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Thymic selection depends on positive and negative selective mechanisms based on the avidity of T cell interaction with antigen-major histocompatibility complex complexes. However, peripheral mechanisms for the recruitment and clonal expansion of the responding T cell repertoire remain obscure. Here we provide evidence for an avidity-based model of peripheral T cell clonal expansion in response to antigenic challenge. We have used the encephalitogenic, H-2 A(u)-restricted, acetylated NH(2)-terminal nonameric peptide (Ac1-9) epitope from myelin basic protein as our model antigen. Peptide analogues were generated that varied in antigenic strength (as assessed by in vitro assay) based on differences in their binding affinity for A(u). In vivo, these analogues elicited distinct repertoires of T cells that displayed marked differences in antigen sensitivity. Immunization with the weakest (wild-type) antigen expanded the high affinity T cells required to induce encephalomyelitis. In contrast, immunization with strongly antigenic analogues led to the elimination of T cells bearing high affinity T cell receptors by apoptosis, thereby preventing disease development. Moreover, the T cell repertoire was consistently tuned to respond to the immunizing antigen with the same activation threshold. This tuning mechanism provides a peripheral control against the expansion of autoreactive T cells and has implications for immunotherapy and vaccine design.

• The immune cell repertoire is controlled through T cell receptor and costimulatory molecule dependant selection of CD4+ and Foxp3+ T cells in the thymus. • The immune response to antigen is fine-tuned through activation induced cell death. • Chronic exposure to antigen leads to desensitization of immune responses through anergy and the generation of regulatory type 1 responses. • Differentiation of anergic Tr1 cells arises from radically reduced cell signalling and epigenetic priming of tolerance associated genes. • Autoimmune responses are successfully controlled by chronic exposure to antigen processing independent T cell epitopes. Lymphocyte receptors confer antigen specificity on the adaptive immune response. Increasing evidence points to the role of adaptive tuning particularly amongst CD4+ T cell responses. This review summarises how T cell tuning impacts on critically important aspects of immune regulation including thymic selection, the immune response to chronic antigen exposure and antigen-specific immunotherapy of autoimmune conditions. Recent work has revealed a novel mechanism for T cell anergy and regulatory type 1 T cell differentiation through a limitation of T cell receptor mediated signalling combined with epigenetic priming of tolerance associated genes. [ABSTRACT FROM AUTHOR]

The immune response to exogenous proteins can overcome the therapeutic benefits of immunotherapies and hamper the treatment of protein replacement therapies. One clear example of this is haemophilia A resulting from deleterious mutations in the FVIII gene. Replacement with serum derived or recombinant FVIII protein can cause anti-drug antibodies in 20-50% of individuals treated. The resulting inhibitor antibodies override the benefit of treatment and, at best, make life unpredictable for those treated. The only way to overcome the inhibitor issue is to reinstate immunological tolerance to the administered protein. Here we compare the various approaches that have been tested and focus on the use of antigen-processing independent T cell epitopes (apitopes) for tolerance induction. Apitopes are readily designed from any protein whether this is derived from a clotting factor, enzyme replacement therapy, gene therapy or therapeutic antibody. [ABSTRACT FROM AUTHOR]

Allergen‐specific immunotherapy (AIT) is the only means of altering the natural immunological course of allergic diseases and achieving long‐term remission. Pharmacological measures are able to suppress the immune response and/or ameliorate the symptoms but there is a risk of relapse soon after these measures are withdrawn. Current AIT approaches depend on the administration of intact allergens, often comprising crude extracts of the allergen. We propose that the challenges arising from current approaches, including the risk of serious side‐effects, burdensome duration of treatment, poor compliance and high cost, are overcome by application of peptides based on CD4+ T cell epitopes rather than whole allergens. Here we describe evolving approaches, summarize clinical trials involving peptide AIT in allergic rhinitis and asthma, discuss the putative mechanisms involved in their action, address gaps in evidence and propose future directions for research and clinical development. [ABSTRACT FROM AUTHOR]

