Expanded Regulatory T Cells in Chronically Friend Retrovirus-Infected Mice Suppress Immunity to a Murine Cytomegalovirus Superinfection

It is still unclear whether expanded and activated regulatory T cells (Tregs) in chronic viral infections can influence primary immune responses against superinfections with unrelated viruses. Expanded Tregs found in the spleens of chronically Friend virus (FV)-infected mice decreased murine cytomegalovirus (mCMV)-specific CD8⁺ T cell responses during acute mCMV superinfection. This suppression of mCMV-specific T cell immunity was found only in organs with FV-induced Treg expansion. Surprisingly, acute mCMV infection itself did not expand or activate Tregs.     

B-Cell Control of Regulatory T Cells in Friend Virus Infection

B lymphocytes have well-established effector roles during viral infections, including production of antibodies and functioning as antigen-presenting cells for CD4 + and CD8 + T cells. B cells have also been shown to regulate immune responses and induce regulatory T cells (Tregs). In the Friend virus (FV) model, Tregs are known to inhibit effector CD8 + T-cell responses and contribute to virus persistence. Recent work has uncovered a role for B cells in the induction and activation of Tregs during FV infection. In addition to inducing Tregs, B cell antibody production and antigen-presenting cell activity is a target of Treg suppression. This review focuses on the dynamic interactions between B cells and Tregs during FV infection.     

Suppression of HIV-specific and allogeneic T cell activation by human regulatory T cells is dependent on the strength of signals

Regulatory T cells (Tregs) suppress immune responses against both self and non-self antigens. Tregs require activation through the T cell receptor (TCR) and IL-2 to exert their suppressive functions. However, how strength of TCR signals modulate the potency of Treg-mediated suppression of antigen-specific T cell activation remain unclear. We found that both strength of TCR signals and ratios of Tregs to target cells, either through superantigen, allogeneic antigens or HIV-specific peptides, modified the suppressive ability of Tregs. While human Tregs were able to mediate suppression in the presence of only autologous antigen-presenting cells, this was much less efficient as compared to when Tregs were activated by allogeneic dendritic cells. In another physiologically relevant system, we show that the strength of peptide stimulation, high frequency of responder CD8+ T cells or presence of high IL-2 can override the suppression of HIV-specific CD8+ T cells by Tregs. These findings suggest that ratios and TCR activation of human Tregs, are important parameters to overcome robust immune responses to pathogens or allogeneic antigens. Modulating the strength of T cell signals and selective enhancement or depletion of antigen-specific Tregs thus may have implications for designing potent vaccines and regulating immune responses during allogeneic transplantation and chronic infections.     

B7-deficient autoreactive T cells are highly susceptible to suppression by CD4(+)CD25(+) regulatory T cells

CD4(+)CD25(+) regulatory T cells (Tregs) suppress immunity to infections and tumors as well as autoimmunity and graft-vs-host disease. Since Tregs constitutively express CTLA-4 and activated T cells express B7-1 and B7-2, it has been suggested that the interaction between CTLA-4 on Tregs and B7-1/2 on the effector T cells may be required for immune suppression. In this study, we report that autopathogenic T cells from B7-deficient mice cause multiorgan inflammation when adoptively transferred into syngeneic RAG-1-deficient hosts. More importantly, this inflammation is suppressed by adoptive transfer of purified wild-type (WT) CD4(+)CD25(+) T cells. WT Tregs also inhibited lymphoproliferation and acquisition of activation markers by the B7-deficient T cells. An in vitro suppressor assay revealed that WT and B7-deficient T cells are equally susceptible to WT Treg regulation. These results demonstrate that B7-deficient T cells are highly susceptible to immune suppression by WT Tregs and refute the hypothesis that B7-CTLA-4 interaction between effector T cells and Tregs plays an essential role in Treg function.     

Essential Roles for CD8+ T Cells and Gamma Interferon in Protection of Mice against Retrovirus-Induced Immunosuppression

It is known that both animal and human retroviruses typically cause immunosuppression in their respective hosts, but the mechanisms by which this occurs are poorly understood. The present study uses Friend virus (FV) infections of mice as a model to determine how major histocompatibility complex (MHC) genes influence immunosuppression. Previously, MHC-I genes were shown to influence antibody responses to potent antigenic challenges given during acute FV infection. The mapping of an immune response to an MHC-I gene implicated CD8+ T cells in the mechanism, so we directly tested for their role by using in vivo CD8+ T-cell depletions. Mice resistant to FV-induced immunosuppression became susceptible when they were depleted of CD8+ T cells. Resistance also required gamma interferon (IFN-gamma), as in vivo neutralization of IFN-gamma converted mice from a resistant to susceptible phenotype. On the other hand, susceptibility to FV-induced immunosuppression was dependent on the immunosuppressive cytokine, interleukin-10 (IL-10), as antibody responses were restored in susceptible mice when IL-10 function was blocked in vivo. Thus, FV-induced immunosuppression of antibody responses involves complex mechanisms controlled at least in part by CD8+ T cells.     

Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients

CD4+CD25+FOXP3+ T regulatory cells (Tregs) are pivotal for the induction and maintenance of peripheral tolerance in both mice and humans. Rapamycin has been shown to promote tolerance in experimental models and to favor CD4+CD25+ Treg-dependent suppression. We recently reported that rapamycin allows in vitro expansion of murine CD4+CD25+FoxP3+ Tregs, which preserve their suppressive function. In the current study, we show that activation of human CD4+ T cells from healthy subjects in the presence of rapamycin leads to growth of CD4+CD25+FOXP3+ Tregs and to selective depletion of CD4+CD25- T effector cells, which are highly sensitive to the antiproliferative effect of the compound. The rapamycin-expanded Tregs suppress proliferation of both syngeneic and allogeneic CD4+ and CD8+ T cells. Interestingly, rapamycin promotes expansion of functional CD4+CD25+FOXP3+ Tregs also in type 1 diabetic patients, in whom a defect in freshly isolated CD4+CD25+ Tregs has been reported. The capacity of rapamycin to allow growth of functional CD4+CD25+FOXP3+ Tregs, but also to deplete T effector cells, can be exploited for the design of novel and safe in vitro protocols for cellular immunotherapy in T cell-mediated diseases.     

The phenotype and activation status of regulatory T cells during Friend retrovirus infection

The suppressive capacity of regulatory T cells (Tregs) has been extensively studied and is well established for many diseases. The expansion, accumulation, and activation of Tregs in viral infections are of major interest in order to find ways to alter Treg functions for therapeutic benefit. Tregs are able to dampen effector T cell responses to viral infections and thereby contribute to the establishment of a chronic infection. In the Friend retrovirus (FV) mouse model, Tregs are known to expand in all infected organs. To better understand the characteristics of these Treg populations, their phenotype was analyzed in detail. During acute FV-infection, Tregs became activated in the spleen and bone marrow, as indicated by various T cell activation markers, such as CD43 and CD103. Interestingly, Tregs in the bone marrow, which contains the highest viral loads during acute infection, displayed greater levels of activation than Tregs from the spleen. Treg expansion was driven by proliferation but no FV-specific Tregs could be detected. Activated Tregs in FV-infection did not produce Granzyme B (GzmB) or tumor necrosis factor α (TNFα), which are thought to be a potential mechanism for their suppressive activity. Furthermore, Tregs expressed inhibitory markers, such as TIM3, PD-1 and PD-L1. Blocking TIM3 and PD-L1 with antibodies during chronic FV-infection increased the numbers of activated Tregs. These data may have important implications for the understanding of Treg functions during chronic viral infections.     

Double-negative T regulatory cells can develop outside the thymus and do not mature from CD8+ T cell precursors

Recent studies have demonstrated that activated peripheral alphabeta TCR+ CD3+ CD4- CD8- NK1.1- (double-negative, DN) regulatory T cells (Tregs) from both mice and humans are able to down-regulate immune responses in vitro and in vivo. However, the origin and developmental requirements of functional DN Tregs remain unclear. In this study, we investigated the requirement for CD8 expression as well as the presence of a thymus for the development of functional DN Tregs. We demonstrate that DN Tregs exist in CD8-deficient mice and that stimulation of CD8+ T cells in vivo with TCR-specific Ag does not convert CD8+ T cells into DN Tregs. In addition, we found that DN T cells are present in the spleens and lymph nodes of thymectomized mice that are irradiated and reconstituted with T cell-depleted bone marrow cells. Interestingly, DN Tregs that develop in thymectomized mice can suppress syngeneic CD8+ T cells more effectively than those that develop in sham-thymectomized mice. Taken together, our data suggest that DN Tregs are not derived from CD8+ T cell precursors and that functional DN Tregs may preferentially develop outside of the thymus. These data suggest that DN Tregs may represent a developmentally and functionally unique cell population.     

