Program death-1 regulates peripheral T cell tolerance via an anergy-independent mechanism

Program death-1 (PD-1) has been documented to negatively regulate immune responses. However, the cellular and molecular mechanisms for PD-1-mediated immune suppression have not been fully elucidated. In this study, we show that loss of PD-1 does not lead to defective induction of CD4(+) T cell anergy in vitro and in vivo. Rather, the absence of PD-1 inhibits the development of inducible CD4(+)Foxp3(+) regulatory T cells (iTregs) induced by TGF-β in vitro. In support of this finding, PD-1 deficiency impairs the generation of iTregs in vivo and leads to development of severe T cell-transfer-induced colitis. Mechanistically, defective iTreg generation in the absence of PD-1 was attributed to the heightened phosphorylation of Akt. Therefore, we first demonstrate that PD-1 controls peripheral T cell tolerance via an anergy-independent but iTreg-dependent mechanism.     

Identification of a CD4+ T cell line with Treg-like activity

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4⁺ CD25^high Foxp3⁺ Tregs and demonstrate they suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3⁺ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4⁺ PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4⁺ PBMC proliferation by MoT cells and Tregs, suggesting cell–cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity. We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner, suggesting that a Foxp3⁺ Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.     

Granulocytic myeloid derived suppressor cells expand in human pregnancy and modulate T‐cell responses

Immune tolerance toward the semiallogeneic fetus plays a crucial role in the maintenance of pregnancy. Myeloid‐derived suppressor cells (MDSCs) are innate immune cells characterized by their ability to modulate T‐cell responses. Recently, we showed that MDSCs accumulate in cord blood of healthy newborns, yet their role in materno–fetal tolerance remained elusive. In the present study, we demonstrate that MDSCs with a granulocytic phenotype (GR‐MDSCs) are highly increased in the peripheral blood of healthy pregnant women during all stages of pregnancy compared with nonpregnant controls, whereas numbers of monocytic MDSCs were unchanged. GR‐MDSCs expressed the effector enzymes arginase‐I and iNOS, produced high amounts of ROS and efficiently suppressed T‐cell proliferation. After parturition, GR‐MDSCs decreased within a few days. In combination, our results show that GR‐MDSCs expand in normal human pregnancy and may indicate a role for MDSCs in materno–fetal tolerance.     

Distinct roles of CTLA-4 and TGF-beta in CD4+CD25+ regulatory T cell function

Both CTLA-4 and TGF-beta have been implicated in suppression by CD4+CD25+ regulatory T cells (Treg). In this study, the relationship between CTLA-4 and TGF-beta in Treg function was examined. Blocking CTLA-4 on wild-type Treg abrogated their suppressive activity in vitro, whereas neutralizing TGF-beta had no effect, supporting a TGF-beta-independent role for CTLA-4 in Treg-mediated suppression in vitro. In CTLA-4-deficient mice, Treg development and homeostasis was normal. Moreover, Treg from CTLA-4-deficient mice exhibited uncompromised suppressive activity in vitro. These CTLA-4-deficient Treg expressed increased levels of the suppressive cytokines IL-10 and TGF-beta, and in vitro suppression mediated by CTLA-4(-/-) Treg was markedly reduced by neutralizing TGF-beta, suggesting that CTLA-4-deficient Treg develop a compensatory suppressive mechanism through CTLA-4-independent production of TGF-beta. Together, these data suggest that CTLA-4 regulates Treg function by two distinct mechanisms, one during functional development of Treg and the other during the effector phase, when the CTLA-4 signaling pathway is required for suppression. These results help explain contradictions in the literature and support the existence of functionally distinct Treg.     

c‐Rel but not NF‐κB1 is important for T regulatory cell development

Regulatory T (Treg) cells are crucial for maintaining peripheral tolerance and controlling T‐cell responses. The generation of Treg in the thymus requires TCR triggering and CD28 costimulation. Engagement of these receptors induces a number of signalling pathways, including the activation of NF‐κB via PKCθ and the Bcl‐10/CARMA1/MALT complex. Previous studies have shown that PKCθ, Bcl‐10 and CARMA1 are important for Treg development. It is unclear, however, whether different members of the NF‐κB family contribute to Treg development or homeostasis. In this study, we show that Treg numbers are reduced in the absence of c‐Rel but not NF‐κB1 (p50). Furthermore, using mixed bone marrow chimeras from WT and KO animals, we demonstrate that the requirement for PKCθ, Bcl‐10 and c‐Rel is T‐cell intrinsic, and cannot be rescued by the presence of WT cells. Therefore, c‐Rel and NF‐κB1 have differential roles in Treg development.     

