An embarrassment of riches: RORγt+ antigen-presenting cells in peripheral tolerance.

RORγt+ regulatory T (Treg) cells are critical toward maintaining gut immune tolerance. In recent studies published in Nature, Kedmi et al., Lyu et al., and Akagbosu et al. describe MHCII+RORγt+ antigen-presenting cells that mediate RORγt+ Treg cell differentiation but propose disparate identities for these cells.

The multiple pathways to autoimmunity.

Efforts to understand autoimmunity have been pursued relentlessly for several decades. It has become apparent that the immune system evolved multiple mechanisms for controlling self-reactivity, and defects in one or more of these mechanisms can lead to a breakdown of tolerance. Among the multitude of lesions associated with disease, the most common seem to affect peripheral tolerance rather than central tolerance. The initial trigger for both systemic autoimmune disorders and organ-specific autoimmune disorders probably involves the recognition of self or foreign molecules, especially nucleic acids, by innate sensors. Such recognition, in turn, triggers inflammatory responses and the engagement of previously quiescent autoreactive T cells and B cells. Here we summarize the most prominent autoimmune pathways and identify key issues that require resolution for full understanding of pathogenic autoimmunity.

Immunometabolism of regulatory T cells.

The bidirectional interaction between the immune system and whole-body metabolism has been well recognized for many years. Via effects on adipocytes and hepatocytes, immune cells can modulate whole-body metabolism (in metabolic syndromes such as type 2 diabetes and obesity) and, reciprocally, host nutrition and commensal-microbiota-derived metabolites modulate immunological homeostasis. Studies demonstrating the metabolic similarities of proliferating immune cells and cancer cells have helped give birth to the new field of immunometabolism, which focuses on how the cell-intrinsic metabolic properties of lymphocytes and macrophages can themselves dictate the fate and function of the cells and eventually shape an immune response. We focus on this aspect here, particularly as it relates to regulatory T cells.

CD4(+) T cell anergy prevents autoimmunity and generates regulatory T cell precursors.

The role of anergy, an acquired state of T cell functional unresponsiveness, in natural peripheral tolerance remains unclear. In this study, we found that anergy was selectively induced in fetal antigen-specific maternal CD4(+) T cells during pregnancy. A naturally occurring subpopulation of anergic polyclonal CD4(+) T cells, enriched for self antigen-specific T cell antigen receptors, was also present in healthy hosts. Neuropilin-1 expression in anergic conventional CD4(+) T cells was associated with hypomethylation of genes related to thymic regulatory T cells (Treg cells), and this correlated with their ability to differentiate into Foxp3(+) Treg cells that suppressed immunopathology. Thus, our data suggest that not only is anergy induction important in preventing autoimmunity but also it generates the precursors for peripheral Treg cell differentiation.

Conditioning of naive CD4(+) T cells for enhanced peripheral Foxp3 induction by nonspecific bystander inflammation.

Inflammation induced during infection can both promote and suppress immunity. This contradiction suggests that inflammatory cytokines affect the immune system in a context-dependent manner. Here we show that nonspecific bystander inflammation conditions naive CD4(+) T cells for enhanced peripheral Foxp3 induction and reduced effector differentiation. This results in inhibition of immune responses in vivo via a Foxp3-dependent effect on antigen-specific naive CD4(+) T cell precursors. Such conditioning may have evolved to allow immunity to infection while limiting subsequent autoimmunity caused by release of self-antigens in the wake of infection. Furthermore, this phenomenon suggests a mechanistic explanation for the idea that early tuning of the immune system by infection affects the long-term quality of immune regulation.

Control of peripheral tolerance by regulatory T cell-intrinsic Notch signaling.