Probing the Effector and Suppressive Functions of Human T Cell Subsets Using Antigen-Specific Engineered T Cell Receptors

Activation of T cells through the engagement of the T cell receptors (TCRs) with specific peptide-MHC complexes on antigen presenting cells (APCs) is the major determinant for their proliferation, differentiation and display of effector functions. To assess the role of quantity and quality of peptide-MHC presentation in eliciting T cell activation and suppression functions, we genetically engineered human T cells with two TCRs that recognize HLA-A*0201-restricted peptides derived from either HIV or melanoma antigens. The engineered-TCRs are highly functional in both CD8⁺ and CD4⁺ T cells as assessed by the upregulation of activation markers, induction of cytokine secretion and cytotoxicity. We further demonstrated that engineered-TCRs can also be expressed on naïve human T cells, which are stimulated through APCs presenting specific peptides to induce T cell proliferation and acquire effector functions. Furthermore, regulatory T cells (Tregs) ectopically expressing the engineered-TCRs are activated in an antigen-specific fashion and suppress T cell proliferation. In this system, the inhibitory activity of peptide-stimulated Tregs require the presence of dendritic cells (DCs) in the culture, either as presenters or as bystander cells, pointing to a critical role for DCs in suppression by Tregs. In conclusion, the engineered-TCR system reported here advances our ability to understand the differentiation pathways of naïve T cells into antigen-specific effector cells and the role of antigen-specific signaling in Treg-mediated immune suppression.     

Combining Regulatory T Cell Depletion and Inhibitory Receptor Blockade Improves Reactivation of Exhausted Virus-Specific CD8+ T Cells and Efficiently Reduces Chronic Retroviral Loads

Chronic infections with human viruses, such as HIV and HCV, or mouse viruses, such as LCMV or Friend Virus (FV), result in functional exhaustion of CD8⁺ T cells. Two main mechanisms have been described that mediate this exhaustion: expression of inhibitory receptors on CD8⁺ T cells and expansion of regulatory T cells (Tregs) that suppress CD8⁺ T cell activity. Several studies show that blockage of one of these pathways results in reactivation of CD8⁺ T cells and partial reduction in chronic viral loads. Using blocking antibodies against PD-1 ligand and Tim-3 and transgenic mice in which Tregs can be selectively ablated, we compared these two treatment strategies and combined them for the first time in a model of chronic retrovirus infection. Blocking inhibitory receptors was more efficient than transient depletion of Tregs in reactivating exhausted CD8⁺ T cells and reducing viral set points. However, a combination therapy was superior to any single treatment and further augmented CD8⁺ T cell responses and resulted in a sustained reduction in chronic viral loads. These results demonstrate that Tregs and inhibitory receptors are non-overlapping factors in the maintenance of chronic viral infections and that immunotherapies targeting both pathways may be a promising strategy to treat chronic infectious diseases.     

Ex vivo activated OVA specific and non-specific CD4+CD25+ regulatory T cells exhibit comparable suppression to OVA mediated T cell responses

CD4+CD25+ regulatory T cells (Tr) are important in maintaining immune tolerance to self-antigen (Ag) and preventing autoimmunity. Reduced number and inadequate function of Tr are observed in chronic autoimmune diseases. Adoptively transferred Tr effectively suppress ongoing autoimmune disease in multiple animal models. Therefore, strategies to modulate Tr have become an attractive approach to control autoimmunity. Activation of Tr is necessary for their optimal immune regulatory function. However, due to the low ratio of Tr to any given antigen (Ag) and the unknown nature of Ag in many autoimmune diseases, specific activation is not practical for potential therapeutic intervention. It has been shown in animal models that once activated, Tr can exhibit immune suppression in a bystander Ag-non-specific fashion, suggesting the effector phase of Tr is Ag independent. To investigate whether the immune suppression by activated bystander Tr is as potent as that of the Ag specific Tr, Tr cells were isolated from BALB/c or ovalbumin (OVA) specific T cell receptor (TCR) transgenic mice (DO11.10) and their immune suppression of an OVA specific T cell response was compared. We found that once activated ex vivo, Tr from BALB/c and DO11.10 mice exhibited comparable inhibition on OVA specific T cell responses as determined by T cell proliferation and cytokine production. Furthermore, their immune suppression function was compared in a delayed type hypersensitivity (DTH) model induced by OVA specific T cells. Again, OVA specific and non-specific Tr exhibited similar inhibition of the DTH response. Taken together, the results indicate that ex vivo activated Ag-non-specific Tr are as efficient as Ag specific Tr in immune suppression, therefore our study provides additional evidence suggesting the possibility of applying ex vivo activated Tr therapy for the control of autoimmunity.     

