Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

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.     

CD8+ Treg cells suppress CD8+ T cell‐responses by IL‐10‐dependent mechanism during H5N1 influenza virus infection

Although Treg‐cell‐mediated suppression during infection or autoimmunity has been described, functions of Treg cells during highly pathogenic avian influenza virus infection remain poorly characterized. Here we found that in Foxp3‐GFP transgenic mice, CD8⁺ Foxp3⁺ Treg cells, but not CD4⁺ Foxp3⁺ Treg cells, were remarkably induced during H5N1 infection. In addition to expressing CD25, the CD8⁺ Foxp3⁺ Treg cells showed a high level of GITR and produced IL‐10. In an adoptive transfer model, CD8⁺ Treg cells suppressed CD8⁺ T‐cell responses and promoted H5N1 virus infection, resulting in enhanced mortality and increased virus load in the lung. Furthermore, in vitro neutralization of IL‐10 and studies with IL‐10R‐deficient mice in vitro and in vivo demonstrated an important role for IL‐10 production in the capacity of CD8⁺ Treg cells to inhibit CD8⁺ T‐cell responses. Our findings identify a previously unrecognized role of CD8⁺ Treg cells in the negative regulation of CD8⁺ T‐cell responses and suggest that modulation of CD8⁺ Treg cells may be a therapeutic strategy to control H5N1 viral infection.     

The role of 1α,25‐dihydroxyvitamin D3 and cytokines in the promotion of distinct Foxp3+and IL‐10+ CD4+ T cells

1α,25‐Dihydroxyvitamin D3 (1α25VitD3) has potent immunomodulatory properties. We have previously demonstrated that 1α25VitD3 promotes human and murine IL‐10‐secreting CD4⁺ T cells. Because of the clinical relevance of this observation, we characterized these cells further and investigated their relationship with Foxp3⁺ regulatory T (Treg) cells. 1α25VitD3 increased the frequency of both Foxp3⁺ and IL‐10⁺ CD4⁺T cells in vitro. However, Foxp3 was increased at high concentrations of 1α25VitD3 and IL‐10 at more moderate levels, with little coexpression of these molecules. The Foxp3⁺ and IL‐10⁺ T‐cell populations showed comparable suppressive activity. We demonstrate that the enhancement of Foxp3 expression by 1α25VitD3 is impaired by IL‐10. 1α25VitD3 enables the selective expansion of Foxp3⁺ Treg cells over their Foxp3^? T‐cell counterparts. Equally, 1α25VitD3 maintains Foxp3⁺ expression by sorted populations of human and murine Treg cells upon in vitro culture. A positive in vivo correlation between vitamin D status and CD4⁺Foxp3⁺ T cells in the airways was observed in a severe pediatric asthma cohort, supporting the in vitro observations. In summary, we provide evidence that 1α25VitD3 enhances the frequency of both IL‐10⁺ and Foxp3⁺ Treg cells. In a translational setting, these data suggest that 1α25VitD3, over a broad concentration range, will be effective in enhancing the frequency of Treg cells.     

CD39 is involved in mediating suppression by Mycobacterium bovis BCG‐activated human CD8+CD39+ regulatory T cells

Regulatory T (Treg) cells can balance normal tissue homeostasis by limiting inflammatory tissue damage, e.g. during pathogen infection, but on the other hand can also limit protective immunity induced during natural infection or following vaccination. Because most studies have focused on the role of CD4⁺ Treg cells, relatively little is known about the phenotype and function of CD8⁺ Treg cells, particularly in infectious diseases. Here, we describe for the first time the expression of CD39 (E‐NTPDase1) on Mycobacterium‐activated human CD8⁺ T cells. These CD8⁺CD39⁺ T cells significantly co‐expressed the Treg markers CD25, Foxp3, lymphocyte activation gene‐3 (LAG‐3), and CC chemokine ligand 4 (CCL4), and suppressed the proliferative response of antigen‐specific CD4⁺ T helper‐1 (Th1) cells. Pharmacological or antibody mediated blocking of CD39 function resulted in partial reversal of suppression. These data identify CD39 as a novel marker of human regulatory CD8⁺ T cells and indicate that CD39 is functionally involved in suppression by CD8⁺ Treg 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.     

