Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

IL‐10 production in murine IgM+CD138hi cells is driven by Blimp‐1 and downregulated in class‐switched cells

In contrast to antibody‐induced inflammatory responses, some B‐cell subpopulations suppress inflammation through the production of interleukin (IL)‐10. However, the mechanisms underlying Il10 gene expression during B‐cell development is elusive. Here, we identify IgM⁺B220^loCD138^hi cells responsible for marked IL‐10 production in the bone marrow and spleen of mice. These murine IL‐10‐producing cells predominantly secrete IgM and have unique characteristics of long‐lived plasma cells in spite of high expression of surface IgM. We found that IL‐10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp‐1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class‐switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp‐1 expression during the formation of IgM⁺CD138^hi cells without class‐switching elicits IL‐10 production. Adoptive transfer of Il10‐deficient B cells into B‐cell‐deficient mice demonstrated that IgM⁺CD138^hi cell‐derived IL‐10 supports the survival of class‐switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL‐10 secretion by IgM⁺CD138^hi cells in the complete and efficient humoral response.     

DX5+CD4+ T cells modulate CD4+ T‐cell response via inhibition of IL‐12 production by DCs

DX5⁺CD4⁺ T cells have been shown to dampen collagen‐induced arthritis and delayed‐type hypersensitivity reactions in mice. These cells are also potent modulators of T‐helper cell responses through direct effects on CD4⁺ T cells in an IL‐4 dependent manner. To further characterize this T‐cell population, we studied their effect on DCs and the potential consequences on T‐cell activation. Here, we show that mouse DX5⁺CD4⁺ T cells modulate DCs by robustly inhibiting IL‐12 production. This modulation is IL‐10 dependent and does not require cell contact. Furthermore, DX5⁺CD4⁺ T cells modulate the surface phenotype of LPS‐matured DCs. DCs modulated by DX5⁺CD4⁺ T‐cell supernatant express high levels of the co‐inhibitor molecules PDL‐1 and PDL‐2. OVA‐specific CD4⁺ T cells primed with DCs exposed to DX5⁺CD4⁺ T‐cell supernatant produce less IFN‐γ than CD4⁺ T cells primed by DCs exposed to either medium or DX5^?CD4⁺ T‐cell supernatant. The addition of IL‐12 to the co‐culture with DX5⁺ DCs restores IFN‐γ production. When IL‐10 present in the DX5⁺CD4⁺ T‐cell supernatant is blocked, DCs re‐establish their ability to produce IL‐12 and to efficiently prime CD4⁺ T cells. These data show that DX5⁺CD4⁺ T cells can indirectly affect the outcome of the T‐cell response by inducing DCs that have poor Th1 stimulatory function.     

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.     

Galectin‐3 negatively regulates the frequency and function of CD4+CD25+Foxp3+ regulatory T cells and influences the course of Leishmania major infection

Galectin‐3, an endogenous glycan‐binding protein, plays essential roles during microbial infection by modulating innate and adaptive immunity. However, the role of galectin‐3 within the CD4⁺CD25⁺Foxp3⁺ T regulatory (T_REG) cell compartment has not yet been explored. Here, we found, in a model of Leishmania major infection, that galectin‐3 deficiency increases the frequency of peripheral T_REG cells both in draining lymph nodes (LNs) and sites of infection. These observations correlated with an increased severity of the disease, as shown by increased footpad swelling and parasite burden. Galectin‐3‐deficient (Lgals3^?/?) T_REG cells displayed higher CD103 expression, showed greater suppressive capacity, and synthesized higher amounts of IL‐10 compared with their wild‐type (WT) counterpart. Furthermore, both T_REG cells and T effector (T_EFF) cells from Lgals3^?/? mice showed higher expression of Notch1 and the Notch target gene Hes‐1. Interestingly, Notch signaling components were also altered in both T_REG and T_EFF cells from uninfected Lgals3^?/? mice. Thus, endogenous galectin‐3 regulates the frequency and function of CD4⁺CD25⁺Foxp3⁺ T_REG cells and alters the course of L. major infection.     

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.     

