Reversal of chronic to resolved infection by IL‐10 blockade is LCMV strain dependent

Chronic viral infections lead to CD8⁺ T‐cell exhaustion, characterized by impaired cytokine secretion. The immune‐regulatory cytokine IL‐10 promotes chronicity of infection with lymphocytic choriomeningitis virus (LCMV) Clone 13, as absence of IL‐10 or blocking of IL‐10R during early LCMV Clone 13 infection results in viral clearance. Thus, treatment of humans suffering from chronic viral infections with IL‐10 neutralizing or IL‐10R blocking antibodies was proposed to boost virus‐specific T‐cell responses to enhance control or even clear the viral infection. Here we demonstrate that although CD4⁺ and CD8⁺ T cells can produce elevated levels of cytokines in IL‐10^?/? mice early after infection compared with WT mice, IL‐10^?/? mice cannot clear an infection with the quicker replicating LCMV strain Docile, eventually resulting in T‐cell exhaustion. These data suggest that the success of IL‐10 blockade to control chronic viral infections may critically depend on the virulence of the infecting strain.     

Inhibition of the Fibrinogen‐Like Protein 2:FcγRIIB/RIII immunosuppressive pathway enhances antiviral T‐cell and B‐cell responses leading to clearance of lymphocytic choriomeningitis virus clone 13

Persistent viruses evade immune detection by interfering with virus‐specific innate and adaptive antiviral immune responses. Fibrinogen‐like protein‐2 (FGL2) is a potent effector molecule of CD4⁺ CD25⁺ FoxP3⁺ regulatory T cells and exerts its immunosuppressive activity following ligation to its cognate receptor, FcγRIIB/RIII. The role of FGL2 in the pathogenesis of chronic viral infection caused by lymphocytic choriomeningitis virus clone‐13 (LCMV cl‐13) was assessed in this study. Chronically infected fgl2^+/+ mice had increased plasma levels of FGL2, with reduced expression of the maturation markers, CD80, CD86 and MHC‐II on macrophages and dendritic cells and impaired production of neutralizing antibody. In contrast, fgl2^?/? mice or fgl2^+/+ mice that had been pre‐treated with antibodies to FGL2 and FcγRIIB/RIII and then infected with LCMV cl‐13 developed a robust CD4⁺ and CD8⁺ antiviral T‐cell response, produced high titred neutralizing antibody to LCMV and cleared LCMV. Treatment of mice with established chronic infection with antibodies to FGL2 and FcγRIIB/RIII was shown to rescue the number and functionality of virus‐specific CD4⁺ and CD8⁺ T cells with reduced total and virus‐specific T‐cell expression of programmed cell death protein 1 leading to viral clearance. These results demonstrate an important role for FGL2 in viral immune evasion and provide a rationale to target FGL2 to treat patients with chronic viral infection.     

Enhancing immunity prevents virus‐induced T‐cell‐mediated immunopathology in B cell‐deficient mice

Hyper‐activated or deviated immune responses can result in immunopathological diseases. Paradoxically, immunodeficiency represents a frequent cause of such immune‐mediated pathologies. Immunopathological manifestations are commonly treated by immunosuppression, but in situations in which immunodeficiency is the basis of disease development, enhancing immunity may represent an alternative treatment option. Here, we tested this counterintuitive concept in a preclinical model using infection of mice with lymphocytic choriomeningitis virus (LCMV). Firstly, we demonstrate that infection of B‐cell‐deficient (B^?/?) but not of wild‐type (WT) mice with the LCMV strain Docile induced a rapid and fatal CD8⁺ T‐cell‐mediated immunopathological disease. Similar to WT mice, LCMV‐infected B^?/? mice generated a potent, functional LCMV‐specific CD8⁺ T‐cell response but exhibited prolonged viral antigen presentation and increased vascular leakage in liver and lungs. Secondly, we were able to prevent this virus‐induced immunopathology in B^?/? mice by active or passive T‐cell immunizations or by treatment with LCMV‐specific virus neutralizing or non‐neutralizing monoclonal antibodies (mAb). Thus, boosting antiviral immunity did not aggravate immunopathology in this model, but prevented it by decreasing the formation of target structures for damage‐causing CD8⁺ T cells.     

Nucleoprotein‐specific nonneutralizing antibodies speed up LCMV elimination independently of complement and FcγR

CD8⁺ T cells have an essential role in controlling lymphocytic choriomeningitis virus (LCMV) infection in mice. Here, we examined the contribution of humoral immunity, including nonneutralizing antibodies (Abs), in this infection induced by low virus inoculation doses. Mice with impaired humoral immunity readily terminated infection with the slowly replicating LCMV strain Armstrong but showed delayed virus elimination after inoculation with the faster replicating LCMV strain WE and failed to clear the rapidly replicating LCMV strain Docile, which is in contrast to the results obtained with wild‐type mice. Thus, the requirement for adaptive humoral immunity to control the infection was dependent on the replication speed of the LCMV strains used. Ab transfers further showed that LCMV‐specific IgG Abs isolated from LCMV immune serum accelerated virus elimination. These Abs were mainly directed against the viral nucleoprotein (NP) and completely lacked virus neutralizing activity. Moreover, mAbs specific for the LCMV NP were also able to decrease viral titers after transfer into infected hosts. Intriguingly, neither C3 nor Fcγ receptors were required for the antiviral activity of the transferred Abs. In conclusion, our study suggests that rapidly generated nonneutralizing Abs specific for the viral NP speed up virus elimination and thereby may counteract T‐cell exhaustion.     