Receptors of the Notch family direct the differentiation of helper T cell subsets, but their influence on regulatory T cell (T(reg) cell) responses is obscure. We found here that lineage-specific deletion of components of the Notch pathway enhanced T(reg) cell-mediated suppression of type 1 helper T cell (T(H)1 cell) responses and protected against their T(H)1 skewing and apoptosis. In contrast, expression in T(reg) cells of a gain-of-function transgene encoding the Notch1 intracellular domain resulted in lymphoproliferation, exacerbated T(H)1 responses and autoimmunity. Cell-intrinsic canonical Notch signaling impaired T(reg) cell fitness and promoted the acquisition by T(reg) cells of a T(H)1 cell-like phenotype, whereas non-canonical Notch signaling dependent on the adaptor Rictor activated the kinase AKT-transcription factor Foxo1 axis and impaired the epigenetic stability of Foxp3. Our findings establish a critical role for Notch signaling in controlling peripheral T(reg) cell function.

How persistent infection overcomes peripheral tolerance mechanisms to cause T cell-mediated autoimmune disease.

T cells help orchestrate immune responses to pathogens, and their aberrant regulation can trigger autoimmunity. Recent studies highlight that a threshold number of T cells (a quorum) must be activated in a tissue to mount a functional immune response. These collective effects allow the T cell repertoire to respond to pathogens while suppressing autoimmunity due to circulating autoreactive T cells. Our computational studies show that increasing numbers of pathogenic peptides targeted by T cells during persistent or severe viral infections increase the probability of activating T cells that are weakly reactive to self-antigens (molecular mimicry). These T cells are easily re-activated by the self-antigens and contribute to exceeding the quorum threshold required to mount autoimmune responses. Rare peptides that activate many T cells are sampled more readily during severe/persistent infections than in acute infections, which amplifies these effects. Experiments in mice to test predictions from these mechanistic insights are suggested.

B cell peripheral tolerance is promoted by cathepsin B protease.

B cells that bind soluble autoantigens receive chronic signaling via the B cell receptor (signal-1) in the absence of strong costimulatory signals (signal-2), and this leads to their elimination in peripheral tissues. The factors determining the extent of soluble autoantigen-binding B cell elimination are not fully understood. Here we demonstrate that the elimination of B cells chronically exposed to signal-1 is promoted by cathepsin B (Ctsb). Using hen egg lysozyme-specific (HEL-specific) immunoglobulin transgenic (MD4) B cells and mice harboring circulating HEL, we found improved survival and increased proliferation of HEL-binding B cells in Ctsb-deficient mice. Bone marrow chimera experiments established that both hematopoietic and nonhematopoietic sources of Ctsb were sufficient to promote peripheral B cell deletion. The depletion of CD4+ T cells overcame the survival and growth advantage provided by Ctsb deficiency, as did blocking CD40L or removing CD40 from the chronically antigen-engaged B cells. Thus, we suggest that Ctsb acts extracellularly to reduce soluble autoantigen-binding B cell survival and that its actions restrain CD40L-dependent pro-survival effects. These findings identify a role for cell-extrinsic protease activity in establishing a peripheral self-tolerance checkpoint.

Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery.

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.

PD-1 cooperates with AIRE-mediated tolerance to prevent lethal autoimmune disease.

Immunological tolerance is established and maintained by a diverse array of safeguards that work together to protect against autoimmunity. Despite the identification of numerous tolerogenic processes, the basis for cooperation among them remains poorly understood. We sought to identify synergy among several well-defined tolerance mediators that alone provide protection only from mild autoimmune symptoms in C57BL/6 mice: BIM, AIRE, CBL-B, and PD-1. Survey of a range of compound mutant mice revealed that the combined loss of the autoimmune regulator, AIRE, with PD-1 unleashed a spontaneous, lethal autoimmune disease. Pdcd1−/−Aire−/− mice succumbed to cachexia before adulthood, with near-complete destruction of the exocrine pancreas. Such fatal autoimmunity was not observed in Pdcd1−/−Bim−/−, Bim−/−Aire−/−, or Cblb−/−Bim−/− mice, suggesting that the cooperation between AIRE-mediated and PD-1­mediated tolerance was particularly potent. Immune profiling revealed largely normal development of FOXP3+ regulatory T (Treg) cells in Pdcd1−/−Aire−/− mice, yet excessive, early activation of effector T cells. Adoptive transfer experiments demonstrated that autoimmune exocrine pancreatitis was driven by conventional CD4+ T cells and could not be prevented by the cotransfer of Treg cells from wild-type mice. The development of autoimmunity in mixed bone marrow chimeras supported these observations, indicating that failure of recessive tolerance was responsible for disease. These findings reveal a potent tolerogenic axis between AIRE and PD-1 that has implications for our understanding of how immune checkpoint blockade might synergize with subclinical defects in central tolerance to elicit autoimmune disease.

