared the recruitment of signaling molecules to the immunological synapse after TCR triggering in T reg and T eff cells. Although T reg cells do form synapses, signaling molecules that promote T eff activation, such as protein kinase C-θ (PKC-θ), were not recruited. Inhibition or depletion of PKC-θ in T reg cells led to suppressive activity against T eff cells, whereas costimulation enhanced PKC-θ recruitment and less suppression. Together, this suggests that PKC-θ is inflammatory in both T reg and T eff cells; however, by excluding it from the synapse, T reg cells are able to maintain suppression in the face of TCR signaling.

Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) cellswhich provide help necessary to generate antibody-producing B cells in normal and autoimmune conditionsWe found that targeting ROCK2 in normal human T cells or peripheral blood mononuclear cells (PBMCs) from patients with active systemic lupus erythematosus (SLE) decreased the number and function of Tfh cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity concomitantly decreased the abundance of the transcriptional regulator Bcl6 and increased that of Blimp1 by xxx concurrent regulation of the binding of signal transducer and activator of transcription 3 (STAT3) and STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a two-fold reduction in the numbers of Tfh cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice, and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of Tfh cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.

Introduction: cGVHD remains a leading cause of post-transplant morbidity / mortality, exhibiting features of both autoimmune and fibrotic diseases across multiple organ systems. cGVHD is characterized by an imbalance between effector and regulatory arms of the immune system that results in over production of inflammatory cytokines including IL-17 and IL-21. Moreover, a persistent reduction in the number of regulatory T (Treg) cells limits the ability of the immune system to re-calibrate this pro-inflammatory environment. KD025 is an orally available Rho-associated coiled-coil kinase 2 (ROCK2) selective inhibitor. In vitro data have demonstrated that KD025 (1) attenuates IL-21 and IL-17 secretion in human CD4+ T cells via STAT3, IRF4 and RORγt regulation, and (2) leads to increased percentages of Foxp3+ CD4+ T cells via a STAT5-dependent mechanism and upregulates the suppressive function of human Tregs. These MOA suggest that KD025 modulates immune homeostasis by shifting the Th17/Treg balance towards a Treg phenotype. No studies have examined KD025 effects in patients with dysregulation associated with high Th17 and low Tregs. Methods: KD025-208 enrolled 3 cohorts (C1: 200 mg QD, C2: 200 mg BID, and C3: 400 mg QD) of patients (pts) with cGVHD after 1-3 prior lines of systemic therapy. Treatment is in 28-Day cycles until disease progression or unacceptable toxicity. The primary endpoint is the overall response rate (ORR) (% pts achieving partial response (PR) or complete response (CR)) as per 2014 NIH response criteria. Secondary endpoints include duration of response (DOR) and corticosteroid (CS) dose reductions. Exploratory endpoints include Lee Symptom Scale (LSS) score and pharmacodynamics (PD). Peripheral blood samples were collected from all pts at Cycle 1 Day 1 (C1D1), C2D1, C4D1 and C6D25, and shipped at ambient temperature. PBMC were isolated and kept frozen until analysis. The intracellular expression of IL-17A and FOXP3 on viable CD3+ CD4+ cells was determined by using multicolor flow cytometry. Results: Data as of 6-June-2018 for C1 and C2 are included; C3 data will be available 4Q18. 