Elevated expression of interleukin‐7 receptor in inflamed joints mediates interleukin‐7–induced immune activation in rheumatoid arthritis

Objective

To evaluate the expression and functional ability of the high‐affinity interleukin‐7 receptor (IL‐7Rα) in patients with rheumatoid arthritis (RA).

Methods

Expression of IL‐7Rα and IL‐7 was determined in synovial tissue from RA patients and was compared with that in synovial tissue from patients with undifferentiated arthritis (UA) and osteoarthritis (OA). IL‐7Rα expression on CD4 T cells, CD19 B cells, and CD14 monocyte/macrophages from RA synovial tissue, synovial fluid, and peripheral blood was also assessed. The proliferative capacity of IL‐7Rα^bright and IL‐7Rα^dim/− T cells was measured. In addition, we examined IL‐7R blockade with soluble human IL‐7Rα (hIL‐7Rα) in the prevention of immune activation of peripheral blood mononuclear cells.

Results

We found significantly higher IL‐7Rα expression in RA and UA synovial tissue than in OA synovial tissue, and the level of IL‐7Rα expression correlated significantly with the levels of CD3 and IL‐7 expression. CD4 T cells from RA synovial fluid and synovial tissue strongly expressed IL‐7Rα. A substantial percentage of B cells and macrophages from RA synovial fluid and synovial tissue also expressed IL‐7Rα, although less prominently than T cells. We found that peripheral blood IL‐7Rα^bright T cells that did not express FoxP3 were highly proliferative as compared with IL‐7Rα^dim/− T cells that did express high levels of FoxP3. Soluble hIL‐7Rα inhibited IL‐7–induced proliferation and interferon‐γ production by mononuclear cells from RA patients.

Conclusion

Our data suggest that enhanced expression of IL‐7Rα and IL‐7 in RA patients contributes significantly to the joint inflammation by activating T cells, B cells, and macrophages. The inhibition of IL‐7R–mediated immune activation by soluble hIL‐7Rα further indicates an important role of IL‐7Rα in inflammatory responses in RA, suggesting IL‐7Rα as a therapeutic target for immunotherapy in RA.

     

The response of T cells to interleukin‐6 is differentially regulated by the microenvironment of the rheumatoid synovial fluid and tissue

Objective

Interleukin‐6 (IL‐6) is a proinflammatory cytokine with regulatory effects on the survival and differentiation of T cells. It exerts its biologic function in 2 ways: by directly binding to the IL‐6 receptor (IL‐6R; CD126) or via trans‐signaling, in which soluble IL‐6R/IL‐6 complexes bind to the signaling component CD130. This study was undertaken to assess the expression and regulation of CD126 and CD130 and determine how these affect the response of CD4+ T cells to IL‐6 in the joints of patients with rheumatoid arthritis (RA).

Methods

Flow cytometry and immunofluorescence microscopy were used to determine the expression, function, and regulation of CD126 and CD130 in CD4+ T cells from the peripheral blood (PB), synovial fluid (SF), and synovial tissue of RA patients.

Results

Compared to the findings in RA PB, CD4+ T cells in the SF and synovial tissue expressed low levels of CD126. In contrast, whereas CD4+ T cell expression of CD130 was minimal in the SF, its level in the synovial tissue was high. Consistent with this phenotype, synovial tissue T cells responded to trans‐signaling by soluble IL‐6R/IL‐6 complexes, whereas no response was evident in CD4+ T cells from the SF. Down‐regulation of both receptor components in SF T cells could be explained by exposure to high levels of IL‐6. Increased levels of CD130 messenger RNA and protein in synovial tissue CD4+ T cells suggested that CD130 is up‐regulated locally. Among a range of cytokines tested, only IL‐10 induced CD130 expression in T cells.

Conclusion

The inflamed microenvironment in the synovial tissue maintains responsiveness to IL‐6 trans‐signaling through the up‐regulation of CD130 expression in CD4+ T cells, and this process may be driven by IL‐10.

