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.     

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+ 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.     

IL‐4 and IL‐15 promotion of virtual memory CD8+ T cells is determined by genetic background

Virtual memory (VM) CD8⁺ T cells are present in unimmunized mice, yet possess T‐cell receptors specific for foreign antigens. To date, VM cells have only been characterized in C57BL/6 mice. Here, we assessed the cytokine requirements for VM cells in C57BL/6 and BALB/c mice. As reported previously, VM cells in C57BL/6 mice rely mostly on IL‐15 and marginally on IL‐4. In stark contrast, VM cells in BALB/c mice rely substantially on IL‐4 and marginally on IL‐15. Further, NKT cells are the likely source of IL‐4, because CD1d‐deficient mice on a BALB/c background have significantly fewer VM cells. Notably, this NKT/IL‐4 axis contributes to appropriate effector and memory T‐cell responses to infection in BALB/c mice, but not in C57BL/6 mice. However, the effects of IL‐4 are manifest prior to, rather than during, infection. Thus, cytokine‐mediated control of the precursor population affects the development of virus‐specific CD8⁺ T‐cell memory. Depending upon the genetic background, different cytokines encountered before infection may influence the subsequent ability to mount primary and memory anti‐viral CD8⁺ T‐cell responses.     

Emergence of a distinct HIV‐specific IL‐10‐producing CD8+ T‐cell subset with immunomodulatory functions during chronic HIV‐1 infection

Interleukin‐10 (IL‐10) plays a key role in regulating proinflammatory immune responses to infection but can interfere with pathogen clearance. Although IL‐10 is upregulated throughout HIV‐1 infection in multiple cell subsets, whether this is a viral immune evasion strategy or an appropriate response to immune activation is unresolved. Analysis of IL‐10 production at the single cell level in 51 chronically infected subjects (31 antiretroviral (ART) naïve and 20 ART treated) showed that a subset of CD8⁺ T cells with a CD25^neg FoxP3^neg phenotype contributes substantially to IL‐10 production in response to HIV‐1 gag stimulation. The frequencies of gag‐specific IL‐10‐ and IFN‐γ‐producing T cells in ART‐naïve subjects were strongly correlated and the majority of these IL‐10⁺ CD8⁺ T cells co‐produced IFN‐γ; however, patients with a predominant IL‐10⁺/IFN‐γ^neg profile showed better control of viraemia. Depletion of HIV‐specific CD8⁺ IL‐10⁺ cells from PBMCs led to upregulation of CD38 on CD14⁺ monocytes together with increased IL‐6 production, in response to gag stimulation. Increased CD38 expression was positively correlated with the frequency of the IL‐10⁺ population and was also induced by exposure of monocytes to HIV‐1 in vitro. Production of IL‐10 by HIV‐specific CD8⁺ T cells may represent an adaptive regulatory response to monocyte activation during chronic infection.     

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.     

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.     

Impact of changes in antigen level on CD38/PD‐1 co‐expression on HIV‐specific CD8 T cells in chronic,untreated HIV‐1 infection

Excessive immune activation is a hallmark of chronic uncontrolled HIV infection. During the past years, growing evidence suggests that immune inhibitory signals also play an important role in progressive disease. However, the relationship between positive and negative immune signals on HIV‐specific CD8 T cells has not been studied in detail so far in chronic HIV‐1 infection. In this study, the expression of markers of positive (CD38) and negative (PD‐1) immune signals on virus‐specific CD8 T cells in chronic, untreated HIV‐1 infection was evaluated using intracellular cytokine staining. Viral escape mutations were assessed by autologous virus sequence analysis and subsequent peptide titration assays. Single‐epitope CD8 T‐cell responses toward Gag, Pol, and Nef were compared in 12 HIV‐1 controllers (viral load <5,000 cp/ml) and 12 HIV‐1 progressors (viral load >50,000 cp/ml) and a highly significant increase of CD38/PD‐1 co‐expression on virus‐specific CD8 T cells in progressors was found (P < 0.0001). The level of CD38/PD‐1 co‐expression was independent of epitope specificity. Longitudinal follow‐up revealed a clear drop in CD38/PD‐1 co‐expression on virus‐specific CD8 T cells after the suppression of antigen following either viral escape mutation or the initiation of HAART (P = 0.004). Antigen persistence with a fluctuating viral load revealed stable levels of CD38/PD‐1 co‐expression whereas significant rises in viral load were accompanied or even preceded by substantial increases in CD38/PD‐1 co‐expression. The CD38/PD‐1 phenotype clearly distinguishes HIV‐specific CD8 T‐cell responses between controllers and progressors. Whether it plays a causative role in disease progression remains debatable. J. Med. Virol. 82:358–370, 2010. © 2010 Wiley‐Liss, Inc.     