17 and 16 pts were enrolled in C1 and C2 with median age 52 years, median time from cGVHD diagnosis to KD025 treatment of 19 months, and a median of 3 prior treatment regimens. Median duration of treatment was 37 (C1) and 33 (C2) weeks (wks). KD025 demonstrated an ORR of 65% in C1 and 63% in C2. ORR in key subgroups is shown below. Responses were rapid (76% achieved at first assessment at 8 wks) and durable with 82% (C1) and 50% (C2) of responders sustaining response for ≥20 wks. Responses including CRs were observed across all affected organ systems except lung (mouth, eyes, joints/ fascia, skin, upper GI, lower GI, esophagus, liver). A previously reported lung PR was deemed on review to be IPF with no cGVHD involvement. During treatment with KD025, median CS dose was reduced by 44% (C1) and 26% (C2). 76% and 56% of pts achieved CS dose reductions. 5/33 (15%) pts discontinued CS. 65% (C1) and 50% (C2) achieved a meaningful improvement (≥7 point reduction) in the LSS score, including both responders (67%) and non-responders (42%). KD025 has been well tolerated. AEs reported were generally consistent with those expected in pts with advanced cGVHD treated with CS. Common AEs were increased LFTs (42%), URI (33%), anemia (27%), nausea (24%), diarrhea (24%) and fatigue (21%). Increased LFTs did not lead to treatment discontinuation, commonly occurred prior to KD025 dosing and were often considered attributable to cGVHD. Grade 3+ LFT increases were reported for 6 (18%) pts. 2 pts discontinued treatment due to AEs possibly related to KD025 (headache, diarrhea). SAEs were reported for 11/33 (33%) pts, none considered related to KD025 treatment. There was no apparent increased risk of infection. Exploratory PD analyses of PBMCs across C1 and C2 revealed an early increase in the percentage of CD4+ Treg cells by C2D1 of KD025 treatment with a simultaneous decrease in Th17 cells. The percentage of CD4+ Tregs continued to increase, and the Th17 cells continued to decrease through C4D1 and C6D25. Conclusion: Treatment with KD025 has demonstrated encouraging clinical responses in cGVHD pts with little toxicity. Responses have been clinically meaningful as evidenced by durability, reductions in CS doses and improvement in LSS score. Exploratory PD data indicate treatment with KD025 may modulate immune homeostasis by restoring the Th17/Treg balance. Figure. Figure. Disclosures Jagasia: Incyte Corporation: Membership on an entity's Board of Directors or advisory committees. Salhotra:Kadmon Corporation, LLC: Consultancy. Bachier:Sanofi: Speakers Bureau; Seattle Genetics: Speakers Bureau. Weisdorf:Seattle Genetics: Consultancy; Pharmacyclics: Consultancy; Equillium: Consultancy; SL Behring: Consultancy; FATE: Consultancy. Green:Kadmon Corporation, LLC: Employment. Schueller:Kadmon Corporation, LLC: Employment. Zanin-Zhorov:Kadmon Corporation, LLC: Employment. Weiss:Kadmon Corporation, LLC: Employment. Eiznhamer:Kadmon Corporation, LLC: Employment. Aggarwal:Kadmon Corporation, LLC: Employment. Blazar:Kadmon Corporation, LLC: Consultancy, Research Funding. Lee:Takeda: Research Funding; Kadmon: Research Funding; Amgen: Consultancy, Research Funding; Mallinckrodt: Honoraria; Incyte: Consultancy; Pfizer: Consultancy; Onyx: Research Funding.

LPS, a molecule produced by Gram-negative bacteria, is known to activate both innate immune cells such as macrophages and adaptive immune B cells via TLR4 signaling. Although TLR4 is also expressed on T cells, LPS was observed not to affect T cell proliferation or cytokine secretion. We now report, however, that LPS can induce human T cells to adhere to fibronectin via TLR4 signaling. This response to LPS was confirmed in mouse T cells; functional TLR4 and MyD88 were required, but T cells from TLR2 knockout mice could respond to LPS. The human T cell response to LPS depended on protein kinase C signaling and involved the phosphorylation of the proline-rich tyrosine kinase (Pyk-2) and p38. LPS also up-regulated the T cell expression of suppressor of cytokine signaling 3, which led to inhibition of T cell chemotaxis toward the chemokine stromal cell-derived factor 1α (CXCL12). Thus, LPS, through TLR4 signaling, can affect T cell behavior in inflammation.