Immunotherapy with antigen-processing independent T cell epitopes (apitopes) targeting autoreactive CD4+ T cells has translated to the clinic and been shown to modulate progression of both Graves' disease and multiple sclerosis. The model apitope (Ac1-9[4Y]) renders antigen-specific T cells anergic while repeated administration induces both Tr1 and Foxp3+ regulatory cells. Here we address why CD4+ T cell epitopes should be designed as apitopes to induce tolerance and define the antigen presenting cells that they target in vivo. Furthermore, we reveal the impact of treatment with apitopes on CD4+ T cell signaling, the generation of IL-10-secreting regulatory cells and the systemic migration of these cells. Taken together these findings reveal how apitopes induce tolerance and thereby mediate antigen-specific immunotherapy of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Secretion of interleukin-10 (IL-10) by CD4+ T cells is an essential immunoregulatory mechanism. The work presented here assesses the role of the signaling molecule protein kinase C theta (PKCθ) in the induction of IL-10 expression in CD4+ T cells. Using wildtype and PKCθ-deficient Tg4 T cell receptor transgenic mice, we implemented a well-described protocol of repeated doses of myelin basic protein (MBP)Ac1-9[4Y] antigen to induce Tr1-like IL-10+ T cells. We find that PKCθ is required for the efficient induction of IL-10 following antigen administration. Both serum concentrations of IL-10 and the proportion of IL-10+ T cells were reduced in PKCθ-deficient mice relative to wildtype mice following [4Y] treatment. We further characterized the T cells of [4Y] treated PKCθ-deficient Tg4 mice and found reduced expression of the transcription factors cMaf, Nfil3 and FoxP3 and the surface receptors PD-1 and Tim3, all of which have been associated with the differentiation or function of IL-10+ T cells. Finally, we demonstrated that, unlike [4Y] treated wildtype Tg4 T cells, cells from PKCθ-deficient mice were unable to suppress the priming of naïve T cells in vitro and in vivo. In summary, we present data demonstrating a role for PKCθ in the induction of suppressive, IL-10-secreting T cells induced in TCR-transgenic mice following chronic antigen administration. This should be considered when contemplating PKCθ as a suitable drug target for inducing immune suppression and graft tolerance. [ABSTRACT FROM AUTHOR]

The immune system evolved to respond to foreign invaders and prevent autoimmunity to self-antigens. Several types of regulatory T cells facilitate the latter process. These include a subset of Foxp3− CD4+ T cells able to secrete IL-10 in an antigen-specific manner, type 1 regulatory (Tr1) T cells. Although their suppressive function has been confirmed both in vitro and in vivo, their phenotype remains poorly defined. It has been suggested that the surface markers LAG-3 and CD49b are biomarkers for murine and human Tr1 cells. Here, we discuss these findings in the context of our data regarding the expression pattern of inhibitory receptors (IRs) CD49b, TIM-3, PD-1, TIGIT, LAG-3, and ICOS on Tr1-like human T cells generated in vitro from CD4+ memory T cells stimulated with αCD3 and αCD28 antibodies. We found that there were no differences in IR expression between IL-10+ and IL-10− T cells. However, CD4+IL-10+ T cells isolated ex vivo, following a short stimulation and cytokine secretion assay, contained significantly higher proportions of TIM-3+ and PD-1+ cells. They also expressed significantly higher TIGIT mRNA and showed a trend toward increased TIM-3 mRNA levels. These data led us to conclude that large pools of IRs may be stored intracellularly; hence, they may not represent ideal candidates as cell surface biomarkers for Tr1-like T cells. [ABSTRACT FROM AUTHOR]

Adoptive transfer of antigen-specific, in vitro-induced Foxp3 + Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3 + iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3 + iTreg cells. This increased efficacy not only boosts the yield of Foxp3 + iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy. [ABSTRACT FROM AUTHOR]