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.     

Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent

Host protection to helminth infection requires IL‐4 receptor α chain (IL‐4Rα) signalling and the establishment of finely regulated Th2 responses. In the current study, the role of IL‐4Rα‐responsive T cells in Schistosoma mansoni egg‐induced inflammation was investigated. Egg‐induced inflammation in IL‐4Rα‐responsive BALB/c mice was accompanied with Th2‐biased responses, whereas T‐cell‐specific IL‐4Rα‐deficient BALB/c mice (iLck^creIl4ra^?/lox) developed Th1‐biased responses with heightened inflammation. The proportion of Foxp3⁺ Treg in the draining LN of control mice did not correlate with the control of inflammation and was reduced in comparison to T‐cell‐specific IL‐4Rα‐deficient mice. This was due to IL‐4‐mediated inhibition of CD4⁺Foxp3⁺ Treg conversion, demonstrated in adoptively transferred Rag2^?/? mice. Interestingly, reduced footpad swelling in Il4ra^?/lox mice was associated with the induction of IL‐4 and IL‐10‐secreting CD4⁺CD25^?CD103⁺Foxp3^? cells, confirmed in S. mansoni infection studies. Transfer of IL‐4Rα‐responsive CD4⁺CD25^?CD103⁺ cells, but not CD4⁺CD25^high or CD4⁺CD25^?CD103^? cells, controlled inflammation in iLck^creIl4ra^?/lox mice. The control of inflammation depended on IL‐10, as transferred CD4⁺CD25^?CD103⁺ cells from IL‐10‐deficient mice were not able to effectively downregulate inflammation. Together, these results demonstrate that IL‐4 signalling in T cells inhibits Foxp3⁺ Treg in vivo and promotes CD4⁺CD25^?CD103⁺Foxp3^? cells that control S. mansoni egg‐induced inflammation via IL‐10.     

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3⁺ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.     

Beta2‐adrenergic receptor signaling in CD4+ Foxp3+ regulatory T cells enhances their suppressive function in a PKA‐dependent manner

Beta2‐adrenergic receptor (B2AR) signaling is known to impair Th1‐cell differentiation and function in a cAMP‐dependent way, leading to inhibition of cell proliferation and decreased production of IL‐2 and IFN‐γ. CD4⁺ Foxp3⁺ Treg cells play a key role in the regulation of immune responses and are essential for maintenance of self‐tolerance. Nevertheless, very little is known about adrenergic receptor expression in Treg cells or the influence of noradrenaline on their function. Here we show that Foxp3⁺ Treg cells express functional B2AR. B2AR activation in Treg cells leads to increased intracellular cAMP levels and to protein kinase A (PKA)‐dependent CREB phosphorylation. We also found that signaling via B2AR enhances the in vitro suppressive activity of Treg cells. B2AR‐mediated increase in Treg‐cell suppressive function was associated with decreased IL‐2 mRNA levels in responder CD4⁺ T cells and improved Treg‐cell‐induced conversion of CD4⁺ Foxp3^? cells into Foxp3⁺ induced Treg cells. Moreover, B2AR signaling increased CTLA‐4 expression in Treg cells in a PKA‐dependent way. Finally, we found that PKA inhibition totally prevented the B2AR‐mediated increase in Treg‐cell suppressive function. Our data suggest that sympathetic fibers are able to regulate Treg‐cell suppressive activity in a positive manner through B2AR signaling.     

Donor bone marrow cells are essential for iNKT cell‐mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti‐CD40 ligand (anti‐CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub‐lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3‐positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)‐γ‐producing CD8⁺T cells, expand host Ki67⁺CD4⁺CD25⁺Foxp3⁺ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC‐recipients accepted skin grafts in an allo‐specific manner. Myeloid‐derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.     

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.     

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.     