Peptide p277 is a 24-amino acid fragment of the heat shock protein 60 molecule, first discovered to be an antigen for diabetogenic T-cell clones in non-obese diabetic (NOD) mice. Therapeutic vaccination with p277 can arrest the spontaneous diabetogenic process both in NOD mice and in humans associated with a T(h)1 to T(h)2 cytokine shift specific for the autoimmune T cells. We now report that p277 can directly signal human T cells via innate toll-like receptor (TLR)-2, leading to up-regulation of integrin-mediated adhesion to fibronectin, and inhibition of chemotaxis to the chemokine SDF-1alpha in vitro. Resting CD45RA(+) T cells responded to lower concentrations of p277 than resting CD45RO(+) T cells, but activation of CD45RO(+) T cells greatly increased their sensitivity to p277. Mouse T cells, but not macrophages, were also sensitive to the innate effects of peptide p277, and adoptive transfer of diabetes by splenic T cells from NOD mice could be inhibited by p277 treatment before transfer. Thus, T cells do respond innately to p277, and signaling by soluble p277 through TLR2 could contribute to the treatment of type 1 diabetes; p277 may stop the destruction of beta cells by signaling in concert both innate and adaptive receptors on T cells.

Halofuginone, a low molecular weight plant alkaloid, inhibits collagen α1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-β (TGF-β), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-κB activity, production of tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10–40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-κB activity (80% inhibition following incubation with 40 ng halofuginone, P=0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-α, IFN-γ, interleukin (IL)-4, IL-13, and TGF-β (P

Extracellular heat shock protein 60 (HSP60) has been considered a proinflammatory danger signal. Yet, HSP60 can also down-regulate experimental immune arthritis and diabetes models by specific inhibition of Th1-like responses. We now report that HSP60 in vitro differentially modulates the expression of Th1/Th2 transcription factors in human T cells: HSP60 down-regulates T-bet, NF-κB, and NFATp and up-regulates GATA-3, leading to decreased secretion of TNF-α and IFN-γ and enhanced secretion of IL-10. These effects depended on TLR2 signaling and could not be attributed to LPS or to other contaminants. In BALB/c mice, HSP60 in vivo inhibited the clinical, histological, and serological manifestations of Con A-induced hepatitis associated with up-regulated T cell expression of suppressor of cytokine signaling 3 and GATA-3 and down-regulated T-bet expression. These results provide a molecular explanation for the effects of HSP60 treatment on T cell inflammation via innate regulation of the inflammatory response.

Soluble 60 kDa heat shock protein (HSP60) activates macrophages via TLR4. We now report that soluble HSP60 activates T cells via the innate receptor TLR2. HSP60 activated T cell adhesion to fibronectin to a degree similar to other activators: IL-2, SDF-1alpha, and RANTES. T cell type and state of activation was important; nonactivated CD45RA+ and IL-2-activated CD45RO+ T cells responded optimally (1 h) at low concentrations (0.1-1 ng/ml), but nonactivated CD45RO+ T cells required higher concentrations (approximately 1 microg/ml) of HSP60. T cell HSP60 signaling was inhibited specifically by monoclonal antibodies (mAb) to TLR2 but not by a mAb to TLR4. Indeed, T cells from mice with mutated TLR4 could still respond to HSP60, whereas Chinese hamster T cells with mutated TLR2 did not respond. The human T cell response to soluble HSP60 depended on phosphatidylinositol 3-kinase and protein kinase C signaling and involved the phosphorylation of Pyk-2. Soluble HSP60 also inhibited actin polymerization and T cell chemotaxis through extracellular matrix-like gels toward the chemokines SDF-1alpha (CXCL12) or ELC (CCL19). Exposure to HSP60 for longer times (18 h) down-regulated chemokine receptor expression: CXCR4 and CCR7. These results suggest that soluble HSP60, through TLR2-dependent interactions, can regulate T cell behavior in inflammation.