In vitro induced Foxp3+ T regulatory (iTreg) cells form a novel and promising target for therapeutic tolerance induction. However, the potential of these cells as a target for the treatment of various immune diseases, as well as the factors involved in their development and function, remain debated. Here, we demonstrate in a myelin basic protein (MBP)-specific murine model of CNS autoimmune disease that adoptive transfer of antigen-specific iTreg cells ameliorates disease progression. Moreover, we show that the co-stimulatory molecule CTLA-4 mediates in vitro differentiation of iTreg cells. Finally, we demonstrate that the secreted, immunosuppressive cytokine IL-10 controls the ability of antigen-specific iTreg cells to suppress autoimmune disease. Overall, we conclude that antigen-specific iTreg cells, which depend on various immune regulatory molecules for their differentiation and function, represent a major target for effective immunotherapy of autoimmune disease. [ABSTRACT FROM AUTHOR]

T regulatory (Treg) cells expressing the transcription factor FoxP3 play a key role in protection against autoimmune disease. GFP-FoxP3 reporter mice have been used widely to study the induction, function and stability of both thymically- and peripherally-induced Treg cells. The N-terminal modification of FoxP3, however, affects its interaction with transcriptional co-factors; this can alter Treg cell development and function in certain self-antigen specific animal models. Interestingly, Treg cell function can be negatively or positively affected, depending on the nature of the model. In this study, we focused on the effect of the GFP-FoxP3 reporter on Treg cell development and function in the Tg4 mouse model. In this model, T cells express a transgenic T cell receptor (TCR) specific for the Myelin Basic Protein (MBP) peptide Ac1-9, making the animals susceptible to experimental autoimmune encephalomyelitis (EAE), a disease akin to multiple sclerosis in humans. Unlike diabetes-susceptible mice, Tg4 FoxP3gfp mice did not develop spontaneous autoimmune disease and did not demonstrate augmented susceptibility to induced disease. Concurrently, thymic generation of natural Treg cells was not negatively affected. The induction of FoxP3 expression in naive peripheral T cells was, however, significantly impaired as a result of the transgene. This study shows that the requirements for the interaction of FoxP3 with co-factors, which governs its regulatory ability, differ not only between natural and inducible Treg cells but also between animal models of diseases such as diabetes and EAE. [ABSTRACT FROM AUTHOR]

Approximately 1 in 20 individuals in Europe and North America suffers from an autoimmune disease. Current treatments are nonspecific and fail to prevent the underlying autoimmune response. Regulatory T cells have been shown to control autoimmunity in healthy individuals. Here we focus on strategies developed to promote immune regulation through the administration of antigenic peptides. [Copyright &y& Elsevier]

The immune system is a delicately balanced network of interacting cells. In recent years, the concept of immune regulation/suppression has been firmly established, and both natural and induced regulatory cells play vital roles in protection from autoimmune disease. Recent work has revealed the diverse nature of regulatory CD4+ T (Treg) cells and the molecules involved in their function. Innate and adaptive responses to infection are able to override the suppressive properties of such regulatory cells, whereas several reports point to deficiencies in regulatory cell function in autoimmune disease. Protocols have been developed that allow the expansion of Treg cells in vitro and their antigen-specific induction in vivo. A full understanding of Treg differentiation and function will facilitate the development of improved strategies for prevention and treatment of autoimmune diseases. [Copyright &y& Elsevier]

T-cells provide antigen specific focus to the adaptive immune response. However, the receptors that initiate functional responses are not in themselves specific, as they can recognise many different ligands. In this paper, we propose that the immune system deals with TCR degeneracy by an adaptive process that effects both T-cell activation and T-cell function, and exploits TCR degeneracy to limit pathogenic autoimmune disease. [Copyright &y& Elsevier]