CD8+ T cells producing IL‐3 and IL‐5 in non‐IgE‐mediated eosinophilic diseases

The cytokines IL‐5, IL‐3, and GM‐CSF are crucial for eosinophil development, survival, and function. To better understand their role in non‐IgE‐mediated eosinophilic diseases, we investigated plasma levels of these cytokines as well as cytokine expression in peripheral blood T cells. While we did not find any evidence for an involvement of T‐cell‐derived GM‐CSF, some of these patients did show an increased proportion of IL‐5‐ or IL‐3‐producing CD4⁺ T cells. However, in a significant proportion of patients, IL‐5‐producing CD8⁺ T cells, so‐called Tc2 cells, which in healthy donors can only be detected at very low levels, were prominent. Furthermore, increased IL‐3 production by CD8⁺ T cells was also observed, strongly supporting the notion that CD8⁺ T cells, not just CD4⁺ T cells, must also be considered as a potential source of the cytokines promoting eosinophilia.     

Zinc signals promote IL‐2‐dependent proliferation of T cells

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T‐cell activation by IL‐2 was investigated in the murine cytotoxic T‐cell line CTLL‐2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin‐3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL‐2‐receptor. Activation of the ERK‐pathway was blocked by chelation of free zinc with N,N,N′,N′‐tetrakis‐2(pyridyl‐methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK‐specific gene expression of c‐fos and IL‐2‐induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK‐dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL‐2‐induced ERK signaling and proliferation of T cells.     

IL‐12 and IL‐4 activate a CD39‐dependent intrinsic peripheral tolerance mechanism in CD8+ T cells

Immune responses to protein antigens involve CD4⁺ and CD8⁺ T cells, which follow distinct programs of differentiation. Naïve CD8 T cells rapidly develop cytotoxic T‐cell (CTL) activity after T‐cell receptor stimulation, and we have previously shown that this is accompanied by suppressive activity in the presence of specific cytokines, i.e. IL‐12 and IL‐4. Cytokine‐induced CD8⁺ regulatory T (Treg) cells are one of several Treg‐cell phenotypes and are Foxp3^? IL‐10⁺ with contact‐dependent suppressive capacity. Here, we show they also express high level CD39, an ecto‐nucleotidase that degrades extracellular ATP, and this contributes to their suppressive activity. CD39 expression was found to be upregulated on CD8⁺ T cells during peripheral tolerance induction in vivo, accompanied by release of IL‐12 and IL‐10. CD39 was also upregulated during respiratory tolerance induction to inhaled allergen and on tumor‐infiltrating CD8⁺ T cells. Production of IL‐10 and expression of CD39 by CD8⁺ T cells was independently regulated, being respectively blocked by extracellular ATP and enhanced by an A2A adenosine receptor agonist. Our results suggest that any CTL can develop suppressive activity when exposed to specific cytokines in the absence of alarmins. Thus negative feedback controls CTL expansion under regulation from both nucleotide and cytokine environment within tissues.     

IL‐10 interferes directly with TCR‐induced IFN‐γ but not IL‐17 production in memory T cells

IL‐10 is a potent immunoregulatory and anti‐inflammatory cytokine. However, therapeutic trials in chronic inflammation have been largely disappointing. It is well established that IL‐10 can inhibit Th1 and Th2 cytokine production via indirect effects on APC. Less data are available about the influence of IL‐10 on IL‐17 production, a cytokine which has been recently linked to chronic inflammation. Furthermore, there are only few reports about a direct effect of IL‐10 on T cells. We demonstrate here that IL‐10 can directly interfere with TCR‐induced IFN‐γ production in freshly isolated memory T cells in the absence of APC. This effect was independent of the previously described effects of IL‐10 on T cells, namely inhibition of IL‐2 production and inhibition of CD28 signaling. In contrast, IL‐10 did not affect anti‐CD3/anti‐CD28‐induced IL‐17 production from memory T cells even in the presence of APC. This might have implications for the interpretation of therapeutic trials in patients with chronic inflammation where Th17 cells contribute to pathogenesis.     