     

Macrophage subpopulations in rheumatoid synovium: Reduced CD163 expression in CD4+ T lymphocyte–rich microenvironments

Objective

The cell surface glycoprotein CD163 is a member of the cysteine‐rich scavenger receptor family, highly specific for leukocytes of the mononuclear phagocyte lineage. In vitro, it is induced by glucocorticoids, interleukin‐6 (IL‐6), and IL‐10 and down‐regulated by interferon‐γ (IFNγ), indicating that it has a role in antiinflammatory or other immunomodulatory pathways. We assessed CD163 expression in microenvironments within rheumatoid arthritis (RA) synovium to clarify the relationships among CD4+ T lymphocytes, IFNγ, and macrophage function in RA.

Methods

Double immunofluorescence and serial immunoenzymatic studies were performed on normal, osteoarthritic, and RA synovium and tonsil with antibodies to CD163, CD45, CD68, CD14, CD3, CD4, CD8, CD19, and IFNγ.

Results

CD163 was observed on all CD14+ cells in synovium and tonsil with the exception of cells within larger T lymphocyte clusters in synovium and within tonsillar follicles. All brightly CD14+ cells in or around vessel walls (interpreted as immigrant monocytes) were CD163+. CD163 labeled fewer cells than did CD68 in synovial intima, but all CD45+ intimal cells were CD163+. CD4+,IFNγ+ T lymphocytes in RA synovium were chiefly localized within clusters containing CD68+, CD163− cells.

Conclusion

Within RA synovium, CD163 has major advantages as a macrophage marker and does not appear to be restricted to “mature” macrophages. CD163 discriminates between synovial macrophages and synovial intimal fibroblasts, which also stain positively for CD68 in diseased tissue.

     

Proinflammatory mediator–induced reversal of CD4+,CD25+ regulatory T cell–mediated suppression in rheumatoid arthritis

Objective

We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg–mediated suppression.

Methods

Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme‐linked immunosorbent assay. Magnetically sorted CD4+,CD25– and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti‐CD3 monoclonal antibody (mAb) and autologous antigen‐presenting cells, in the absence or presence of anti‐CD28 mAb or the proinflammatory cytokines interleukin‐6 (IL‐6), tumor necrosis factor α (TNFα), or IL‐7.

Results

Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB‐derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti‐CD28 mAb to cocultures of CD4+,CD25– and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg–mediated suppression in both PB and SF. Furthermore, IL‐7 and, to a limited extent, TNFα, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg–mediated suppression. In contrast, IL‐6 did not influence Treg‐mediated suppression.

Conclusion

Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.

     

Impact of the CTLA‐4/CD28 axis on the processes of joint inflammation in rheumatoid arthritis

Objective

The importance of the costimulatory molecules CD28 and CTLA‐4 in the pathologic mechanism of rheumatoid arthritis (RA) has been demonstrated by genetic associations and the successful clinical application of CTLA‐4Ig for the treatment of RA. This study was undertaken to investigate the role of the CTLA‐4/CD28 axis in the local application of CTLA‐4Ig in the synovial fluid (SF) of RA patients.

Methods

Quantitative polymerase chain reaction was used to analyze the expression of proinflammatory and antiinflammatory cytokines in ex vivo fluorescence‐activated cell sorted CTLA‐4+ and CTLA‐4− T helper cells from the peripheral blood and SF of RA patients. T helper cells were also analyzed for cytokine expression in vitro after the blockade of CTLA‐4 by anti–CTLA‐4 Fab fragments or of B7 (CD80/CD86) molecules by CTLA‐4Ig.

Results

CTLA‐4+ T helper cells were unambiguously present in the SF of all RA patients examined, and they expressed increased amounts of interferon‐γ (IFNγ), interleukin‐17 (IL‐17), and IL‐10 as compared to CTLA‐4− T helper cells. The selective blockade of CTLA‐4 in T helper cells from the SF in vitro led to increased levels of IFNγ, IL‐2, and IL‐17. The concomitant blockade of CD28 and CTLA‐4 in T helper cells from RA SF by CTLA‐4Ig in vitro resulted in reduced levels of the proinflammatory cytokines IFNγ and IL‐2 and increased levels of the antiinflammatory cytokines IL‐10 and transforming growth factor β.

Conclusion

Our ex vivo and in vitro results demonstrate that the CTLA‐4/CD28 axis constitutes a drug target for not only the systemic, but potentially also the local, application of the costimulation blocking agent CTLA‐4Ig for the treatment of RA.