Uptake of membrane molecules from T cells endows antigen-presenting cells with novel functional properties

Although intercellular transfer of cell surface molecules has been observed between several cells of the immune system, the physiological relevance of this phenomenon remained obscure. Until now the transfer of molecules between antigen-presenting cells (APC) and T cells has been described as a unidirectional process from APC to T cells. However, here we show that T cells in turn donate molecules to APC, and that T cell-derived vesicles can mediate this transfer. The transferred proteins are incorporated into the APC as active molecules. Our data provide evidence that T cells use intercellular molecule transfer to mediate cell contact-dependent regulation of T cell responses via modulation of the APC.     

Induced regulatory T cells: mechanisms of conversion and suppressive potential

Thymus-derived, naturally occurring CD4(+) Forkhead Box P3(+) regulatory T cells (nTreg) have suppressive activity that is important for the establishment and maintenance of immune homeostasis in the healthy state. Abundant reports have demonstrated that they can suppress pathogenic processes in autoimmune diseases and inhibit transplant rejection and graft-versus-host disease. Far less is known about induced regulatory T cells (iTreg) that are generated from naive T cells in the periphery or in vitro by directing naive T cells to acquire suppressive function under the influence of transforming growth factor-β and other factors. In this review, we describe mechanisms by which naive T cells are thought to be converted into iTreg. We also discuss the suppressive potential of iTreg, particularly in comparison with their naturally occurring counterparts, focusing on those reports in which direct comparisons have been made. Based on current knowledge, we consider the rationale for using iTreg versus nTreg in clinical trials.     

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.     

Autoreactive human T cell lines recognizing ribosomal protein L7

Sera of patients suffering from systemic lupus erythematosus (SLE) frequently contain oligoclonal IgG autoantibodies with high affinity for the ribosomal protein L7 (rpL7). The humoral autoimmune response to rpL7 apparently is driven by antigen and T cell dependent. In order to analyze the T cell response to rpL7 we cultured peripheral blood lymphocytes of healthy individuals and SLE patients in the presence of recombinant rpL7. After 10 days, the cytokine response to re-stimulation with rpL7 was examined using a spot-ELISA. Measuring IFN-gamma secretion, the T cells of two patients and four healthy donors showed a significant increase in the number of spots as compared to control cells. Secretion of IL-4 or IL-10 was not detected. From the antigen-stimulated primary cultures we established by limiting dilution cloning six rpL7-reactive, IFN-gamma-secreting T cell lines which show a CD3+CD4+CD8- phenotype. One line additionally was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. The cell lines carry alphabeta TCR chains which differ from each other in sequence and specificity. rpL7 fragments rich in basic amino acids could be identified as epitopes recognized by the TCR of three cell lines. Recognition of rpL7 is HLA-DR6 restricted or respectively HLA-DP restricted in the two cell lines analyzed.     

Ganglioside-exposed dendritic cells inhibit T-cell effector function by promoting regulatory cell activity

Tumour pathogenesis is characterized by an immunosuppressive microenvironment that limits the development of effective tumour‐specific immune responses. This is in part the result of tumour‐dependent recruitment and activation of regulatory cells, such as myeloid‐derived suppressor cells and regulatory T cells in the tumour microenvironment and draining lymph nodes. Shedding of gangliosides by tumour cells has immunomodulatory properties, suggesting that gangliosides may be a critical factor in initiating an immunosuppressive microenvironment. To better define the immunomodulatory properties of gangliosides on antigen‐specific T‐cell activation and development we have developed an in vitro system using ganglioside‐treated murine bone‐marrow‐derived dendritic cells to prime and activate antigen‐specific CD4⁺ T cells from AND T‐cell receptor transgenic mice. Using this system, ganglioside treatment promotes the development of a dendritic cell population characterized by decreased CD86 (B7‐2) expression, and decreased interleukin‐12 and interleukin‐6 production. When these cells are used as antigen‐presenting cells, CD4 T cells are primed to proliferate normally, but have a defect in T helper (Th) effector cell development. This defect in Th effector cell responses is associated with the development of regulatory T‐cell activity that can suppress the activation of previously primed Th effector cells in a contact‐dependent manner. In total, these data suggest that ganglioside‐exposed dendritic cells promote regulatory T‐cell activity that may have long‐lasting effects on the development of tumour‐specific immune responses.     

Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

CD4(+) CD25(+) regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4(+) CD25(+) regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4(+) CD25(+) regulatory T cells anergize CD4(+) T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4(+) CD25(+) T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4(+) and CD8(+) T cells. This suppression only occurs when CD4(+) CD25(+) T cells are preactivated. Furthermore, we could demonstrate that CD4(+) T-cell clones stop secreting interferon-gamma (IFN-gamma), start to produce interleukin-10 and transforming growth factor-beta after coculture with preactivated CD4(+) CD25(+) T cells and become suppressive themselves. Surprisingly preactivated CD4(+) CD25(+) T cells affect CD8(+) T cells differently, leading to reduced proliferation and reduced production of IFN-gamma. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8(+) T cells, unlike CD4(+) T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4(+) CD25(+) T cells.     

Impaired regulatory T cell function in germ-free mice

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto-antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4+CD25+ T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ-free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4+CD25+ T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4+CD25+ T cells from conv animals. Intracellular staining for Foxp3 and CTLA-4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4+CD25+ T cells in GF MLN expressed Foxp3 and CTLA-4, while the expression of these markers was similar amongst the CD4+CD25+ T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population.     

Circumvention of regulatory CD4(+) T cell activity during cross-priming strongly enhances T cell-mediated immunity

Immunization with purified antigens is a safe and practical vaccination strategy but is generally unable to induce sustained CD8(+) T cell-mediated protection against intracellular pathogens. Most efforts to improve the CD8(+) T cell immunogenicity of these vaccines have focused on co-administration of adjuvant to support cross-presentation and dendritic cell maturation. In addition, it has been shown that CD4(+) T cell help during the priming phase contributes to the generation of protective CD8(+) memory T cells. In this report we demonstrate that the depletion of CD4(+) T cells paradoxically enhances long-lasting CD8-mediated protective immunity upon protein vaccination. Functional and genetic in vivo inactivation experiments attribute this enhancement primarily to MHC class II-restricted CD4(+) regulatory T cells (Treg), which appear to physiologically suppress the differentiation process towards long-living effector memory T cells. Since, in functional terms, this suppression by Treg largely exceeds the positive effects of conventional CD4(+) T cell help, even the absence of all CD4(+) T cells or lack of MHC class II-mediated interactions on priming dendritic cells result in enhanced CD8(+) T cell immunogenicity. These findings have important implications for the improvement of vaccines against intracellular pathogens or tumors, especially in patients with highly active Treg.     

Induction of peripheral CD4+ T‐cell tolerance and CD8+ T‐cell cross‐tolerance by dendritic cells

DC can present and cross‐present self‐antigens to autoreactive CD4⁺ and CD8⁺ T cells, respectively, and incapacitate them by inducing anergy, deletion or converting them into Treg. In this review, we summarize the recent progress in immune tolerance research, which has been achieved by employing antigen‐ and TCR‐transgenic mice. We cover the numerous discoveries that have furthered our knowledge of the DC subsets and maturation pathways involved in tolerance; the signals, such as CD70, TGF‐β, B7‐H1/PD‐L1, which dictate the decision between immunity and tolerance; and the in vivo role of DC in the maintenance of CD4⁺ T‐cell tolerance and CD8⁺ T‐cell cross‐tolerance.     

Complement and human T cell metabolism: Location,location, location

The complement system represents one of the evolutionary oldest arms of our immune system and is commonly recognized as a liver-derived and serum-active system critical for providing protection against invading pathogens. Recent unexpected findings, however, have defined novel and rather “uncommon” locations and activities of complement. Specifically, the discovery of an intracellularly active complement system—the complosome—and its key role in the regulation of cell metabolic pathways that underly normal human T cell responses have taught us that there is still much to be discovered about this system. Here, we summarize the current knowledge about the emerging functions of the complosome in T cell metabolism. We further place complosome activities among the non-canonical roles of other intracellular innate danger sensing systems and argue that a “location-centric” view of complement evolution could logically justify its close connection with the regulation of basic cell physiology.