Targeted inhibition of Rho-associated kinase 2 (ROCK2) down-regulates pro-inflammatory T-cell response, while increasing the regulatory arm of immune response in animals models of autoimmunity and T helper 17 (Th17)-skewing human cell culture in vitro. We conducted a Phase 2, open-label, dose-finding study to evaluate the safety, tolerability, and activity of selective ROCK2 inhibitor, KD025, in subjects with Psoriasis Vulgaris who failed first-line therapy (NCT02317627 at ClinicalTrials.gov). Here, we report that oral administration of KD025 reduces Psoriasis Area and Severity Index (PASI) scores in 85% of patients completing the study, with minimal side effects. In the 400 mg QD and 200 mg BID cohorts 42% and 71% of patients respectively achieved at least a 50% decrease in PASI score (PASI 50) after three month of treatment. KD025 treatment reduced levels of both IL-17 and IL-23, but no IL-6 and TNF-a in the peripheral blood of clinical responders, whereas IL-10 levels were increased at the end of the study. The clinical improvement and changes in cytokine levels were associated with decreased epidermal thickness, T-cell infiltration and down-regulation of key molecules implicated in the regulation of the Th17 pathway, such as ROCK2, pSTAT3, RORgt and IRF4 in the skin. Collectively, these data demonstrate that orally available selective ROCK2 inhibitor down-regulates Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.

Rho-associated kinase 2 (ROCK2) was recently shown to be implicated in regulation of IL-17 and IL-21 secretion in both mice and humans via down-regulation of STAT3 phosphorylation and subsequent transcription activity. Here we report that the ROCK2, but not ROCK1 protein binds phosphorylated-STAT3 (pSTAT3) in both cytoplasmic and nuclear compartments in human CD4+ T cells during T helper 17 (TH17)-skewing activation. The interaction of ROCK2 with pSTAT3 was partially abrogated by treatment of cells with selective ROCK2 inhibitor suggesting that ROCK2 kinase activity is required for formation of JAK-STAT complex and STAT3 phosphorylation. Further analysis by chromatin-immunoprecipitation sequencing (ChIP-seq) revealed that ROCK2 binding is significantly enriched toward promoter regions and peaks at transcription start sites (TSS) relative to genomic DNA. Moreover, 60–70% of ROCK2 and STAT3 peaks are overlapped genome-wide and co-localized to several key genes that control TH17 and T follicular helper (TFH) cell functions. Specifically, the binding of ROCK2 and STAT3 to the IRF4 and Bcl6 genes was validated by ChIP-qPCR analysis performed in human CD4+ T cells activated by TH17-skewing conditions. Finally, siRNA-mediated inhibition of STAT3 attenuated ROCK2 binding to the IRF4 and Bcl6 promoters indicating the critical role of STAT3 in the recruitment of ROCK2 to chromatin and ROCK2-mediated regulation of TH17/TFH gene transcription. Together, the present work provides previously unidentified insights into the molecular mechanism of specific involvement of ROCK2 isoform in regulating STAT3 phosphorylation and transcriptional activity in TH17-driven autoimmune responses.

Balanced regulation of cytokine secretion in T cells is critical for maintenance of immune homeostasis and prevention of autoimmunity. The Rho-associated kinase (ROCK) 2 signaling pathway was previously shown to be involved in controlling of cellular movement and shape. However, recent work from our group and others has demonstrated a new and important role of ROCK2 in regulating cytokine secretion in T cells. We found that ROCK2 promotes pro-inflammatory cytokines such as IL-17 and IL-21, whereas IL-2 and IL-10 secretion are negatively regulated by ROCK2 under Th17-skewing activation. Also, in disease, but not in steady state conditions, ROCK2 contributes to regulation of IFN-γ secretion in T cells from rheumatoid arthritis patients. Thus, ROCK2 signaling is a key pathway in modulation of T-cell mediated immune responses underscoring the therapeutic potential of targeted inhibition of ROCK2 in autoimmunity.