The immune system must avoid aggressive T-cell responses against self-antigens. But, paradoxically, exposure to self-peptides seems to have an important role in positive selection in the thymus and the maintenance of a broad T-cell repertoire in the periphery. Recent experiments have highlighted situations that allow high-avidity self-reactive T cells to avoid negative selection in the thymus. Accumulating evidence indicates that other, non-deleting mechanisms control the avidity with which T cells recognize self-antigens ? a phenomenon that is known as 'tuning'. This might maximize the peripheral T-cell repertoire by allowing the survival of T cells that can respond to self, but only at concentrations that are not normally reached in vivo. [ABSTRACT FROM AUTHOR]

The rules governing which T cells are inactivated during peptide-induced tolerance are unclear. Here we show that MBP(89?101) contains three overlapping but distinct T cell epitopes that are restricted by a single major histocompatibility complex (MHC) class II molecule. The dominant epitope is not processed from MBP and is not relevant to the induction of autoimmunity. Pathogenic T cells recognize two minor epitopes that are processed from MBP but are presented only poorly after exposure to MBP(89?101). Induction of immunological tolerance by MBP(89?101) therefore inactivates T cells that recognize the dominant epitope and disease-relevant T cells escape tolerance. The topology of the three epitopes implicates asparagine endopeptidase as the enzyme that controls recognition of this region of MBP. Our results highlight the need to use peptides that mimic the binding of processed antigen fragments to MHC molecules for successful modulation of disease-relevant T cells. [ABSTRACT FROM AUTHOR]

This study explores antigen administration via mucosal surfaces as a potential means of inducing antigen-specific non-responsiveness in experimental autoimmune encephalomyelitis (EAE). In the H-2 mouse model of EAE, the acetylated N-termlnal peptide of myelin basic protein represents a dominant T cell epitope which on its own is sufficient to induce disease. Oral administration of the encephalitogenic peptide over a wide range of doses failed to induce oral tolerance to EAE. In marked contrast, a single intranasal dose of this peptide (Ac1–9 or Ac1–11) profoundly inhibited EAE when administered prior to disease induction. We investigated this phenomen further by using two analogues of Ac1–11 with alanlne or tyroslne at position 4 which display higher affinity binding to the I-A molecule than the original peptide with lysine at this position. There was a positive correlation between the degree of protection from EAE and the affinity of individual peptides for class II MHC. Peptide inhalation inhibited not only EAE induced by subcutaneous injection of the encephalitogenic peptide but also disease induced by a complex mixture of potential auto-antigens such as spinal cord homogenate. Thus, in contrast to oral tolerance, nonresponsiveness by peptide inhalation is induclble with the encephalitogenic peptide in the absence of additional regulatory eprtopes. The finding that a single epitope may protect against EAE induced with whole spinal cord homogenate implies, however, that regulatory mechanisms affecting additional potential self-epltopes may play a significant role. [ABSTRACT FROM PUBLISHER]

Peripheral immune regulation is critical for the maintenance of self-tolerance. Here we have investigated signaling processes that distinguish T cells with regulatory capability from effector T cells. The murine Tg4 T cell receptor recognizes a peptide derived from the self-antigen myelin basic protein. T cells from Tg4 T cell receptor transgenic mice can be used to generate effector T cells and three types of T cells with regulatory capability, inducible regulatory T cells, T cells tolerized by repeated in vivo antigenic peptide exposure or T cells treated with the tolerogenic drug UCB9608 (a phosphatidylinositol 4 kinase IIIβ inhibitor). We comparatively studied signaling in all of these T cells by activating them with the same antigen presenting cells presenting the same myelin basic protein peptide. Supramolecular signaling structures, as efficiently detected by large-scale live cell imaging, are critical mediators of T cell activation. The formation of a supramolecular signaling complex anchored by the adaptor protein linker for activation of T cells (LAT) was consistently terminated more rapidly in Tg4 T cells with regulatory capability. Such termination could be partially reversed by blocking the inhibitory receptors CTLA-4 and PD-1. Our work suggests that attenuation of proximal signaling may favor regulatory over effector function in T cells. [ABSTRACT FROM AUTHOR]

This article focuses on the role of T-cells in autoimmune disease. The presentation to T cells of complexes between MHC molecules and peptide fragments of foreign antigens lies at the heart of the immune response. Classically, those peptides displaying the highest affinity for their MHC ligand are considered immunodominant, outcompeting subdominant or cryptic determinants for interaction with the peptide-binding groove. Autoimmunity, however, represents the failure of tolerance to establish a state of non-responsiveness to self, and is characterized by the persistence of autoreactive T cells.