Mycobacterium tuberculosis‐specific CD4+ T‐cell response is increased,and Treg cells decreased,in anthelmintic‐treated patients with latent TB

In many settings, adults with active or latent tuberculosis will also be coinfected with helminths. Our study aimed to investigate how anthelmintic treatment modulates antimycobacterial immunity, in a setting where helminth reinfection should not occur. We investigated the potential impact of helminth infection on immune responses to Mycobacterium tuberculosis (Mtb) in patients with latent Mtb infection with or without helminth infection (Strongyloides or Schistosoma), and tested T‐cell responses before and after anthelmintic treatment. The study was performed in migrants resident in the United Kingdom, where reexposure and reinfection following anthelmintic treatment would not occur. The frequency of CD4⁺IFN‐γ⁺ T cells was measured following stimulation with Mtb Purified Protein Derivative or ESAT‐6/CFP‐10 antigen, and concentrations of IFN‐γ in culture supernatants measured by ELISA and multiplex bead array. Helminth infection was associated with a lower frequency of CD4⁺IFN‐γ⁺ T cells, which increased following treatment. Patients with helminth infection showed a significant increase in CD4⁺FoxP3⁺ T cells (Treg) compared to those without helminth infection. There was a decrease in the frequency of Treg cells, and an associated increase in CD4⁺IFN‐γ⁺ T cells after the anthelmintic treatment. Here, we show a potential role of Treg cells in reducing the frequency and function of antimycobacterial CD4⁺IFN‐γ⁺ T cells, and that these effects are reversed after anthelmintic treatment.     

CTLA‐4 is required by CD4+CD25+ Treg to control CD4+ T‐cell lymphopenia‐induced proliferation

CTLA‐4 is constitutively expressed by CD4⁺CD25⁺Foxp3⁺ Treg but its precise role in Treg function is not clear. Although blockade of CTLA‐4 interferes with Treg function, studies using CTLA‐4‐deficient Treg have failed to reveal an essential requirement for CTLA‐4 in Treg suppression in vivo. Conditional deletion of CTLA‐4 in Foxp3⁺ T cells disrupts immune homeostasis in vivo but the immune processes disrupted by CTLA‐4 deletion have not been determined. We demonstrate that Treg expression of CTLA‐4 is essential for Treg control of lymphopenia‐induced CD4 T‐cell expansion. Despite IL‐10 expression, CTLA‐4‐deficient Treg were unable to control the expansion of CD4⁺ target cells in a lymphopenic environment. Moreover, unlike their WT counterparts, CTLA‐4‐deficient Treg failed to inhibit cytokine production associated with homeostatic expansion and were unable to prevent colitis. Thus, while Treg developing in the absence of CTLA‐4 appear to acquire some compensatory suppressive mechanisms in vitro, we identify a non‐redundant role for CTLA‐4 in Treg function in vivo.     

Serotonin decreases the production of Th1/Th17 cytokines and elevates the frequency of regulatory CD4+ T‐cell subsets in multiple sclerosis patients

Excessive levels of proinflammatory cytokines in the CNS are associated with reduced serotonin (5‐HT) synthesis, a neurotransmitter with diverse immune effects. In this study, we evaluated the ability of exogenous 5‐HT to modulate the T‐cell behavior of patients with MS, a demyelinating autoimmune disease mediated by Th1 and Th17 cytokines. Here, 5‐HT attenuated, in vitro, T‐cell proliferation and Th1 and Th17 cytokines production in cell cultures from MS patients. Additionally, 5‐HT reduced IFN‐γ and IL‐17 release by CD8⁺ T cells. By contrast, 5‐HT increased IL‐10 production by CD4⁺ T cells from MS patients. A more accurate analysis of these IL‐10‐secreting CD4⁺ T cells revealed that 5‐HT favors the expansion of FoxP3⁺CD39⁺ regulatory T cells (Tregs) and type 1 regulatory T cells. Notably, this neurotransmitter also elevated the frequency of Treg17 cells, a novel regulatory T‐cell subset. The effect of 5‐HT in upregulating CD39⁺ Treg and Treg17 cells was inversely correlated with the number of active brain lesions. Finally, in addition to directly reducing cytokine production by purified Th1 and Th17 cells, 5‐HT enhanced in vitro Treg function. In summary, our data suggest that serotonin may play a protective role in the pathogenesis of MS.     