Toll-like receptors (TLRs) are widely expressed and play an essential role in the activation of innate immune cells. However, certain TLRs are also expressed on T cells, and TLR ligands can directly modulate T cell functions. Here, we discuss findings indicating that T cells directly respond to Heat Shock Protein (HSP) 60, a self molecule, or to the HSP60-derived peptide, p277, via a TLR2-dependent mechanism. HSP60 has been considered to be a “danger signal” for the immune system because of its ability to induce pro-inflammatory phenotypes in innate immune cells – in this case via TLR4 activation; nevertheless, TLR2 engagement by HSP60 on T cells can lead to resolution of inflammation by up-regulating the suppression function of regulatory T cells and shifting the resulting cytokine secretion balance toward a Th2 phenotype. Moreover, T cell TLR4 engagement by LPS leads to up-regulation of suppressor of cytokine signaling 3 expression and consequently down-regulates T cell chemotaxis. Thus, TLR2 and TLR4 activation can contribute to both induction and termination of effector immune responses by controlling the activities of both innate and adaptive immune cells.

Foxp3 + CD4 + CD25 high regulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4 + T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.

The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound directly to tumor necrosis factor receptors (TNFRs) and disturbed the TNFα-TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling, and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.

Exposing skin to ultraviolet (UV) radiation contributes to photoaging and to the development of skin cancer by DNA lesions and triggering inflammatory and other harmful cellular cascades. The present study tested the ability of unique lipid molecules, polyhydroxylated fatty alcohols (PFA), extracted from avocado, to reduce UVB-induced damage and inflammation in skin. Introducing PFA to keratinocytes prior to their exposure to UVB exerted a protective effect, increasing cell viability, decreasing the secretion of IL-6 and PGE(2), and enhancing DNA repair. In human skin explants, treating with PFA reduced significantly UV-induced cellular damage. These results support the idea that PFA can play an important role as a photo-protective agent in UV-induced skin damage.

T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.

Heat shock protein (HSP) 60 functions as a key signal to the immune system: its expression is up-regulated under inflammation and HSP60-reactive T and B cells were observed in almost all inflammatory diseases. Moreover, HSP60 induces pro-inflammatory phenotype in innate immune cells via Toll-like receptors (TLRs). Accordingly, HSP60 has been considered a pro-inflammatory “danger signal”. However, HSP60 have immunoregulatory potential and could arrest inflammatory damage. In this chapter we discuss recent findings indicating that T and B cells may directly respond to HSP60 via TLR-2 and TLR4 respectively. HSP60 inhibits T-cell chemotaxis, shift the cytokine secretion balance towards a Th2 phenotype, and activates the suppression ability of Treg. B cells responding to HSP60 secreted also high levels of IL-10. Then, these innate effects of HSP60 on adaptive immune system lead to resolution of inflammation. Thus, HSP60, which is up-regulated by stress and inflammation, can innately resolve it

PD1+CXCR5+ T follicular helper (Tfh) cells are essential for generating high-affinity antibodies and B cell memory. But aberrant Tfh activity can drive autoimmune disease such as systemic lupus erythematosus (SLE) which is characterized by high levels of auto-antibodies and increased frequencies of IL-21+ T cells. Rho-associated kinase (ROCK) 2 was recently shown to be critical in regulating IL-21 secretion in both mice and humans. Here, we show that the PD1+CXCR5+ subset is a major source of IL-21 in human CD4+ T cells stimulated in vitro. By using KD025, a Kadmon orally available, selective ROCK2 inhibitor, we attenuated the development of PD1+CXCR5+ T cells by over 60% and the perpetuation of existing Tfh cells in a dose dependent manner. Targeting of ROCK2 concurrently regulates STAT3/STAT5 phosphorylation and transcription activity followed by a reduction of Bcl6 and up-regulation of Blimp1 protein levels in human T cells. Oral administration of KD025 reduced the percent of both Tfh and plasma B cells in spleens by more than 2 fold as well as germinal center size in the Mrl/lpr murine model of SLE. KD025 treated animals had a substantial improvement in both histological and clinical scores complimented by a reduction in pSTAT3 and BCL6 expression in addition to an increase in STAT5 phosphorylation. This data suggests that ROCK2 controls the function of Tfh cells and underscores the therapeutic potential of targeted ROCK2 inhibition in the clinic.

