TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

Immature dendritic cells convert anergic nonregulatory T cells into Foxp3−IL‐10+ regulatory T cells by engaging CD28 and CTLA‐4

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide‐specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL‐10^?Foxp3^?CTLA‐4⁺CD25^lowEgr2⁺ T cells could be converted into suppressive IL‐10⁺Foxp3^?CTLA‐4⁺CD25^highEgr2⁺ cells resembling type‐1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF‐β during anergy induction favored Foxp3⁺ Treg‐cell induction, while TGF‐β had little effect when added to the second stimulation. Expression of both CD28 and CTLA‐4 molecules on anergic T cells was required to allow their conversion into Tr1‐like cells. Suppressor activity was enabled via CD28‐mediated CD25 upregulation, acting as an IL‐2 sink, together with a CTLA‐4‐mediated inhibition of NFATc1/α activation to shut down IL‐2‐mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1‐like cells.     

Expansion of regulatory T cells via IL‐2/anti‐IL‐2 mAb complexes suppresses experimental myasthenia

Human autoimmune diseases are often characterized by a relative deficiency in CD4⁺CD25⁺ regulatory T cells (Treg). We therefore hypothesized that expansion of Treg can ameliorate autoimmune pathology. We tested this hypothesis in an experimental model for autoimmune myasthenia gravis (MG), a B‐cell‐mediated disease characterized by auto‐Ab directed against the acetylcholine receptor within neuromuscular junctions. We showed that injection of immune complexes composed of the cytokine IL‐2 and anti‐IL‐2 mAb (JES6‐1A12) induced an effective and sustained expansion of Treg, via peripheral proliferation of CD4⁺CD25⁺Foxp3⁺ cells and peripheral conversion of CD4⁺CD25^?Foxp3^? cells. The expanded Treg potently suppressed autoreactive T‐ and B‐cell responses to acetylcholine receptor and attenuated the muscular weakness that is characteristic of MG. Thus, IL‐2/anti‐IL‐2 mAb complexes can expand functional Treg in vivo, providing a potential clinical application of this modality for treatment of MG and other autoimmune disorders.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Synergistic effect of TGF‐β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3⁺ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4⁺CD25^? cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3⁺ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3⁺ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.     

TGF‐β‐producing regulatory B cells induce regulatory T cells and promote transplantation tolerance

Regulatory B (Breg) cells have been shown to play a critical role in immune homeostasis and in autoimmunity models. We have recently demonstrated that combined anti‐T cell immunoglobulin domain and mucin domain‐1 and anti‐CD45RB antibody treatment results in tolerance to full MHC‐mismatched islet allografts in mice by generating Breg cells that are necessary for tolerance. Breg cells are antigen‐specific and are capable of transferring tolerance to untreated, transplanted animals. Here, we demonstrate that adoptively transferred Breg cells require the presence of regulatory T (Treg) cells to establish tolerance, and that adoptive transfer of Breg cells increases the number of Treg cells. Interaction with Breg cells in vivo induces significantly more Foxp3 expression in CD4⁺CD25^? T cells than with naive B cells. We also show that Breg cells express the TGF‐β associated latency‐associated peptide and that Breg‐cell mediated graft prolongation post‐adoptive transfer is abrogated by neutralization of TGF‐β activity. Breg cells, like Treg cells, demonstrate preferential expression of both C‐C chemokine receptor 6 and CXCR3. Collectively, these findings suggest that in this model of antibody‐induced transplantation tolerance, Breg cells promote graft survival by promoting Treg‐cell development, possibly via TGF‐β production.     

Curcumin induces the tolerogenic dendritic cell that promotes differentiation of intestine‐protective regulatory T cells

The gut is home to a large number of Treg, with both CD4⁺ CD25⁺ Treg and bacterial antigen‐specific Tr1 cells present in normal mouse intestinal lamina propria. It has been shown recently that intestinal mucosal DC are able to induce Foxp3⁺ Treg through production of TGF‐β plus retinoic acid (RA). However, the factors instructing DC toward this mucosal phenotype are currently unknown. Curcumin has been shown to possess a number of biologic activities including the inhibition of NF‐κB signaling. We asked whether curcumin could modulate DC to be tolerogenic whose function could mimic mucosal DC. We report here that curcumin modulated BM‐derived DC to express ALDH1a and IL‐10. These curcumin‐treated DC induced differentiation of naïve CD4⁺ T cells into Treg resembling Treg in the intestine, including both CD4⁺CD25⁺ Foxp3⁺ Treg and IL‐10‐producing Tr1 cells. Such Treg induction required IL‐10, TGF‐β and retinoic acid produced by curcumin‐modulated DC. Cell contact as well as IL‐10 and TGF‐β production were involved in the function of such induced Treg. More importantly, these Treg inhibited antigen‐specific T‐cell activation in vitro and inhibited colitis due to antigen‐specific pathogenic T cells in vivo.     