CD5 instructs extrathymic regulatory T cell development in response to self and tolerizing antigens.

Self-reactive T cells can escape thymic deletion and therefore some of these potentially autoaggressive T cells need to convert into regulatory T (Treg) cells to help control responses against self. However, it remains unknown how peripheral self-reactive T cells are specifically instructed to become Treg cells. We report that CD5, whose expression is upregulated in T cells by self and tolerizing antigens in the thymus and periphery, governed extrathymic Treg cell development. CD5 modified effector cell-differentiating signals that inhibit Treg cell induction. Treg cell conversion of Cd5(-/-) and CD5(lo) T cells was inhibited by even small amounts of interleukin-4 (IL-4), IL-6, and interferon-γ (IFN-γ) produced by bystander lymphocytes, while CD5(hi) T cells resisted this inhibition of Treg cell induction. Our findings further revealed that CD5 promoted Treg cell induction by blocking mechanistic target of rapamycin (mTOR) activation. Therefore CD5 instructs extrathymic Treg cell development in response to self and tolerizing antigens.

Small spleen peptides prevent development of psoriatic arthritis via restoration of peripheral tolerance.

The major challenge in the treatment of autoimmune diseases is the restoration of the impaired peripheral immune tolerance that always accompanies the development of such diseases. Here, we show that small splenic peptides (SSPs) of whole spleen extract efficiently suppress the development of psoriatic arthritis in vivo, even in the presence of sustained levels of pro-inflammatory cytokines. SSPs target dendritic cells (DCs) and convert them into tolerogenic cells, which in turn differentiate naive CD4+ cells into Foxp3-expressing T regulatory cells (Tregs). The latter requires direct contact between SSP-activated DCs and naive CD4+ T cells via PD-1 and CTLA4 immune checkpoint receptors of T cells. Finally, depletion of Foxp3+ Tregs in vivo abrogated the protective effect of SSPs on psoriatic arthritis development. We hypothesize that SSPs represent an intrinsic component of the adaptive immune system responsible for the physiological maintenance of peripheral tolerance and that therapeutically administered SSPs are able to restore imbalanced peripheral tolerance in autoimmune diseases.

Treg cell therapy: How cell heterogeneity can make the difference.

CD4+ CD25high CD127low/- FOXP3+ T regulatory cells are responsible for maintaining immune tolerance and controlling excessive immune responses. Treg cell use in pre-clinical animal models showed the huge therapeutic potential of these cells in immune-mediated diseases and laid the foundations for their applications in therapy in humans. Currently, there are several clinical trials utilizing the adoptive transfer of Treg cells to reduce the morbidity in autoimmune disorders, allogeneic HSC transplantation, and solid organ transplantation. However, a large part of them utilizes total Treg cells without distinction of their biological variability. Many studies on the heterogeneity of Treg cell population revealed distinct subsets with different functions in the control of the immune response and induction of peripheral tolerance. Some of these subsets also showed a role in controlling the general homeostasis of non-lymphoid tissues. All these Treg cell subsets and their peculiar properties can be therefore exploited to develop novel therapeutic approaches. This review describes these functionally distinct subsets, their phenotype, homing properties and functions in lymphoid and non-lymphoid tissues. In addition, we also discuss the limitations in using Treg cells as a cellular therapy and the strategies to enhance their efficacy.

Peripheral Tolerance Checkpoints Imposed by Ubiquitous Antigen Expression Limit Antigen-Specific B Cell Responses under Strongly Immunogenic Conditions.