DX5+CD4+ T cells modulate cytokine production by CD4+ T cells towards IL‐10 via the production of IL‐4

CD4⁺ Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune‐modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5⁺CD4⁺ T cells, which upon adoptive transfer show potent regulatory properties in murine collagen‐induced arthritis as well as in delayed‐hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5⁺CD4⁺ T cells on other CD4⁺ T cells in vitro. Although proliferation of naïve CD4⁺ T cells upon antigenic triggering was not altered in the presence of DX5⁺CD4⁺ T cells, there was a striking difference in cytokine production. In the presence of DX5⁺CD4⁺ T cells, an IL‐10‐producing CD4⁺ T‐cell response was induced instead of a predominant IFN‐γ‐producing Th1 response. This modulation did not require cell–cell contact. Instead, IL‐4 produced by DX5⁺CD4⁺ T cells was primarily involved in the inhibition of IFN‐γ and promotion of IL‐10 production by CD4⁺ T cells. Together, our data indicate that DX5⁺CD4⁺ T cells modulate the outcome of Th‐responses by diverting Th1‐induction into Th responses characterized by the production of IL‐10.     

Ribavirin modulates the conversion of human CD4+ CD25− T cell to CD4+ CD25+ FOXP3+ T cell via suppressing interleukin‐10‐producing regulatory T cell

Because regulatory T (Treg) cells play an important role in modulating the immune system response against both endogenous and exogenous antigens, their control is critical to establish immunotherapy against autoimmune disorders, chronic viral infections and tumours. Ribavirin (RBV), an antiviral reagent used with interferon, is known to polarize the T helper (Th) 1/2 cell balance toward Th1 cells. Although the immunoregulatory mechanisms of RBV are not fully understood, it has been expected that RBV would affect T reg cells to modulate the Th1/2 cell balance. To confirm this hypothesis, we investigated whether RBV modulates the inhibitory activity of human peripheral CD4⁺ CD25⁺ CD127⁻ T cells in vitro. CD4⁺ CD25⁺ CD127⁻ T cells pre-incubated with RBV lose their ability to inhibit the proliferation of CD4⁺ CD25⁻ T cells. Expression of Forkhead box P3 (FOXP3) in CD4⁺ CD25⁻ T cells was down-modulated when they were incubated with CD4⁺ CD25⁺ CD127⁻ T cells pre-incubated with RBV without down-modulating CD45RO on their surface. In addition, transwell assays and cytokine-neutralizing assays revealed that this effect depended mainly on the inhibition of interleukin-10 (IL-10) produced from CD4⁺ CD25⁺ CD127⁻ T cells. These results indicated that RBV might inhibit the conversion of CD4⁺ CD25⁻ FOXP3⁻ naive T cells into CD4⁺ CD25⁺ FOXP3⁺ adaptive Treg cells by down-modulating the IL-10-producing Treg 1 cells to prevent these effector T cells from entering anergy and to maintain Th1 cell activity. Taken together, our findings suggest that RBV would be useful for both elimination of long-term viral infections such as hepatitis C virus infection and for up-regulation of tumour-specific cellular immune responses to prevent carcinogenesis, especially hepatocellular carcinoma.     

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.     

CD4+ Foxp3+ regulatory T cells mediate Toxoplasma gondii-induced T-cell suppression through an IL-2-related mechanism but independently of IL-10

Acute Toxoplasma gondii infection comprises an immunosuppression stage, characterized by a reduction in T-cell proliferation in vitro. Treg cells maintain the homeostasis of the immune system, but their role in T. gondii-induced suppression has not been addressed. We show herein that immunosuppression, affecting both CD4(+) and CD8(+) T-cell proliferation, concurs with a reduction in Treg-cell number. The residual Treg cells, however, are activated and display an increased suppressive capacity. We show that selective elimination of Treg cells using Foxp3(EGFP) mice leads to a full recovery of CD4(+) and CD8(+) T-cell proliferation. After Treg-cell removal, a reduced production of IL-10 was observed, but IL-2 levels were unchanged. The numbers of IL-10-producing Treg cells also increased during infection, although the in vitro neutralization of this cytokine did not modify T-cell proliferation, suggesting that IL-10 does not mediate the Treg-mediated suppression. However, addition of rIL-2 in vitro fully restored T-cell proliferation from infected animals. Thus, we show that Treg cells mediate the T-cell suppression observed during acute T. gondii infection through an IL-2-dependent mechanism. Our results provide novel insights into the regulation of the immune response against T. gondii.