Expression of IL‐15RA or an IL‐15/IL‐15RA fusion on CD8+ T cells modifies adoptively transferred T‐cell function in cis

IL‐15 and IL‐15 receptor alpha (IL‐15RA) play a significant role in multiple aspects of T‐cell biology. However, given the evidence that IL‐15RA can present IL‐15 in trans, the functional capacity of IL‐15RA expressed on CD8⁺ T cells to modify IL‐15 functions in cis is currently unclear. In the current study, we explore the functional consequences of IL‐15RA, expression on T cells using a novel method to transfect naive CD8⁺ T cells. We observed that RNA nucleofection led to highly efficient, non‐toxic, and rapid manipulation of protein expression levels in unstimulated CD8⁺ T cells. We found that transfection of unstimulated CD8⁺ T cells with IL‐15RA RNA led to enhanced viability of CD8⁺ T cells in response to IL‐15. Transfection with IL‐15RA enhanced IL‐15‐mediated phosphorylation of STAT5 and also promoted IL‐15‐mediated proliferation in vivo of adoptively transferred naïve CD8⁺ T cells. We demonstrated that IL‐15RA can present IL‐15 via cis‐presentation on CD8⁺ T cells. Finally, we showed that transfection with a chimeric construct linking IL‐15 to IL‐15RA cell autonomously enhances the viability and proliferation of primary CD8⁺ T cells and cytotoxic potential of antigen‐specific CD8⁺ T cells. The clinical implications of the current study are discussed.     

Commensal A4 bacteria inhibit intestinal Th2‐cell responses through induction of dendritic cell TGF‐β production

It has been shown that while commensal bacteria promote Th1, Th17 and Treg cells in lamina propria (LP) in steady‐state conditions, they suppress mucosal Th2 cells. However, it is still unclear whether there are specific commensal organisms down‐regulating Th2 responses, and the mechanism involved. Here we demonstrate that commensal A4 bacteria, a member of the Lachnospiraceae family, which produce an immunodominant microbiota CBir1 antigen, inhibits LP Th2‐cell development. When transferred into the intestines of RAG^?/? mice, CBir1‐specific T cells developed predominately towards Th1 cells and Th17 cells, but to a lesser extent into Th2 cells. The addition of A4 bacterial lysates to CD4⁺ T‐cell cultures inhibited production of IL‐4. A4 bacteria stimulated dendritic cell production of TGF‐β, and blockade of TGF‐β abrogated A4 bacteria inhibition of Th2‐cell development in vitro and in vivo. Collectively, our data show that A4 bacteria inhibit Th2‐cell differentiation by inducing dendritic cell production of TGF‐β.     

Regulatory effects of IFN‐β on production of osteopontin and IL‐17 by CD4+ T Cells in MS

IFN‐β currently serves as one of the major treatments for MS. Its anti‐inflammatory mechanism has been reported as involving a shift in cytokine balance from Th1 to Th2 in the T‐cell response against elements of the myelin sheath. In addition to the Th1 and Th2 groups, two other important pro‐inflammatory cytokines, IL‐17 and osteopontin (OPN), are believed to play important roles in CNS inflammation in the pathogenesis of MS. In this study, we examined the potential effects of IFN‐β on the regulation of OPN and IL‐17 in MS patients. We found that IFN‐β used in vitro at 0.5–3 ng/mL significantly inhibited the production of OPN in primary T cells derived from PBMC. The inhibition of OPN was determined to occur at the CD4⁺ T‐cell level. In addition, IFN‐β inhibited the production of IL‐17 and IL‐21 in CD4⁺ T cells. It has been described that IFN‐β suppresses IL‐17 production through the inhibition of a monocytic cytokine, the intracellular translational isoform of OPN. Our further investigation demonstrated that IFN‐β also acted directly on the CD4⁺ T cells to regulate OPN and IL‐17 expression through the type I IFN receptor‐mediated activation of STAT1 and suppression of STAT3 activity. Administration of IFN‐β to EAE mice ameliorated the disease severity. Furthermore, spinal cord infiltration of OPN⁺ and IL‐17⁺ cells decreased in IFN‐β‐treated EAE mice along with decreases in serum levels of OPN and IL‐21. Importantly, decreased OPN production by IFN‐β treatment contributes to the reduced migratory activity of T cells. Taken together, the results from both in vitro and in vivo experiments indicate that IFN‐β treatment can down‐regulate the OPN and IL‐17 production in MS. This study provides new insights into the mechanism of action of IFN‐β in the treatment of MS.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