A series of layered peripheral checkpoints maintain self-reactive B cells in an unresponsive state. Autoantibody production occurs when these checkpoints are breached; however, when and how this occurs is largely unknown. In particular, how self-reactive B cells are restrained during bystander inflammation in otherwise healthy individuals is poorly understood. A weakness has been the unavailability of methods capable of dissecting physiologically relevant B cell responses without the use of an engineered BCR. Resolving this will provide insights that decipher how this process goes awry during autoimmunity or could be exploited for therapy. In this study, we use a strong adjuvant to provide bystander innate and adaptive signals that promote B cell responsiveness in conjunction with newly developed B cell detection tools to study in detail the ways that peripheral tolerance mechanisms limit the expansion and function of self-reactive B cells activated under these conditions. We show that although self-reactive B cells are recruited into the germinal center, their development does not proceed, possibly because of rapid counterselection. Consequently, differentiation of plasma cells is blunted, and Ab responses are transient and devoid of affinity maturation. We propose this approach, and these tools can be more widely applied to track Ag-specific B cell responses to more disease-relevant Ags, without the need for BCR transgenic mice, in settings where tolerance pathways are compromised or have been genetically manipulated to drive stronger insights into the biology underlying B cell-mediated autoimmunity.

sLeX Expression Delineates Distinct Functional Subsets of Human Blood Central and Effector Memory T Cells.

Sialyl Lewis X (sLeX) regulates T cell trafficking from the vasculature into skin and sites of inflammation, thereby playing a critical role in immunity. In healthy persons, only a small proportion of human blood T cells express sLeX, and their function is not fully defined. Using a combination of biochemical and functional studies, we find that human blood sLeX+CD4+T cells comprise a subpopulation expressing high levels of Th2 and Th17 cytokines, chemokine receptors CCR4 and CCR6, and the transcription factors GATA-3 and RORγT. Additionally, sLeX+CD4+T cells exclusively contain the regulatory T cell population (CD127lowCD25high and FOXP3+) and characteristically display immune-suppressive molecules, including the coinhibitor receptors PD-1 and CTLA-4. Among CD8+T cells, sLeX expression distinguishes a subset displaying low expression of cytotoxic effector molecules, perforin and granzyme ß, with reduced degranulation and CD57 expression and, consistently, marginal cytolytic capacity after TCR engagement. Furthermore, sLeX+CD8+T cells present a pattern of features consistent with Th cell-like phenotype, including release of pertinent Tc2 cytokines and elevated expression of CD40L. Together, these findings reveal that sLeX display is associated with unique functional specialization of both CD4+ and CD8+T cells and indicate that circulating T cells that are primed to migrate to lesional sites at onset of inflammation are not poised for cytotoxic function.

Innate-like CD27+CD45RBhigh γδ T Cells Require TCR Signaling for Homeostasis in Peripheral Lymphoid Organs.

TCR signaling is required for homeostasis of naive αß T cells. However, whether such a signal is necessary for γδ T cell homeostasis in the periphery remains unknown. In this study, we present evidence that a portion of Vγ2+ γδ T cells, one of the major γδ T cell subsets in the secondary lymphoid organs, requires TCR signaling for homeostasis. To attenuate γδTCR signals, we generated mice lacking Eγ4 (Eγ4-/-), an enhancer located at the 3'-most end of the TCRγ locus. Overall, we found that in thymus, Eγ4 loss altered V-J rearrangement, chromatin accessibility, and transcription of the TCRγ locus in a distance-dependent manner. Vγ2+ γδ T cells in Eγ4-/- mice developed normally both fetal and adult mouse thymi but were relatively reduced in number in spleen and lymph nodes. Although Vγ2 TCR transcription decreased in all subpopulations of Eγ4-/- mice, the number of Vγ2+ γδ T cells decreased and TCR signaling was attenuated only in the innate-like CD27+CD45RBhigh subpopulation in peripheral lymphoid organs. Consistently, CD27+CD45RBhigh Vγ2+ γδ T cells from Eγ4-/- mice transferred into Rag2-deficient mice were not efficiently recovered, suggesting that continuous TCR signaling is required for their homeostasis. Finally, CD27+CD45RBhigh Vγ2+ γδ T cells from Eγ4-/- mice showed impaired TCR-induced activation and antitumor responses. These results suggest that normal homeostasis of innate-like CD27+CD45RBhigh Vγ2+ γδ T cells in peripheral lymphoid organs requires TCR signaling.