Influenza A virus infection results in a robust, antigen-responsive, and widely disseminated Foxp3+ regulatory T cell response

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.     

Antigen, in the presence of TGF-beta, induces up-regulation of FoxP3gfp+ in CD4+ TCR transgenic T cells that mediate linked suppression of CD8+ T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) inhibit immune responses to a variety of Ags, but their specificity and mechanism of suppression are controversial. This controversy is largely because many studies focused on natural Tregs with undefined specificities and suppression has frequently been measured on polyclonal T cell responses. To address the issue of specificity further, we have bred K(d)-specific, CD4(+) TCR (TCR75) transgenic mice to Foxp3(gfp) knockin reporter mice to permit sorting of Tregs with a known specificity. Foxp3(gfp).TCR75 mice did not express significant numbers of natural FoxP3(+) Tregs expressing the TCR75 transgenes, but FoxP3 expression was induced by stimulating with K(d) plus TGF-beta. The resulting GFP(+) TCR75 cells were anergic, whereas the GFP(-) TCR75 cells proliferated upon restimulation with K(d) peptide. Yet both exhibited severely reduced expression of intracellular IFN-gamma and TNF-alpha upon restimulation. GFP(+), but not GFP(-), TCR75 T cells suppressed responses by naive TCR75 T cells and by nontransgenic spleen cells stimulated with anti-CD3. GFP(+) TCR75 cells also inhibited polyclonal C57BL/6 anti-K(d) CTL responses if the APC expressed K(d) and both MHC class I and class II, and responses by OT1 T cells to B6.K(d).OVA but not B6.K(d) plus OVA expressing APC, demonstrating linked-suppression of CD8 responses. Thus, Tregs exhibit a greater degree of specificity in vitro than previously appreciated. The observation that Tregs and responder T cells must recognize the same APC provides a mechanistic explanation for the observation that Tregs must be in direct contact with effector T cells to suppress their responses.     

Regulation of granulomatous inflammation in murine schistosomiasis. IV. Antigen-induced suppressor T cells down-regulate proliferation and IL-2 production

In murine schistosomiasis mansoni the cell-mediated immune response to the deposited eggs is mediated by CD4+ delayed-type hypersensitivity effector T (TDH) cells that produce vigorous granulomatous responses in the liver and intestines of acutely infected animals. The response is significantly down-modulated in chronically infected mice by Ag-specific Ts cells. The present study was undertaken to establish an in vitro model by which TDH-Ts cell interactions could be analyzed. To this end, Ts cells were induced in vitro by preculture of chronic or acute infection spleen cells with soluble egg Ag (SEA) for 48 h. The induced cells suppressed the SEA-specific proliferation of acute infection spleen cells by 80 to 95%. The induced suppressor cells were Ag specific in both induction and elicitation of function, and were not cytotoxic to the acute infection splenic target cells. Suppression by the induced cells was manifested within the first 24 h of the SEA-induced response as IL-2 produced by acute infection spleen cells was suppressed 62%. Phenotypic analysis by flow cytometry of the induced suppressor cells showed that CD8+ cells from acute infection spleens and CD4+ and CD8+ cells from chronic infection spleens were effector Ts cells. Taken together, CD4+ and CD8+ SEA-specific Ts cells can be induced in vitro to effectively suppress the SEA-specific lymphoproliferation and IL-2 production of acute infection spleen cells. Establishment of this in vitro model will allow us to further analyze the mechanisms of Ts cell-mediated suppression of TDH cells.     

Characterization of the Treg Response in the Hepatitis B Virus Hydrodynamic Injection Mouse Model

Regulatory T cells (Tregs) play an important role in counter-regulating effector T cell responses in many infectious diseases. However, they can also contribute to the development of T cell dysfunction and pathogen persistence in chronic infections. Tregs have been reported to suppress virus-specific T cell responses in hepatitis B virus (HBV) infection of human patients as well as in HBV animal models. However, the phenotype and expansion of Tregs has so far only been investigated in other infections, but not in HBV. We therefore performed hydrodynamic injections of HBV plasmids into mice and analyzed the Treg response in the spleen and liver. Absolute Treg numbers significantly increased in the liver but not the spleen after HBV injection. The cells were natural Tregs that surprisingly did not show any activation or proliferation in response to the infection. However, they were able to suppress effector T cell responses, as selective depletion of Tregs significantly increased HBV-specific CD8+ T cell responses and accelerated viral antigen clearance. The data implies that natural Tregs infiltrate the liver in HBV infection without further activation or expansion but are still able to interfere with T cell mediated viral clearance.     