TGF‐β regulation of encephalitogenic and regulatory T cells in multiple sclerosis

Transforming growth factor beta (TGF‐β) is a pleiotropic cytokine that has been shown to influence the differentiation and function of T cells. The role that TGF‐β plays in immune‐mediated disease, such as multiple sclerosis (MS), has become a major area of investigation since CD4⁺ T cells appear to be a major mediator of autoimmunity. This review provides an analysis of the literature on the role that TGF‐β plays in the generation and regulation of encephalitogenic and regulatory T cells (Treg) in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, as well as in T cells of MS patients. Since TGF‐β plays a major role in the development and function of both CD4⁺ effector and Treg, which are defective in MS patients, recent studies have found potential mechanisms to explain the basis for these T‐cell defects to establish a foundation for potentially modulating TGF‐β signaling to restore normal T‐cell function in MS patients.     

TRAF2 regulates peripheral CD8+ T‐cell and NKT‐cell homeostasis by modulating sensitivity to IL‐15

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8⁺ T‐cell and NKT‐cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8⁺ T‐cell subsets. IL‐15‐dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8⁺CD44^hiCD122⁺ T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8⁺CD44^hi T cells exhibited impaired dose‐dependent proliferation to exogenous IL‐15. In contrast, TRAF2TKO CD8⁺ T cells proliferated normally to anti‐CD3 and TRAF2TKO CD8⁺CD44^hi T cells exhibited normal proliferation to exogenous IL‐2. TRAF2TKO CD8⁺ T cells expressed normal levels of IL‐15‐associated receptors and possessed functional IL‐15‐mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8⁺CD44^hiCD122⁺ and NKT cells was mechanistically linked to an inability to respond to IL‐15. The reduced CD8⁺CD44^hiCD122⁺ T‐cell and NKT‐cell populations in TRAF2TKO mice were rescued in the presence of high dose IL‐15 by IL‐15/IL‐15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8⁺ CD44^hiCD122⁺ T‐cell and NKT‐cell homeostasis by modulating sensitivity to T‐cell intrinsic growth factors such as IL‐15.     

β‐cell‐specific IL‐35 therapy suppresses ongoing autoimmune diabetes in NOD mice

IL‐35 is a recently identified cytokine exhibiting potent immunosuppressive properties. The therapeutic potential and effects of IL‐35 on pathogenic T effector cells (Teff) and Foxp3⁺ Treg, however, are ill defined. We tested the capacity of IL‐35 to suppress ongoing autoimmunity in NOD mice. For this purpose, an adeno‐associated virus vector in which IL‐35 transgene expression is selectively targeted to β cells via an insulin promoter (AAV8mIP‐IL35) was used. AAV8mIP‐IL35 vaccination of NOD mice at a late preclinical stage of type 1 diabetes (T1D) suppressed β‐cell autoimmunity and prevented diabetes onset. Numbers of islet‐resident conventional CD4⁺ and CD8⁺ T cells, and DCs were reduced within 4 weeks of AAV8mIP‐IL35 treatment. The diminished islet T‐cell pool correlated with suppressed proliferation, and a decreased frequency of IFN‐γ‐expressing Teff. Ectopic IL‐35 also reduced islet Foxp3⁺ Treg numbers and proliferation, and protection was independent of induction/expansion of adaptive islet immunoregulatory T cells. These findings demonstrate that IL‐35‐mediated suppression is sufficiently robust to block established β‐cell autoimmunity, and support the use of IL‐35 to treat T1D and other T‐cell‐mediated autoimmune diseases.     