The natural history of naive T cells from birth to maturity.

Generating and maintaining a diverse repertoire of naive T cells is essential for protection against pathogens, and developing a mechanistic and quantitative description of the processes involved lies at the heart of our understanding of vertebrate immunity. Here, we review the biology of naive T cells from birth to maturity and outline how the integration of mathematical models and experiments has helped us to develop a full picture of their life histories.

Enteric α-defensins on the verge of intestinal immune tolerance and inflammation.

The gut is the biggest immune organ in the body that encloses commensal microbiota which aids in food digestion. Paneth cells, positioned at the frontline of host-microbiota interphase, can modulate the composition of microbiota. Paneth cells achieve this via the delivery of microbicidal substances, among which enteric α-defensins play the primary role. If microbiota is dysregulated, it can impact the function of the local mucosal immune system. Importantly, this system is also exposed to an enormous number of antigens which are derived from the gut-resident microbiota and processed food, and may potentially trigger undesirable local inflammatory responses. To understand the intricate regulations and liaisons between Paneth cells, microbiota and the immune system in this intestinal-specific setting, one must consider their mode of interaction in a wider context of regulatory processes which impose immune tolerance not only to self, but also to microbiota and food-derived antigens. These include, but are not limited to, tolerogenic mechanisms of central tolerance in the thymus and peripheral tolerance in the secondary lymphoid organs, and the intestine itself. Defects in these processes can compromise homeostasis in the intestinal mucosal immunity. In this review, which is focused on tolerance to intestinal antigens and its relevance for the pathogenesis of gut immune diseases, we provide an outline of such multilayered immune control mechanisms and highlight functional links that underpin their cooperative nature.

Tissue-specific autoimmunity controlled by Aire in thymic and peripheral tolerance mechanisms.

Tissue-specific autoimmune diseases are assumed to arise through malfunction of two checkpoints for immune tolerance: defective elimination of autoreactive T cells in the thymus and activation of these T cells by corresponding autoantigens in the periphery. However, evidence for this model and the outcome of such alterations in each or both of the tolerance mechanisms have not been sufficiently investigated. We studied these issues by expressing human AIRE (huAIRE) as a modifier of tolerance function in NOD mice wherein the defects of thymic and peripheral tolerance together cause type I diabetes (T1D). Additive huAIRE expression in the thymic stroma had no major impact on the production of diabetogenic T cells in the thymus. In contrast, huAIRE expression in peripheral antigen-presenting cells (APCs) rendered the mice resistant to T1D, while maintaining other tissue-specific autoimmune responses and antibody production against an exogenous protein antigen, because of the loss of Xcr1+ dendritic cells, an essential component for activating diabetogenic T cells in the periphery. These results contrast with our recent demonstration that huAIRE expression in both the thymic stroma and peripheral APCs resulted in the paradoxical development of muscle-specific autoimmunity. Our results reveal that tissue-specific autoimmunity is differentially controlled by a combination of thymic function and peripheral tolerance, which can be manipulated by expression of huAIRE/Aire in each or both of the tolerance mechanisms.

A broad range of self-reactivity drives thymic regulatory T cell selection to limit responses to self.

The degree of T cell self-reactivity considered dangerous by the immune system, thereby requiring thymic selection processes to prevent autoimmunity, is unknown. Here, we analyzed a panel of T cell receptors (TCRs) with a broad range of reactivity to ovalbumin (OVA(323-339)) in the rat insulin promoter (RIP)-mOVA self-antigen model for their ability to trigger thymic self-tolerance mechanisms. Thymic regulatory T (Treg) cell generation in vivo was directly correlated with in vitro TCR reactivity to OVA-peptide in a broad ~1,000-fold range. Interestingly, higher TCR affinity was associated with a larger Treg cell developmental "niche" size, even though the amount of antigen should remain constant. The TCR-reactivity threshold to elicit thymic negative selection and peripheral T cell responses was ~100-fold higher than that of Treg cell differentiation. Thus, these data suggest that the broad range of self-reactivity that elicits thymic Treg cell generation is tuned to secure peripheral tolerance to self.