Altered peptide ligands (APL) with increased MHC-binding properties are highly effective at inducing T cell tolerance after systemic administration in soluble form, preventing experimental autoimmune encephalomyelitis (EAE) induced with the myelin basic protein (MBP) Ac1-9 peptide. We have previously described a diverse Ac1-9-reactive T cell repertoire with differing TCR affinities. A remaining question is what proportion of this repertoire is silenced by peptide therapy? Here, we show that the sensitivity of a T cell to peptide-induced tolerance is related to its avidity for native Ac1-9. These data provide new evidence that self-reactive T cells bearing low-affinity TCRs are able to escape therapeutic induction of tolerance. [Copyright &y& Elsevier]

CD4+ CD25+ Foxp3+ regulatory T cells (Tregs) have a profound ability to control immune responses. We have previously shown that the liver is a major source of peripherally induced Tregs. Here, we investigate the liver cell types and molecular mechanisms responsible for hepatic Treg induction. Methods To assess the Treg-inducing potential of liver resident antigen-presenting cell types, we studied the conversion of Foxp3− non-Tregs into Foxp3+ Tregs induced by liver dendritic cells (DCs), liver sinusoidal endothelial cells (LSECs), or Kupffer cells (KCs). The dependency of Treg induction on TGF-β was tested in Treg conversion assays using T cells with reduced TGF-β sensitivity. The suppressive potential of liver cell-induced Tregs was assessed by an in vitro suppression assay and in vivo, in the model of experimental autoimmune encephalomyelitis (EAE). Results All tested liver cell types were capable of inducing Foxp3+ Tregs; however, LSECs were most efficient in inducing Tregs. Treg-induction was antigen-specific and depended on TGF-β. LSECs featured membrane-bound LAP/TGF-β and the anchor molecule GARP, which is required for tethering LAP/TGF-β to the cell membrane. LSEC-induced Tregs suppressed proliferation and cytokine secretion of effector T cells in vitro. LSEC-induced Tregs were also functional suppressors in vivo, as neuroantigen-specific Tregs induced by LSECs were able to suppress EAE. Conclusions We demonstrate that LSECs are the major liver cell type responsible for TGF-β dependent hepatic Treg induction. The extraordinary capacity of LSECs to induce Tregs was associated with their unique ability to tether TGF-β to their membrane. [ABSTRACT FROM AUTHOR]

Today it is generally accepted that B cells require cognate interactions with CD4+ T cells to develop high-affinity antibodies against proteins. CD4+ T ceils recognize peptides (epitopes) presented by MHC class II molecules that are expressed on antigen-presenting cells. Structural features of both the MHC class II molecule and the peptide determine the specificity of CD4+ T cells that can bind to the MHC class ll-peptide complex. We used a new humanized hemophilic mouse model to identify FVIII peptides presented by HLA-DRB1*1501. This model carries a knockout of all murine MHC class II molecules and expresses a chimeric murine-human MHC class II complex that contains the peptide-binding sites of the human HLA-DRB1*1501. When mice were treated with human FVIII, the proportion of mice that developed antibodies depended on the application route of FVIII and the activation state of the innate immune system. We identified 8 FVIII peptide regions that contained CD4+ T-ceil epitopes presented by HLA-DRB1*1501 to CD4+ T cells during immune responses against FVIII. CD4+ T-cell responses after intravenous and subcutaneous application of FVIII involved the same immunodominant FVIII epitopes. Interestingly, most of the 8 peptide regions contained promiscuous epitopes that bound to several different HLA-DR proteins in in vitro binding assays. [ABSTRACT FROM AUTHOR]