Membrane‐bound Dickkopf‐1 in Foxp3+ regulatory T cells suppresses T‐cell‐mediated autoimmune colitis

Induction of tolerance is a key mechanism to maintain or to restore immunological homeostasis. Here we show that Foxp3⁺ regulatory T (Treg) cells use Dickkopf‐1 (DKK‐1) to regulate T‐cell‐mediated tolerance in the T‐cell‐mediated autoimmune colitis model. Treg cells from DKK‐1 hypomorphic doubleridge mice failed to control CD4⁺ T‐cell proliferation, resulting in CD4 T‐cell‐mediated autoimmune colitis. Thymus‐derived Treg cells showed a robust expression of DKK‐1 but not in naive or effector CD4 T cells. DKK‐1 expression in Foxp3⁺ Treg cells was further increased upon T‐cell receptor stimulation in vitro and in vivo. Interestingly, Foxp3⁺ Treg cells expressed DKK‐1 in the cell membrane and the functional inhibition of DKK‐1 using DKK‐1 monoclonal antibody abrogated the suppressor function of Foxp3⁺ Treg cells. DKK‐1 expression was dependent on de novo protein synthesis and regulated by the mitogen‐activated protein kinase pathway but not by the canonical Wnt pathway. Taken together, our results highlight membrane‐bound DKK‐1 as a novel Treg‐derived mediator to maintain immunological tolerance in T‐cell‐mediated autoimmune colitis.     

ICOS controls Foxp3+ regulatory T‐cell expansion,maintenance and IL‐10 production during helminth infection

Foxp3⁺ regulatory T (Treg) cells are key immune regulators during helminth infections, and identifying the mechanisms governing their induction is of principal importance for the design of treatments for helminth infections, allergies and autoimmunity. Little is yet known regarding the co‐stimulatory environment that favours the development of Foxp3⁺ Treg‐cell responses during helminth infections. As recent evidence implicates the co‐stimulatory receptor ICOS in defining Foxp3⁺ Treg‐cell functions, we investigated the role of ICOS in helminth‐induced Foxp3⁺ Treg‐cell responses. Infection of ICOS^?/? mice with Heligmosomoides polygyrus or Schistosoma mansoni led to a reduced expansion and maintenance of Foxp3⁺ Treg cells. Moreover, during H. polygyrus infection, ICOS deficiency resulted in increased Foxp3⁺ Treg‐cell apoptosis, a Foxp3⁺ Treg‐cell specific impairment in IL‐10 production, and a failure to mount putatively adaptive Helios^?Foxp3⁺ Treg‐cell responses within the intestinal lamina propria. Impaired lamina propria Foxp3⁺ Treg‐cell responses were associated with increased production of IL‐4 and IL‐13 by CD4⁺ T cells, demonstrating that ICOS dominantly downregulates Type 2 responses at the infection site, sharply contrasting with its Type 2‐promoting effects within lymphoid tissue. Thus, ICOS regulates Type 2 immunity in a tissue‐specific manner, and plays a key role in driving Foxp3⁺ Treg‐cell expansion and function during helminth infections.     

Foxp3+ regulatory T cells are activated in spite of B7‐CD28 and CD40‐CD40L blockade

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)‐cell activity. We previously reported that the blockade of the B7‐CD28 and CD40L‐CD40 interaction efficiently suppresses allogeneic T‐cell activation in vivo. This was characterized by an initial rise in Foxp3⁺ cells, followed by depletion of host‐reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4⁺ cell activation. When CTLA‐4Ig and anti‐CD40L mAb (MR1) were added to the cultures, T‐cell proliferation and IL‐2 production were strongly reduced. However, Foxp3⁺ cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4⁺ cells much more efficiently than did freshly isolated Treg cells. CD4⁺ cells activated by allogeneic cells in the presence of MR1 and CTLA‐4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4⁺ cells when Foxp3⁺ Treg cells were removed. We conclude that natural Treg cells are less dependent on B7‐CD28 or CD40‐CD40L costimulation compared with Foxp3^? T cells. Reduced costimulation therefore alters the balance between Teff and Treg‐cell activation in favor of Treg‐cell activity.