     

Increased macrophage activation mediated through toll‐like receptors in rheumatoid arthritis

Objective

Macrophages are the major source of inflammation mediators that are important in the pathogenesis of rheumatoid arthritis (RA). This study was undertaken to analyze macrophages obtained from the joints of RA patients in order to characterize the expression of Toll‐like receptor 2 (TLR‐2) and TLR‐4 and the responses to TLR ligation.

Methods

Cells were isolated from the synovial fluid (SF) of RA patients or patients with other forms of inflammatory arthritis. Cell surface TLR‐2 and TLR‐4 expression and intracellular tumor necrosis factor α (TNFα) and interleukin‐8 (IL‐8) expression by CD14+ macrophages were determined by flow cytometry. Peptidoglycan (PG) and lipopolysaccharide (LPS) were used as ligands for TLR‐2 and TLR‐4, respectively.

Results

The expression of TLR‐2 and TLR‐4 was increased on CD14+ macrophages from the joints of RA patients compared with that on control in vitro–differentiated macrophages or control peripheral blood monocytes. Neither TLR‐2 expression nor TLR‐4 expression differed between RA and other forms of inflammatory arthritis. However, PG‐ and LPS‐induced TNFα expression and IL‐8 expression were greater with RA SF macrophages than with those obtained from the joints of patients with other forms of inflammatory arthritis or with control macrophages. PG‐induced TNFα expression and IL‐8 expression were highly correlated with TLR‐2 expression in normal macrophages, but not with that in macrophages obtained from joints of RA patients or patients with other forms of inflammatory arthritis.

Conclusion

TLR‐2 and TLR‐4 ligation resulted in increased activation of RA synovial macrophages compared with those from patients with other forms of inflammatory arthritis or compared with control macrophages. Factors other than the level of TLR‐2 and TLR‐4 expression contributed to the increased activation of RA SF macrophages. These observations support the notion of a potential role for activation through TLR‐2 and TLR‐4 in the inflammation and joint destruction of RA.

     

CTLA‐4IG suppresses reactive oxygen species by preventing synovial adherent cell–induced inactivation of rap1, a ras family GTPASE mediator of oxidative stress in rheumatoid arthritis T cells

Objective

Oxidative stress contributes to the inflammatory properties of rheumatoid arthritis (RA) synovial T lymphocytes. This study was undertaken to investigate the mechanisms leading to production of reactive oxygen species (ROS) and oxidative stress in RA synovial T lymphocytes.

Methods

ROS production in T lymphocytes from the peripheral blood (PB) of healthy donors and from the PB and synovial fluid (SF) of RA patients was measured by ROS‐dependent fluorescence of 6‐carboxy‐2′,7′‐dichlorofluorescein. Rap1 GTPase activation was assessed by activation‐specific probe precipitation. Proliferation of RA PB and SF T lymphocytes was assayed by ³H‐thymidine incorporation. In some experiments, RA PB T cells were preincubated with autologous SF or with PB or SF adherent cells. Experiments were performed in the absence or presence of transwell membranes or CTLA‐4Ig fusion proteins. Short‐ and long‐term stimulations of healthy donor PB T lymphocytes were performed with inflammatory cytokines, in the absence or presence of activating anti‐CD28 antibodies.

Results

T lymphocyte ROS production and Rap1 inactivation were mediated by cell–cell contact with RA synovial adherent cells, and this correlated with T cell mitogenic hyporesponsiveness. CTLA4‐Ig blockade of synovial adherent cell signaling to CD28 T cells reversed the inhibition of Rap1 activity and prevented induction of ROS. Introduction of active RapV12 into T cells also prevented induction of ROS production. Coincubation of T cells with stimulating anti‐CD28 antibodies and inflammatory cytokines synergistically increased T cell ROS production.

Conclusion

Cell–cell contact between T cells and RA synovial adherent cells mediates Rap1 inactivation and subsequent ROS production in T lymphocytes following exposure to inflammatory cytokines. This process can be blocked by CTLA4‐Ig fusion protein.

     

Selective elimination of synovial inflammatory macrophages in rheumatoid arthritis by an Fcγ receptor I–directed immunotoxin

Objective

To determine whether monocyte/macrophages from rheumatoid arthritis (RA) patients can be selectively eliminated by a toxin‐conjugated antibody CD64–ricin A (CD64‐RiA) directed toward the high‐affinity receptor for IgG (FcγRI), exploiting the capacity of FcγRI to efficiently endocytose antibody which it has bound.