Protection from type 1 diabetes by invariant NK T cells requires the activity of CD4+CD25+ regulatory T cells

Invariant NK T (iNKT) cells regulate immune responses, express NK cell markers and an invariant TCR, and recognize lipid Ags in a CD1d-restricted manner. Previously, we reported that activation of iNKT cells by alpha-galactosylceramide (alpha-GalCer) protects against type 1 diabetes (T1D) in NOD mice via an IL-4-dependent mechanism. To further investigate how iNKT cells protect from T1D, we analyzed whether iNKT cells require the presence of another subset(s) of regulatory T cells (Treg), such as CD4+ CD25+ Treg, for this protection. We found that CD4+ CD25+ T cells from NOD.CD1d(-/-) mice deficient in iNKT cell function similarly in vitro to CD4+ CD25+ T cells from wild-type NOD mice and suppress the proliferation of NOD T responder cells upon alpha-GalCer stimulation. Cotransfer of NOD diabetogenic T cells with CD4+ CD25+ Tregs from NOD mice pretreated with alpha-GalCer demonstrated that activated iNKT cells do not influence the ability of T(regs) to inhibit the transfer of T1D. In contrast, protection from T1D mediated by transfer of activated iNKT cells requires the activity of CD4+ CD25+ T cells, because splenocytes pretreated with alpha-GalCer and then inactivated by anti-CD25 of CD25+ cells did not protect from T1D. Similarly, mice inactivated of CD4+ CD25+ T cells before alpha-GalCer treatment were also not protected from T1D. Our data suggest that CD4+ CD25+ T cells retain their function during iNKT cell activation, and that the activity of CD4+ CD25+ Tregs is required for iNKT cells to transfer protection from T1D.     

CD8+ T cell responses in bronchoalveolar lavage fluid and peripheral blood mononuclear cells of infants with severe primary respiratory syncytial virus infections

A protective role for CD8+ T cells during viral infections is generally accepted, but little is known about how CD8+ T cell responses develop during primary infections in infants, their efficacy, and how memory is established after viral clearance. We studied CD8+ T cell responses in bronchoalveolar lavage (BAL) samples and blood of infants with a severe primary respiratory syncytial virus (RSV) infection. RSV-specific CD8+ T cells with an activated effector cell phenotype: CD27+CD28+CD45RO+CCR7-CD38+HLA-DR+Granzyme B+CD127- could be identified in BAL and blood. A high proportion of these CD8+ T cells proliferated and functionally responded upon in vitro stimulation with RSV Ag. Thus, despite the very young age of the patients, a robust systemic virus-specific CD8+ T cell response was elicited against a localized respiratory infection. RSV-specific T cell numbers as well as the total number of activated effector type CD8+ T cells peaked in blood around day 9-12 after the onset of primary symptoms, i.e., at the time of recovery. The lack of a correlation between RSV-specific T cell numbers and parameters of disease severity make a prominent role in immune pathology unlikely, in contrast the T cells might be involved in the recovery process.     

Enhanced cytotoxic T cell activity in IL-4-deficient mice

CD8+ effectors are critical components of type 1 responses against viral infections as well as for antiviral vaccines. IL-4 plays a clear role as an inhibitor of CD4+ Th1 cells; however, its role in CD8+ T cell regulation appears to be more complex. Thus, IL-4 may augment CD8+ T cell growth, but also limit effector function. Moreover, abundant IL-4 is inhibitory for viral clearance, but the lack of IL-4 appears not to affect CTL-mediated immunity. This report investigates these disparate roles of IL-4 in CD8+ T lymphocyte regulation by comparing T cell responses specific for a single HIV-IIIIB gp120-derived epitope in BALB/c mice deficient in IL-4 to those in wild-type controls. CTL activation was monitored during the acute and memory phases following immunization with recombinant vaccinia virus. Similar frequencies of gp120-specific CTL precursors in splenocytes from both groups indicated that IL-4 plays no significant role in either CTL priming or the establishment of memory. However, cytolytic activity in cultures derived from IL-4-deficient mice developed earlier and was strikingly enhanced following in vitro restimulation, an effect exhibited by both primary and memory T cells. Secretion of IL-2 and IFN-gamma by CD8+ T cells from IL-4-deficient mice was also elevated, reflecting their enhanced activation. Thus, IL-4 appears to limit the activation, expansion, and differentiation of CD8+ T cells with high cytolytic potential.