HCV‐infected hepatocytes drive CD4+CD25+Foxp3+ regulatory T‐cell development through the Tim‐3/Gal‐9 pathway

HCV is remarkable at disrupting human immunity to establish chronic infection. The accumulation of Treg cells at the site of infection and upregulation of inhibitory signaling pathways (such as T‐cell Ig and mucin domain protein‐3 (Tim‐3) and galectin‐9 (Gal‐9)) play pivotal roles in suppressing antiviral effector T (Teff) cells that are essential for viral clearance. While Tim‐3/Gal‐9 interactions have been shown to negatively regulate Teff cells, their role in regulating Treg cells is poorly understood. To explore how Tim‐3/Gal‐9 interactions regulate HCV‐mediated Treg‐cell development, here we provide pilot data showing that HCV‐infected human hepatocytes express higher levels of Gal‐9 and TGF‐β, and upregulate Tim‐3 expression and regulatory cytokines TGF‐β/IL‐10 in co‐cultured human CD4⁺ T cells, driving conventional CD4⁺ T cells into CD25⁺Foxp3⁺ Treg cells. Additionally, recombinant Gal‐9 protein can transform TCR‐activated CD4⁺ T cells into Foxp3⁺ Treg cells in a dose‐dependent manner. Importantly, blocking Tim‐3/Gal‐9 ligations abrogates HCV‐mediated Treg‐cell induction by HCV‐infected hepatocytes, suggesting that Tim‐3/Gal‐9 interactions may regulate human Foxp3⁺ Treg‐cell development and function during HCV infection.     

Overcoming regulatory T‐cell suppression by a lyophilized preparation of Streptococcus pyogenes

Cancer vaccines have yet to yield clinical benefit, despite the measurable induction of humoral and cellular immune responses. As immunosuppression by CD4⁺CD25⁺ regulatory T (Treg) cells has been linked to the failure of cancer immunotherapy, blocking suppression is therefore critical for successful clinical strategies. Here, we addressed whether a lyophilized preparation of Streptococcus pyogenes (OK‐432), which stimulates Toll‐like receptors, could overcome Treg‐cell suppression of CD4⁺ T‐cell responses in vitro and in vivo. OK‐432 significantly enhanced in vitro proliferation of CD4⁺ effector T cells by blocking Treg‐cell suppression and this blocking effect depended on IL‐12 derived from antigen‐presenting cells. Direct administration of OK‐432 into tumor‐associated exudate fluids resulted in a reduction of the frequency and suppressive function of CD4⁺CD25⁺Foxp3⁺ Treg cells. Furthermore, when OK‐432 was used as an adjuvant of vaccination with HER2 and NY‐ESO‐1 for esophageal cancer patients, NY‐ESO‐1–specific CD4⁺ T‐cell precursors were activated, and NY‐ESO‐1–specific CD4⁺ T cells were detected within the effector/memory T‐cell population. CD4⁺ T‐cell clones from these patients had high‐affinity TCRs and recognized naturally processed NY‐ESO‐1 protein presented by dendritic cells. OK‐432 therefore inhibits Treg‐cell function and contributes to the activation of high‐avidity tumor antigen‐specific naive T‐cell precursors.     

IL‐7 is superior to IL‐2 for ex vivo expansion of tumour‐specific CD4+ T cells

It is well established that tumours hinder both natural and vaccine‐induced tumour‐specific CD4⁺ T‐cell responses. Adoptive T‐cell therapy has the potential to circumvent functional tolerance and enhance anti‐tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8⁺ T cells are currently available, data on tumour‐specific CD4⁺ T cells remain scarce. We report here that CD4⁺ T cells sensitized to tumour‐associated Ag in vivo, proliferate in vitro in response to IL‐7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory‐like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour‐sensitized T cells among other memory and naive lymphocytes following exposure to IL‐7. Also IL‐2, previously used to expand anti‐tumour CTL, promotes tumour‐specific CD4⁺ T‐cell accumulation. However, IL‐7 is superior to IL‐2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4⁺ T cells to confer therapeutic efficacy upon transplantation in tumour‐bearing hosts. Together our data support a unique role for IL‐7 in retrieving memory‐like CD4⁺ T cells suitable for adoptive T‐cell therapy.