Increased expression of interleukin-13 but not interleukin-4 in CD4+ cells from patients with the hyper-IgE syndrome

Hyper IgE syndrome (HIES) is a rare immunodeficiency disorder characterized mainly by high levels of polyclonal IgE in serum and recurrent staphylococcal abscesses of the skin and lungs. The raised IgE levels have led researchers to study the synthesis of cytokines that regulate switching of immunoglobulin production towards IgE such as interleukin-4 (IL-4), IL-12 and interferon-gamma (IFN)-gamma. However, the role of IL-13 in the disease pathogenesis has not been investigated extensively. In this study, we investigated intracellular expression of IL-4 and IL-13 in mononuclear cells and CD4+ cells isolated from patients with HIES and healthy controls. Cells were stained intracellularly with antibodies directed against IL-4 and IL-13 and analysed by flow cytometry before and after activation with PMA and calcium ionophore. The mean proportion of resting or activated IL-4 and IL-13 expressing mononuclear cells were comparable in the two groups as well as the proportion of IL-4 expressing CD4+ cells. In contrast, the mean proportion of IL-13 expressing CD4+ cells was increased significantly in patients with HIES in both the resting and the activated state compared to healthy controls. We conclude that increased expression of IL-13 in CD4+ cells from patients with HIES could account, at least partly, for raised IgE levels in those individuals.     

Association of IL‐4 589 C/T promoter and IL‐4RαI50V receptor polymorphism with susceptibility to HIV‐1 infection in North Indians

The clinical course and outcome of HIV‐1 infection are highly variable among individuals. Interleukin 4 (IL‐4) is a key T helper 2 cytokine with various immune‐modulating functions including induction of immunoglobulin E (IgE) production in B cells, downregulation of CCR5 and upregulation of CXCR4, the main co‐receptors for HIV. Our objective is to investigate whether single‐nucleotide polymorphisms (SNPs) in the IL‐4 promoter 589 C/T and IL‐4 Rα I50V affect the susceptibility to HIV infection and its progression to AIDS in North Indian individuals. The study population consisted of 180 HIV‐1 seropositive (HSP) stratified on the basis of disease severity (stage I, II, III), 50 HIV‐1 exposed seronegative (HES), and 305 HIV‐1 seronegative (HSN) individuals. The subjects were genotyped for IL‐4 589 C/T promoter polymorphism and IL‐4 Rα I50V by polymerase chain reaction restriction fragment length polymorphism. The results showed that IL‐4 589 C/T was not associated with the risk of HIV infection and disease progression. However, the IL‐4Rα I50 allele and genotype was significantly increased in HSP compared to HSN and HSP and was associated with risk of HIV infection. The frequency of IL‐4Rα I50 allele in the HSP group was higher than in HSN (76.11 vs. 64.75%; P = 0.000; OR = 1.734) and HES (76.11% vs. 62.00%; P = 0.007; OR = 1.953). Homozygous IL‐4Rα I50I genotype was significantly increased in HSP group compared with HSN (58.88% vs. 44.26%; P = 0.002; OR = 1.804) and HES (58.88% vs. 42.00%; P = 0.038; OR = 1.978). The present study for the first time suggests an association of IL‐4Rα I50 allele with increased likelihood of HIV‐1 infection in North Indian population. Further studies are required to confirm these findings and understand the effect of IL‐4Rα polymorphism on the outcome of HIV‐1 infection. J. Med. Virol. 81:959–965, 2009. © 2009 Wiley‐Liss, Inc.     

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.