Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

T‐cell‐derived,but not B‐cell‐derived,IL‐10 suppresses antigen‐specific T‐cell responses in Litomosoides sigmodontis‐infected mice

IL‐10, a cytokine with pleiotropic functions is produced by many different cells. Although IL‐10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth‐induced immune evasion. Here, we show that B cells and T cells produce IL‐10 during murine Litomosoides sigmodontis infection. IL‐10‐deficient mice produced increased amounts of L. sigmodontis‐specific IFN‐γ and IL‐13 suggesting a suppressive role for IL‐10 in the initiation of the T‐cell response to infection. Using cell type‐specific IL‐10‐deficient mice, we dissected different functions of T‐cell‐ and B‐cell‐derived IL‐10. Litomosoides sigmodontis‐specific IFN‐γ, IL‐5, and IL‐13 production increased in the absence of T‐cell‐derived IL‐10 at early and late time points of infection. In contrast, B‐cell‐specific IL‐10 deficiency did not lead to significant changes in L. sigmodontis‐specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag‐specific cellular responses during L. sigmodontis infection is suppressed by T‐cell‐derived IL‐10 and not by B‐cell‐derived IL‐10.     

Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells

Summary: Th3 CD4⁺ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T‐cell subset which primarily secretes transforming growth factor (TGF)‐β, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4⁺ TGF‐β‐secreting cells with suppressive properties have been generated from interleukin (IL)‐4‐deficient animals. In vitro differentiation of Th3 cells from Th precursors from T‐cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF‐β, IL‐4, IL‐10, and anti‐IL‐12. Th3 CD4⁺ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF‐β with various amounts of IL‐4 and IL‐10. Because Th3 regulatory cells are triggered in an antigen‐specific fashion but suppress in an antigen‐non‐specific fashion, they mediate “bystander suppression” when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL‐4. Anti‐CD86 but not anti‐CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF‐β and has a unique class of dendritic cells. CD4⁺CD25⁺ regulatory T‐cell function also appears related to TGF‐β.     

Regulatory T cells enter the pancreas during suppression of type 1 diabetes and inhibit effector T cells and macrophages in a TGF‐β‐dependent manner

Treg can suppress autoimmune diseases such as type 1 diabetes, but their in vivo activity during suppression remains poorly characterized. In type 1 diabetes, Treg activity has been demonstrated in the pancreatic lymph node, but little has been studied in the pancreas, the site of autoimmune islet destruction. In this study we induced islet‐specific Treg from the BDC‐6.9 TCR transgenic mouse by activation of T cells in the presence of TGF‐β. These Treg can suppress spontaneous diabetes as well as transfer of diabetes into NOD.scid mice by diabetic NOD spleen cells or activated BDC‐2.5 TCR transgenic Th1 effector T cells. In the latter transfer model, we observed infiltration of the pancreas by both effector T cells and Treg, suggesting that Treg are active in the inflammatory site and are not just restricted to the draining lymph node. Within the pancreas, we demonstrate that Treg transfer causes a reduction in the number of effector Th1 T cells and macrophages, and also inhibits effector T‐cell cytokine and chemokine production. Although we found no role for TGF‐β in vitro, transfection of effector T cells with a dominant‐negative TGF‐β receptor demonstrated that in vivo suppression of diabetes by TGF‐β‐induced Treg is TGF‐β‐dependent.     

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.     

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.     

The GARP/Latent TGF‐β1 complex on Treg cells modulates the induction of peripherally derived Treg cells during oral tolerance

Treg cells can secrete latent TGF‐β1 (LTGF‐β1), but can also utilize an alternative pathway for transport and expression of LTGF‐β1 on the cell surface in which LTGF‐β1 is coupled to a distinct LTGF‐β binding protein termed glycoprotein A repetitions predominant (GARP)/LRRC32. The function of the GARP/LTGF‐β1 complex has remained elusive. Here, we examine in vivo the roles of GARP and TGF‐β1 in the induction of oral tolerance. When Foxp3^? OT‐II T cells were transferred to wild‐type recipient mice followed by OVA feeding, the conversion of Foxp3^? to Foxp3⁺ OT‐II cells was dependent on recipient Treg cells. Neutralization of IL‐2 in the recipient mice also abrogated this conversion. The GARP/LTGF‐β1 complex on recipient Treg cells, but not dendritic cell‐derived TGF‐β1, was required for efficient induction of Foxp3⁺ T cells and for the suppression of delayed hypersensitivity. Expression of the integrin αvβ8 by Treg cells (or T cells) in the recipients was dispensable for induction of Foxp3 expression. Transient depletion of the bacterial flora enhanced the development of oral tolerance by expanding Treg cells with enhanced expression of the GARP/LTGF‐β1 complex.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

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.     