Rho-associated kinase 2 (ROCK2) was recently shown to be implicated in regulation of IL-21 and IL-17 secretion in mice and humans. Here, we report that the potent and highly selective ROCK2 inhibitor KD025 effectively reverses an established chronic graft-versus-host (cGVHD) murine model driven by IL-21 and characterized by an aggressive multiorgan system fibrotic disease. KD025 treatment leads to a dose dependent decrease in the development of pathogenic pulmonary function which correlates with a marked reduction of antibody and collagen deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. The spleens of mice treated with KD025 had a decrease in the frequency of germinal centers associated with a reduction in STAT3 and concurrent increase in STAT5 phosphorylation, supporting the molecular mechanism of targeted ROCK2 inhibition in humans. The critical role of STAT3 activation in this cGVHD model was further confirmed by data showing that mice transplanted with inducible STAT3 deficient T cells or BM cells had pulmonary function comparable to the healthy negative controls. Finally, KD025 effectively blocked the progression of sclerodermatous cGVHD model characterized by improvements in weight loss, posture and skin integrity of treated animals. Together, these data demonstrate the importance of ROCK2 signaling in STAT3-dependent regulation of cytokine secretion and highlight the potential of targeted ROCK2 inhibition for cGVHD therapy.

Pro-inflammatory IL-17-producing T cells termed Th17 are actively involved in the pathogenesis of GVHD. The development and function of Th17 cells is dependent on activation of STAT3, RORgt and IRF4 transcription factors. Aberrant activation of Rho-associated kinase 2 (ROCK2) leads to induction of IL-17 and IL-21 secretion via IRF4-dependent mechanism. KD025, is a potent and selective ROCK2 inhibitor, which when given to healthy human subjects down-regulated the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g, in response to TCR stimulation in vitro (Figure 1). KD025 inhibits STAT3 phosphorylation which supports RORgt, Th17 generation, and IL-21 production. Concurrently, KD025 increases STAT5 phosphorylation and Treg suppressor function in a dose-responsive fashion. KD025 treatment therefore shifts Th17/Treg balance. Th17 cells have been linked to in vivo pro-inflammatory responses, antibody production, and fibrosis. Conversely, Tregs can offset these pathogenic responses. Given the profile of KD025, we tested the effects of this inhibitor on cGVHD pathogenesis in a multi-organ system rodent model of disease that is driven by IL-21 responses and is associated with lung, liver and intestinal fibrosis. We observed that Th17/Rorc deficient T cells are unable to mediate cGVHD pathogenesis. In mice with established cGVHD, therapy was initiated with 30, 100, or 150 mg/kg/dose of KD025 daily from d28-56. Treated mice had a dose dependent decrease in the development of pathogenic pulmonary function as determined by whole body plethysmography (Figure 2) which correlated with a marked reduction of antibody deposition in the lungs of treated mice to levels comparable to non-cGVHD controls. KD025 administration also resulted in a 2-fold decrease in collagen deposition in the lungs of mice treated with the highest dose of KD025. The spleens of mice treated with 150 mg/kg dose of KD025 had a decrease in the frequency of germinal centers compared to the vehicle treated mice. To determine the selective role of STAT3 on T cells, mice were transplanted with wildtype (WT) bone marrow (BM) and WT or inducible STAT3 deficient T cells. In parallel cohorts, the role of STAT3 in BM-derived B cells, precursors of germinal center B cells, was examined using WT vs inducible STAT3 deficient BM cells + WT T cells. We demonstrate here that mice transplanted with inducible STAT3 deficient T cells or BM cells had pulmonary function comparable to the healthy negative controls, suggesting that STAT3 is a potential therapeutic target in both T and B cells is necessary for the development of cGVHD and providing mechanistic insight into how KD025 may ameliorate active cGVHD. Studies are in progress to test KD025 administration in a murine scleroderma model using a minor histocompatibility antigen disparate donor-recipient strain that we have shown to be dependent upon STAT3 expressing donor T cells and a STAT3 inhibitor in both cGVHD models described here. Together, these data demonstrate that KD025 is effective at decreasing STAT3-dependent production of IL-21 and IL-17 and the use of KD025 is a potentially novel therapeutic intervention for the treatment of cGVHD. Fig 1 Oral administration of KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs upon stimulation ex vivo. Human PBMCs were purified from healthy human subjects before and after oral administration of KD025 at doses 40, 120, 240, 320 and stimulated ex vivo. Cytokine secretion was determined after 48 hours by ELISA. Fig 1. Oral administration of KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs upon stimulation ex vivo. Human PBMCs were purified from healthy human subjects before and after oral administration of KD025 at doses 40, 120, 240, 320 and stimulated ex vivo. Cytokine secretion was determined after 48 hours by ELISA. Fig 2 KD025 is an effective therapy for established murine cGVHD. Mice were given KD025 (150 mg/kg) d.28-56. PFTs indicate normal resistance, elastance and better compliance. Lung Ig deposition and fibrosis were comparable to BM controls. Fig 2. KD025 is an effective therapy for established murine cGVHD. Mice were given KD025 (150 mg/kg) d.28-56. PFTs indicate normal resistance, elastance and better compliance. Lung Ig deposition and fibrosis were comparable to BM controls. Disclosures No relevant conflicts of interest to declare.