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.     

IL‐17‐producing γδ T cells

IL‐17 is produced not only by CD4⁺ αβ T cells, but also CD8⁺ αβ T cells, NKT cells, and γδ T cells, plus some non‐T cells, including macrophages and neutrophils. The ability of IL‐17 to deploy neutrophils to sites of inflammation imparts this cytokine with a key role in diseases of several types. Surprisingly, γδ T cells are responsible for much of the IL‐17 produced in several disease models, particularly early on.     

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.     

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.     

Interleukin‐17 Facilitates the Immune Suppressor Capacity of High‐Grade Glioma‐Derived CD4 (+) CD25 (+) Foxp3 (+) T Cells Via Releasing Transforming Growth Factor Beta

High‐grade glioma is a malignant tumour; the pathogenesis is to be further investigated. Interleukin (IL)‐17 is an inflammatory cytokine. Chronic inflammation is a pathological feature of cancer. This study aimed to characterize the glioma‐derived IL‐17⁺ regulatory T cells (Treg). In this study, single cells were isolated from surgically removed high‐grade glioma tissue and examined by flow cytometry. The immune suppressor effect of IL‐17⁺ Tregs on CD8⁺ T cells was assessed in vitro. The results showed that abundant IL‐17⁺ Tregs were found in high‐grade glioma tissue. The immune suppressor molecule, transforming growth factor (TGF)‐beta, was detected in the IL‐17⁺ Tregs. The proliferation of CD8⁺ T cells was suppressed by culturing with the IL‐17⁺ Tregs, which was partially abrogated by neutralizing antibodies of either TGF‐beta or IL‐17 and completely abrogated by neutralizing antibodies against both TGF‐beta and IL‐17. In conclusion, IL‐17⁺ Tregs exist in the high‐grade glioma tissue; this subset of T cells can suppress CD8⁺ T cell activities via releasing TGF‐beta and IL‐17.     

Influenza matrix 1‐specific human CD4+FOXP3+ and FOXP3− regulatory T cells can be detected long after viral clearance

Control and termination of infection with Influenza A virus is associated with increased IL‐10 production in mouse models. Notably, IL‐10 can be produced by Treg. Therefore, we investigated whether the population of IL‐10‐producing influenza‐specific CD4⁺ T cells comprised Treg as they are potent suppressors of the adaptive immune response. Influenza‐specific IL‐10‐producing T cells were detected in all human donors displaying influenza‐specific immunity. Isolation of Matrix 1 protein‐specific IL‐10‐producing T‐cell clones revealed that a substantial proportion of these T‐cell clones displayed the capacity to suppress effector cells, functionally identifying them as Treg. Both FOXP3⁺ and FOXP3^? CD4⁺ Treg were isolated and all were able to exert their suppressive capacity when stimulated with cognate antigen, including influenza virus‐infected cells. In vitro suppression was not mediated by IL‐10 but involved interference with the IL‐2 axis. The isolated Treg suppressed amongst others the IL‐2 production of influenza‐specific T‐helper cells as well as partially prevented the upregulation of the high‐affinity IL‐2 receptor on CD8 effector cells. So far the induction of virus‐specific Treg has only been studied in the context of chronic viral infections. This study demonstrates that virus‐specific Treg can also be induced by viruses that are rapidly cleared in humans.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.     

Tracking antigen‐specific CD4+ T cells throughout the course of chronic Leishmania major infection in resistant mice

Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4⁺ T‐cell‐derived IFN‐γ and IL‐10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite‐specific primary CD4⁺ T‐cell response to L. major in mice resistant to infection. Maximal frequencies of IFN‐γ⁺ CD4⁺ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL‐10⁺ CD4⁺ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type‐1 (Th1) cells, not Foxp3⁺ regulatory T cells, were the chief producers of IL‐10 and were not exhausted. Therefore, tracking antigen‐specific CD4⁺ T cells revealed that IL‐10 production by Th1 cells is not due to persistent T‐cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection.     