Methods

Mononuclear cells from peripheral blood (PB) and synovial fluid (SF) obtained from RA patients were cultured in the presence of CD64‐RiA. Cell death of monocyte/macrophages was measured by phenotypic changes (light‐scatter patterns and CD14 and FcγRI expression) and apoptosis (nuclear DNA fragmentation). We then tested whether CD64‐RiA–induced cell death of macrophages affected their capacity to stimulate antigen‐induced lymphocyte proliferation and to secrete cytokines. Additionally, the capacity of CD64‐RiA to inhibit proinflammatory activity and cartilage degradation by RA synovial tissue explants was evaluated.

Results

Inflammatory macrophages from RA SF expressed elevated levels of FcγRI and were selectively eliminated by CD64‐RiA via apoptotic cell death. Monocyte/macrophages from RA PB, which had lower levels of FcγRI expression, were much less affected. Induction of SF macrophage apoptosis was associated with efficient inhibition of antigen‐induced lymphocyte proliferation and a reduction in tumor necrosis factor α (TNFα) release. Consistent with these effects on SF macrophages, CD64‐RiA also inhibited TNFα production, interleukin‐1β production, and cartilage‐degrading activity of RA synovial tissue explants.

Conclusion

Together, these data underscore the crucial role of synovial macrophages in RA joint inflammation and indicate that selective elimination of these cells through FcγRI‐directed immunotoxins could be a novel approach to the treatment of RA.

     

Inflammation and ectopic lymphoid structures in rheumatoid arthritis synovial tissues dissected by genomics technology: Identification of the interleukin‐7 signaling pathway in tissues with lymphoid neogenesis

Objective

In ∼25% of synovial tissues from rheumatoid arthritis (RA) patients, infiltrates of T cells, B cells, and follicular dendritic cells (FDCs) are spatially organized into structures resembling lymph nodes with germinal centers. The remainder of the tissues lack FDCs and show either a diffuse or an aggregated T cell and B cell infiltrate. To gain more insight into this specific disease process, we sought to identify the genes expressed in RA tissues with ectopic lymphoid structures.

Methods

Gene expression profiling of RA synovial tissues was determined by complementary DNA microarray analysis and quantitative real‐time polymerase chain reaction. The presence of lymphoid follicles and localization of interleukin‐7 (IL‐7) in synovial tissue sections was determined by immunofluorescence staining using specific antibodies.

Results

Findings of gene expression analysis confirmed previous reports that tissues with lymphoid structures showed elevated expression of CXCL13, CCL21, CCR7, and lymphotoxin α and β messenger RNA. In addition, the tissues also showed enhanced expression of the chemokines CXCL12 and CCL19 and the associated receptors CXCR4 and CXCR5, which are important for the attraction of T cells, B cells, and dendritic cells. Pathway analysis revealed increased expression of genes involved in JAK/STAT signaling, T cell– and B cell–specific pathways, Fcε receptor type I signaling in mast cells, and IL‐7 signal transduction in the tissues with ectopic lymphoid follicles, accompanied by increased expression of IL‐7 receptor α (IL‐7Rα)/IL‐2Rγ chains and IL‐7. Protein expression of IL‐7 in RA tissues was localized within fibroblast‐like synoviocytes, macrophages, and blood vessels and was colocalized with extracellular matrix structures around the B cell follicles.

Conclusion

Activation of the IL‐7 pathway may play an important role in lymphoid neogenesis, analogous to its role in the development of normal lymphoid tissue.

     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

Monoclonal anti‐CD8 therapy induces disease amelioration in the K/BxN mouse model of spontaneous chronic polyarthritis

Objective

CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8+ T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis.

Methods

CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti‐CD8 mAb (YTS105 and YTS169.4), with and without thymectomy.

Results

CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L−CD27+ T cells expressing proinflammatory cytokines (interferon‐γ [IFNγ], tumor necrosis factor α [TNFα], interleukin‐17a [IL‐17A], and IL‐4), and granzyme B. In mice receiving anti‐CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti‐CD8 mAb–treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti‐CD8 mAb–treated mice. In anti‐CD8 mAb–treated mice, the serologic levels of TNFα, IFNγ, IL‐6, and IL‐5 normalized. The levels of the disease‐related anti–glucose‐6‐phosphate isomerase antibodies did not change.