We recently reported that soluble 60-kDa heat shock protein (HSP60) can directly activate T cells via TLR2 signaling to enhance their Th2 response. In this study we investigated whether HSP60 might also activate B cells by an innate signaling pathway. We found that human HSP60 (but not the Escherichia coli GroEL or the Mycobacterial HSP65 molecules) induced naive mouse B cells to proliferate and to secrete IL-10 and IL-6. In addition, the HSP60-treated B cells up-regulated their expression of MHC class II and accessory molecules CD69, CD40, and B7-2. We tested the functional ability of HSP60-treated B cells to activate an allogeneic T cell response and found enhanced secretion of both IL-10 and IFN-γ by the responding T cells. The effects of HSP60 were found to be largely dependent on TLR4 and MyD88 signaling; B cells from TLR4-mutant mice or from MyD88 knockout mice showed decreased responses to HSP60. Care was taken to rule out contamination of the HSP60 with LPS as a causative factor. These findings add B cells to the complex web of interactions by which HSP60 can regulate immune responses.

Previously, we reported that treatment of T cells with the 60-kDa heat shock protein (HSP60) inhibits chemotaxis. We now report that treatment of purified human T cells with recombinant human HSP60 or its biologically active peptide p277 up-regulates suppressor of cytokine signaling (SOCS)3 expression via TLR2 and STAT3 activation. SOCS3, in turn, inhibits the downstream effects of stromal cell-derived-1α (CXCL12)-CXCR4 interaction in: 1) phosphorylation of ERK1/2, Pyk2, AKT, and myosin L chain, required for cell adhesion and migration; 2) formation of rear-front T cell polarity; and 3) migration into the bone marrow of NOD/SCID mice. HSP60 also activates SOCS3 in mouse lymphocytes and inhibits their chemotaxis toward stromal cell-derived factor-1α and their ability to adoptively transfer delayed-type hypersensitivity. These effects of HSP60 could not be attributed to LPS or LPS-associated lipoprotein contamination. Thus, HSP60 can regulate T cell-mediated inflammation via specific signal transduction and SOCS3 activation.