IL‐33 promotes innate IFN‐γ production and modulates dendritic cell response in LCMV‐induced hepatitis in mice

Recent studies have revealed IL‐33 as a key factor in promoting antiviral T‐cell responses. However, it is less clear as to how IL‐33 regulates innate immunity. In this study, we infected wild‐type (WT) and IL‐33^?/? mice with lymphocytic choriomeningitis virus and demonstrated an essential role of infection‐induced IL‐33 expression for robust innate IFN‐γ production in the liver. We first show that IL‐33 deficiency resulted in a marked reduction in the number of IFN‐γ⁺ γδ T and NK cells, but an increase in that of IL‐17⁺ γδ T cells at 16 h postinfection. Recombinant IL‐33 (rIL‐33) treatment could reverse such deficiency via increasing IFN‐γ‐producing γδ T and NK cells, and inhibiting IL‐17⁺ γδ T cells. We also found that rIL‐33‐induced type 2 innate lymphoid cells were not involved in T‐cell responses and liver injury, since the adoptive transfer of type 2 innate lymphoid cells neither affected the IFN‐γ and TNF‐α production in T cells, nor liver transferase levels in lymphocytic choriomeningitis virus infected mice. Interestingly, we found that while IL‐33 was not required for costimulatory molecule expression, it was critical for DC proliferation and cytokine production. Together, this study highlights an essential role of IL‐33 in regulating innate IFN‐γ‐production and DC function during viral hepatitis.     

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.     

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.     

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice

CD40‐CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T‐cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8⁺ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus‐specific CD8⁺ T‐cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L^?/? mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV‐specific effector and memory T cells consists of CD40L⁺ CD8⁺ T cells. However, deficiency of CD40L in CD8⁺ T cells did influence neither the quantity nor the quality of primary T‐cell responses in LCMV infection. Virus‐specific CD8⁺ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8⁺ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8⁺ T‐cell responses also under inflammatory conditions, CD40L expression by CD8⁺ T cells themselves is dispensable in acute LCMV infection.     

Hypergammaglobulinemia sustains the development of regulatory responses during chronic Leishmania donovani infection in mice

Visceral leishmaniasis, a chronic, potentially fatal disease, is characterized by high production of low‐affinity antibodies. In humans, hypergammaglobulinemia is prediction of disease progression. Nevertheless, the contribution of hypermutated and/or class‐switched immunoglobulins to disease pathogenesis has never been studied. Using Aicda^?/? mice and the experimental model of Leishmania donovani infection, we demonstrate that the absence of hypermutated and/or class‐switched antibodies was associated with increased resistance to disease, stronger protective Th1 responses, and a lower frequency of regulatory IFNγ⁺IL‐10⁺ CD4 T cells. Interestingly, stronger Th1 responses and the absence of IFNγ⁺IL‐10⁺ CD4 T cells during chronic infection in infected Aicda^?/? mice were not caused by a T‐cell intrinsic effect of AID, but by changes in the cytokine environment during chronic disease. Indeed TNF, IL‐10 and IFN‐ß expressions were only upregulated in the presence of hypermutated, class‐switched antibodies and hypergammaglobulinemia at later stages of infection. Taken together, our results suggest that hypergammaglobulinemia sustains inhibitory responses during chronic visceral leishmaniasis.     

Immunoactivation induced by chronic viral infection inhibits viral replication and drives immunosuppression through sustained IFN‐I responses

Acute or chronic viral infections can lead to generalized immunosuppression. Several mechanisms, such as immunopathology of CD8⁺ T cells, inhibitory receptors, or regulatory T (Treg) cells, contribute to immune dysfunction. Moreover, patients with chronic viral infections usually do not respond to vaccination, a finding that has not been previously explained. Recently, we reported that CD169⁺ macrophages enforce viral replication, which is essential for guaranteeing antigen synthesis and efficient adaptive immune responses. In the present study, we used a chronic lymphocytic choriomeningitis virus infection mouse model to determine whether this mechanism is affected by chronic viral infection, which may impair the activation of adaptive immunity. We found that enforced viral replication of a superinfecting virus is completely blunted in chronically infected mice. This absence of enforced viral replication in CD169⁺ macrophages is not explained by CD8⁺ T‐cell‐mediated immunopathology but rather by prolonged IFN‐I responses. Consequently, the absence of viral replication impairs both antigen production and the adaptive immune response against the superinfecting virus. These findings indicate that chronic infection leads to sustained IFN‐I action, which is responsible for the absence of an antiviral immune response against a secondary viral infection.     

Signal 3 requirement for memory CD8+ T-cell activation is determined by the infectious pathogen

The relevance of direct inflammatory signals (signal 3) for the activation of memory CD8⁺ T cells during recall responses is so far unknown. We therefore investigated the direct impact of IL‐12 and type I IFN on the formation, recall potential and protective capacity of memory T cells. Using CD8⁺ T cells deficient for IL‐12 or type I IFN receptors in an adoptive transfer system, we generated memory populations after infection with vaccinia virus, lymphocytic choriomeningitis virus or Listeria monocytogenes. The results demonstrate that in the absence of signal 3 cytokines during primary infection, functional memory T cells were formed. After retransfer into naïve mice, signal 3‐deficient memory T cells were able to specifically lyse target cells in vivo under non‐infectious conditions. However, after reinfection, secondary effector CD8⁺ T cells lacking signal 3 were impaired in expansion and protective capacity dependent on the nature of the pathogen. We conclude that memory CD8⁺ T cells depend on a signal 3 for expansion, independent of signals obtained during priming, thereby being influenced by the pathogen‐induced inflammatory milieu during secondary infection. In summary, our results reveal an essential role for direct inflammatory cytokine signaling in secondary T‐cell responses.