Conclusion

These results indicate that synovial activated effector CD8+ T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL‐17, IL‐6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis.

     

Targeting interleukin‐15 in patients with rheumatoid arthritis: A proof‐of‐concept study

Objective

Interleukin‐15 (IL‐15) is a proinflammatory, innate response cytokine that mediates pleiotropic effector function in rheumatoid arthritis (RA) inflammatory synovitis. Our objective was to study the ability of HuMax‐IL15, a human IgG1 anti–IL‐15 monoclonal antibody, to neutralize exogenous and endogenous IL‐15 activity in vitro and to perform a phase I–II dose‐escalation trial with HuMax‐IL15 in patients with active RA.

Methods

Mononuclear cells from blood and synovial fluid (SF) of RA patients were isolated and cultured in vitro under experimental conditions involving the addition of HuMax‐IL15. HuMax‐IL15 was administered to 30 RA patients who received no other disease‐modifying antirheumatic drugs in a 12‐week, dose‐ascending, placebo‐controlled, double‐blind, phase I–II proof‐of‐concept study.

Results

In vitro studies showed that HuMax‐IL15 suppressed proliferation and induced apoptosis in an IL‐15–dependent cell line, BDB2, and was capable of suppressing the release of interferon‐γ by synovial fluid mononuclear cell (SFMC) cultures induced by exogenous IL‐15. Furthermore, HuMax‐IL15 F(ab′)₂ fragments suppressed exogenous IL‐15–induced CD69 expression in RA peripheral blood mononuclear cells and SFMCs, which indicates that HuMax‐IL15 can specifically neutralize several biologic effects of IL‐15 in synovial tissue in vitro. In a phase I–II clinical trial, HuMax‐IL15 was well tolerated clinically, with no significant effects on T lymphocyte subset and natural killer cell numbers. Substantial improvements in disease activity were observed according to the American College of Rheumatology criteria for 20% improvement (63% of patients), 50% improvement (38%), and 70% improvement (25%).

Conclusion

These clinical data suggest for the first time that IL‐15 could represent a novel therapeutic target in RA.

     

Interleukin‐17–producing T cells are enriched in the joints of children with arthritis,but have a reciprocal relationship to regulatory T cell numbers

Objective

To identify interleukin‐17 (IL‐17)–producing T cells from patients with juvenile idiopathic arthritis (JIA), and investigate their cytokine production, migratory capacity, and relationship to Treg cells at sites of inflammation, as well as to test the hypothesis that IL‐17+ T cell numbers correlate with clinical phenotype in childhood arthritis.

Methods

Flow cytometry was used to analyze the phenotype, cytokine production, and chemokine receptor expression of IL‐17–producing T cells in peripheral blood and synovial fluid mononuclear cells from 36 children with JIA, in parallel with analysis of forkhead box P3 (FoxP3)–positive Treg cells. Migration of IL‐17+ T cells toward CCL20 was assessed by a Transwell assay. Synovial tissue was analyzed by immunohistochemistry for IL‐17 and IL‐22.

Results

IL‐17+ T cells were enriched in the joints of children with JIA as compared with the blood of JIA patients (P = 0.0001) and controls (P = 0.018) and were demonstrated in synovial tissue. IL‐17+ T cell numbers were higher in patients with extended oligoarthritis, the more severe subtype of JIA, as compared with patients with persistent oligoarthritis, the milder subtype (P = 0.046). Within the joint, there was an inverse relationship between IL‐17+ T cells and FoxP3+ Treg cells (r = 0.61, P = 0.016). IL‐17+,CD4+ T cells were uniformly CCR6+ and migrated toward CCL20, but synovial IL‐17+ T cells had variable CCR4 expression. A proportion of IL‐17+ synovial T cells produced IL‐22 and interferon‐γ.

Conclusion

This study is the first to define the frequency and characteristics of “Th17” cells in JIA. We suggest that these highly proinflammatory cells contribute to joint pathology, as indicated by relationships with clinical phenotypes, and that the balance between IL‐17+ T cells and Treg cells may be critical to outcome.