The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor‐β receptor on dendritic cells potentiates tumour antigen‐specific CD8+ T cell immunity

Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune‐priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)‐10 and transforming growth factor (TGF)‐β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL‐10 receptor alpha (siIL‐10RA) initiated the strongest antigen‐specific CD8⁺ T cell immune responses. The potency of siIL‐10RA was enhanced further by combining it with siRNA targeting TGF‐β receptor (siTGF‐βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl‐2‐like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL‐10RA and siTGF‐βR generated the strongest antigen‐specific CD8⁺ T cell immunity. Concordantly, the knock‐down of both IL‐10RA and TGF‐βR in DCs induced the strongest anti‐tumour effects in the TC‐1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)‐16 E7 antigen, and even in the immune‐resistant TC‐1 (P3) tumour model that secretes more IL‐10 and TGF‐β than the parental tumour cells (TC‐1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail‐mediated strategy by co‐targeting immunosuppressive molecules to enhance the potency of DC‐based vaccines.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid

Antigen‐specific CD4⁺ T cells are central to natural and vaccine‐induced immunity. An ongoing antigen‐specific T‐cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4⁺ T cells against TT in vivo would influence injected CD4⁺ TCR transgenic T cells (OT‐II) specific for an unrelated OVA peptide. If OT‐II cells were pre‐activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT‐specific response. Bystander proliferation was dependent on boosting of the TT‐specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT‐specific memory T‐cell response. T cells activated in vitro displayed functional receptors for IL‐2 and IL‐7, suggesting these as potential mediators. This crosstalk between a stimulated CD4⁺ memory T‐cell response and CD4⁺ T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.     

IL‐1‐dependent,IL‐1R1‐independent resistance to gastrointestinal nematodes

IL‐1 null mice are unable to expel the intestinal nematode Trichuris muris; whereas WT littermates exhibit sterile immunity. Intriguingly the essential signalling components IL‐1R1 and IL‐1R accessory protein (AcP) are dispensable for expulsion of this parasite. IL‐1 is thus critical for CD4⁺ Th2‐mediated immunity to T. muris; however, this action is independent of the established IL‐1 signalling receptor. We also present data demonstrating that both IL‐1α and IL‐1β induce measurable effects on T. muris primed cells isolated from IL‐1R1 or IL‐1R AcP null mice. MLN cells from these mice restimulated with parasite antigen proliferated at a greater rate and produced more cytokines in response to exogenous IL‐1. This ability to respond to IL‐1 was restricted to these parasite‐primed cells and importantly was not evident in cells from naïve gene null mice. These in vitro data are consistent with the observed ability of mice with compromised IL‐1 signalling to expel the parasite, bolstering the premise that an alternative IL‐1 signalling mechanism is accessible in the context of an intestinal helminth‐driven Th2 immune response.     

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.     

Inhibition of type 1 diabetes in filaria‐infected non‐obese diabetic mice is associated with a T helper type 2 shift and induction of FoxP3+ regulatory T cells

We sought to determine whether Litomosoides sigmodontis, a filarial infection of rodents, protects against type 1 diabetes in non‐obese diabetic (NOD) mice. Six‐week‐old NOD mice were sham‐infected or infected with either L3 larvae, adult male worms, or adult female worms. Whereas 82% of uninfected NOD mice developed diabetes by 25 weeks of age, no L. sigmodontis‐infected mice developed disease. Although all mice had evidence of ongoing islet cell inflammation by histology, L. sigmodontis‐infected mice had greater numbers of total islets and non‐infiltrated islets than control mice. Protection against diabetes was associated with a T helper type 2 (Th2) shift, as interleukin‐4 (IL‐4) and IL‐5 release from α‐CD3/α‐CD28‐stimulated splenocytes was greater in L. sigmodontis‐infected mice than in uninfected mice. Increased circulating levels of insulin‐specific immunoglobulin G1, showed that this Th2 shift occurs in response to one of the main autoantigens in diabetes. Multicolour flow cytometry studies demonstrated that protection against diabetes in L. sigmodontis‐infected NOD mice was associated with significantly increased numbers of splenic CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells. Interestingly, injection of crude worm antigen into NOD mice also resulted in protection against type 1 diabetes, though to a lesser degree than infection with live L. sigmodontis worms. In conclusion, these studies demonstrate that filarial worms can protect against the onset of type 1 diabetes in NOD mice. This protection is associated with a Th2 shift, as demonstrated by cytokine and antibody production, and with an increase in CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells.     

Reverse plasticity: TGF‐β and IL‐6 induce Th1‐to‐Th17‐cell transdifferentiation in the gut

Th17 cells are a heterogeneous population of pro‐inflammatory T cells that have been shown to mediate immune responses against intestinal bacteria. Th17 cells are highly plastic and can transdifferentiate to Th1/17 cells or unconventional Th1 cells, which are highly pathogenic in animal models of immune‐mediated diseases such as inflammatory bowel diseases. A recent European Journal of Immunology article by Liu et al. (Eur. J. Immunol. 2015. 45:1010–1018) showed, surprisingly, that Th1 cells have a similar plasticity, and could transdifferentiate to Th17 cells. Thus, IFN‐γ‐producing Th1 effector cells specific for an intestinal microbial antigen were shown to acquire IL‐17‐producing capacities in the gut in a mouse model of colitis, and in response to TGF‐β and IL‐6 in vitro. TGF‐β induced Runx1, and together with IL‐6 was shown to render the ROR‐γt and IL‐17 promoters in Th1 cells accessible for Runx1 binding. In this commentary, we discuss how this unexpected plasticity of Th1 cells challenges our view on the generation of Th1/17 cells with the capacity to co‐produce IL‐17 and IFN‐γ, and consider possible implications of this Th1‐to‐Th17‐cell conversion for therapies of inflammatory bowel diseases and protective immune responses against intracellular pathogens.