Abstract: Recent studies have expanded our understanding of the role of the anti-inflammatory cytokine interleukin (IL)–10, produced by multiple lineages of both human and murine T cells, in regulating the immune response. Here, we demonstrate that the small percentage of circulating CD4+ T cells that secrete IL-10 can be isolated from human peripheral blood and, importantly, we have optimized a protocol to expand these cells in both antigen-specific and polyclonal manners. Expanded CD4+IL-10+ T cells abrogate proliferation and T helper (Th) 1–like cytokine production in an antigen-specific manner, and to a lesser extent exhibit bystander suppressive capacity. CD4+IL-10+ T cells are suppressive in a cell contact–dependent way, though they do not require secretion of IL-10 for their suppressive role in vitro. CD4+IL-10+ T cells have an activated phenotype, with high expression of CD25, CD69, and cytotoxic T-lymphocyte antigen-4, and are largely FoxP3 negative. This novel method for the isolation and expansion of suppressive IL-10–secreting T cells has important implications both for further research and clinical therapeutic development. [Copyright &y& Elsevier]

How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3 -Tocky mice, we characterized early quantitative and qualitative changes that occur in CD4+ T cells in relation to TCR signaling strength. We captured how dose- and time-dependent programming of distinct co-inhibitory receptors rapidly recalibrates T cell activation thresholds and visualized the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 immunotherapy leads to an increased TCR signal strength. We defined a strong TCR signal metric of five genes upregulated by anti-PD1 in T cells (TCR.strong), which was superior to a canonical T cell activation gene signature in stratifying melanoma patient outcomes to anti-PD1 therapy. Our study therefore reveals how analysis of TCR signal strength—and its manipulation—can provide powerful metrics for monitoring outcomes to immunotherapy. [Display omitted] • TCR signal strength drives dynamic and time-dependent changes in CD4+ T cells • Inhibitory receptor expression recalibrates T cell activation thresholds • PD1 blockade leads to a strong TCR signal signature in T cells (TCR.strong) • TCR.strong can stratify melanoma patient responses to anti-PD1 therapy How antigen and immune checkpoint engagement regulate T cell function is not completely understood. Elliot et al. reveal how antigen abundance regulates immune checkpoint expression and recalibrates T cell activation thresholds. PD1 blockade lowers the T cell activation threshold, resulting in a transcriptional signature that stratifies responses to immunotherapy. [ABSTRACT FROM AUTHOR]

Tregs are important mediators of immune tolerance to self antigens, and it has been suggested that Treg inactivation may cause autoimmune disease. Therefore, immunotherapy approaches that aim to restore or expand autoantigen-specific Treg activity might be beneficial for the treatment of autoimmune disease. Here we report that Treg-mediated suppression of autoimmune disease can be achieved in vivo by taking advantage of the ability of the liver to promote immune tolerance. Expression of the neural autoantigen myelin basic protein (MBP) in the liver was accomplished stably in liver-specific MBP transgenic mice and transiently using gene transfer to liver cells in vivo. Such ectopic MBP expression induced protection from autoimmune neuroinflammation in a mouse model of multiple sclerosis. Protection from autoimmunity was mediated by MBP-specific CD4+CD25+Foxp3+ Tregs, as demonstrated by the ability of these cells to prevent disease when adoptively transferred into nontransgenic mice and to suppress conventional CD4+CD25- T cell proliferation after antigen-specific stimulation with MBP in vitro. The generation of MBP-specific CD4+CD25+Foxp3+ Tregs in vivo depended on expression of MBP in the liver, but not in skin, and occurred by TGF-beta-dependent peripheral conversion from conventional non-Tregs. Our findings indicate that autoantigen expression in the liver may generate autoantigen-specific Tregs. Thus, targeting of autoantigens to hepatocytes may be a novel approach to prevention or treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]