Recently, it has been shown that Fas ligand (FasL) interacts with the extracellular matrix (ECM) protein fibronectin (FN), and that the bound FasL retains its cytotoxic efficacy. Herein, we examined the ramifications of FasL-ECM protein interactions throughout a specific time period, in the absence or presence of additional activating molecules, assuming that these complexed interactions occur during inflammation. We found that exposure of purified human T cells to FN-associated recombinant FasL for as brief as 5–10 min at 0.1–100 ng/ml induced their adhesion in β1 integrin- and FasR-dependent manners while activating the intracellular protein kinase, Pyk-2. The FN-associated FasL stops the CXCL12 (stromal cell-derived factor 1α)-induced chemotaxis of T cells by inhibiting the chemokine-induced extracellular signal-regulated kinase signaling and cytoskeletal rearrangement. This short term exposure of T cells to the FN-bound FasL (1 ng/ml), which was followed by T cell activation via the CD3 complex, resulted in 1) increased secretion of IFN-γ (measured after 24 h), and 2) enhanced T cell apoptosis (measured after 72 h). Thus, in the context of inflamed ECM and depending on the time after FasL activation, its concentration, and the nature of other contextual mediators, FasL initially retains effector T cells at sites of inflammation and, later, induces T cell apoptosis and return to homeostasis.

This chapter positions HSP60 at the center of inflammation and body maintenance. We shall discuss the following topics: Inflammation: Physiological Inflammation: Pathological - HSP60: Autoimmune target HSP60: Regulator signal HSP60: Innate ligand HSP60 model Signal fidelity and HSP60

Targeting pro-inflammatory cytokines is a promising approach to the treatment of certain autoimmune diseases. However, the signaling mechanisms involved in the initiation and effector phases of auto-aggressive pathways are still an enigma. Rho-associated kinase 2 (ROCK2) regulates the secretion of inflammatory cytokines interleukin (IL)-21 and IL-17 and the development of autoimmunity in mice. Our data from a phase 1 clinical trial demonstrates that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects significantly down-regulates the ability of T cells to secrete IL-21 and IL-17, but not interferon (IFN)-g in response to TCR stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, leads to down-regulation of STAT3 phosphorylation, interferon regulatory factor 4 (IRF4) and steroid receptor-type nuclear receptor RORgt protein levels in T cells derived from healthy subjects or rheumatoid arthritis (RA) patients. Simultaneously, ROCK2 inhibition induces phosphorylation of STAT5 and SMAD2/3, and subsequently increases the percentage of Foxp3+ T cells. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between pro-inflammatory and regulatory T cell subsets in man. Targeted inhibition of ROCK2 may therefore have a role in the treatment of autoimmune disease with disturbed immune homeostasis.

Foxp3+CD4+CD25high regulatory T cell (Treg) suppression of inflammation depends on T cell receptor (TCR)-mediated NFAT activation with reduced Akt activity. We investigated the role of the scaffold protein Dlgh1 in linking the TCR to this unique signaling outcome. The Treg immunological synapse (IS) recruited 4-fold more Dlgh1 than conventional CD4+ T cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, display reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced PTEN levels and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma-1 and thus presents an array of novel targets to selectively manipulate Treg function.

T cell receptor (TCR) dependent regulatory T cell (Treg) activity controls effector T cell (Teff) function and is acutely inhibited by tumor necrosis factor (TNF). Protein kinase C-θ (PKC-θ) recruitment to the immunological synapse is required in effector cells to recruit Carma-1 in the pathway to NF-κB activation. Surprisingly, PKC-θ was sequestered away from the Treg immunological synapse. Furthermore, PKC-θ inhibition or suppression by RNAi enhanced Treg function, demonstrating PKC-θ mediated negative feedback. Treg integrate TCR and TNF signals through control of PKC-θ location such that inhibition of PKC-θ protected Treg from inactivation by TNF, restored activity of defective Treg from rheumatoid arthritis patients, and enhanced protection of mice from inflammatory colitis. Treg freed of PKC-θ mediated negative feedback can function in the presence of TNF and thus have therapeutic potential in control of inflammatory diseases.