Targeted depletion of PD-1-expressing cells induces immune tolerance through peripheral clonal deletion.

Thymic negative selection of the T cell receptor (TCR) repertoire is essential for establishing self-tolerance and acquired allograft tolerance following organ transplantation. However, it is unclear whether and how peripheral clonal deletion of alloreactive T cells induces transplantation tolerance. Here, we establish that programmed cell death protein 1 (PD-1) is a hallmark of alloreactive T cells and is associated with clonal expansion after alloantigen encounter. Moreover, we found that diphtheria toxin receptor (DTR)-mediated ablation of PD-1+ cells reshaped the TCR repertoire through peripheral clonal deletion of alloreactive T cells and promoted tolerance in mouse transplantation models. In addition, by using PD-1-specific depleting antibodies, we found that antibody-mediated depletion of PD-1+ cells prevented heart transplant rejection and the development of experimental autoimmune encephalomyelitis (EAE) in humanized PD-1 mice. Thus, these data suggest that PD-1 is an attractive target for peripheral clonal deletion and induction of immune tolerance.

CD8 T cell tolerance results from eviction of immature autoreactive cells from the thymus.

CD8 T cell tolerance is thought to result from clonal deletion of autoreactive thymocytes before they differentiate into mature CD8 T cells in the thymus. However, we report that, in mice, CD8 T cell tolerance instead results from premature thymic eviction of immature autoreactive CD8 thymocytes into the periphery, where they differentiate into self-tolerant mature CD8 T cells. Premature thymic eviction is triggered by T cell receptor (TCR)-driven down-regulation of the transcriptional repressor Gfi1, which induces expression of sphingosine-1-phosphate receptor-1 (S1P1) on negatively selected immature CD8 thymocytes. Thus, premature thymic eviction is the basis for CD8 T cell tolerance and is the mechanism responsible for the appearance in the periphery of mature CD8 T cells bearing autoreactive TCRs that are absent from the thymus.

How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

Standing on the shoulders of mice.

While inbred mice have informed most of what we know about the immune system in the modern era, they have clear limitations with respect to their ability to be informative regarding genetic heterogeneity or microbial influences. They have also not been very predictive as models of human disease or vaccination results. Although there are concerted attempts to compensate for these flaws, the rapid rise of human studies, driven by both technical and conceptual advances, promises to fill in these gaps, as well as provide direct information about human diseases and vaccination responses. Work on human immunity has already provided important additional perspectives on basic immunology such as the importance of clonal deletion to self-tolerance, and while many challenges remain, it seems inevitable that "the human model" will continue to inform us about the immune system and even allow for the discovery of new mechanisms.

Transplant Tolerance, Not Only Clonal Deletion.

The quest to understand how allogeneic transplanted tissue is not rejected and how tolerance is induced led to fundamental concepts in immunology. First, we review the research that led to the Clonal Deletion theory in the late 1950s that has since dominated the field of immunology and transplantation. At that time many basic mechanisms of immune response were unknown, including the role of lymphocytes and T cells in rejection. These original observations are reassessed by considering T regulatory cells that are produced by thymus of neonates to prevent autoimmunity. Second, we review "operational tolerance" induced in adult rodents and larger animals such as pigs. This can occur spontaneously especially with liver allografts, but also can develop after short courses of a variety of rejection inhibiting therapies. Over time these animals develop alloantigen specific tolerance to the graft but retain the capacity to reject third-party grafts. These animals have a "split tolerance" as peripheral lymphocytes from these animals respond to donor alloantigen in graft versus host assays and in mixed lymphocyte cultures, indicating there is no clonal deletion. Investigation of this phenomenon excludes many mechanisms, including anti-donor antibody blocking rejection as well as anti-idiotypic responses mediated by antibody or T cells. This split tolerance is transferred to a second immune-depleted host by T cells that retain the capacity to effect rejection of third-party grafts by the same host. Third, we review research on alloantigen specific inhibitory T cells that led to the first identification of the CD4+CD25+T regulatory cell. The key role of T cell derived cytokines, other than IL-2, in promoting survival and expansion of antigen specific T regulatory cells that mediate transplant tolerance is reviewed. The precise methods for inducing and diagnosing operational tolerance remain to be defined, but antigen specific T regulatory cells are key mediators.

Type 2 cytokines in the thymus activate Sirpα+ dendritic cells to promote clonal deletion.

The thymus contains a diversity of dendritic cells (DCs) that exist in defined locations and have different antigen-processing and -presenting features. This suggests that they play nonredundant roles in mediating thymocyte selection. In an effort to eliminate SIRPα+ classic DC2 subsets, we discovered that a substantial proportion expresses the surface lectin, CD301b, in the thymus. These cells resemble the CD301b+ type 2 immune response promoting DCs that are present in the skin-draining lymph nodes. Transcriptional and phenotypic comparison to other DC subsets in the thymus revealed that thymic CD301b+ cDCs represent an activated state that exhibits enhanced antigen processing and presentation. Furthermore, a CD301b+ cDC2 subset demonstrated a type 2 cytokine signature and required steady-state interleukin-4 receptor signaling. Selective ablation of CD301b+ cDC2 subsets impaired clonal deletion without affecting regulatory T cells (Treg cells). The T cell receptor α repertoire sequencing confirmed that a cDC2 subset promotes deletion of conventional T cells with minimal effect on Treg cell selection. Together, these findings suggest that cytokine-induced activation of DCs in the thymus substantially enforces central tolerance.

Development of organ-specific autoimmunity by dysregulated Aire expression.

Deficiency for AIRE/Aire in both humans and mice results in the development of organ-specific autoimmune disease. We tested whether augmented and/or dysregulated AIRE/Aire expression might be also prone to the breakdown of self-tolerance. To define the effect of augmented Aire expression on the development of autoimmunity, antigen-specific clonal deletion and production of clonotypic regulatory T cells (Tregs) in the thymus were examined using mice expressing two additional copies of Aire in a heterozygous state (3xAire-knockin mice: 3xAire-KI). We found that both clonal deletion of autoreactive T cells and production of clonotypic Tregs in the thymus from 3xAire-KI were impaired in a T-cell receptor-transgenic system. Furthermore, 3xAire-KI females showed higher scores of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein than wild-type littermates, suggesting that augmented Aire expression exacerbates organ-specific autoimmunity under disease-prone conditions. In humans, we found that one patient with amyopathic dermatomyositis showed CD3- CD19- cells expressing AIRE in the peripheral blood before the treatment but not during the remission phase treated with immunosuppressive drugs. Thus, not only loss of function of AIRE/Aire but also augmented and/or dysregulated expression of AIRE/Aire should be considered for the pathogenesis of organ-specific autoimmunity. We suggest that further analyses should be pursued to establish a novel link between organ-specific autoimmune disease and dysregulated AIRE expression in clinical settings.

Proteasome-Generated cis-Spliced Peptides and Their Potential Role in CD8+ T Cell Tolerance.

The human immune system relies on the capability of CD8+ T cells to patrol body cells, spot infected cells and eliminate them. This cytotoxic response is supposed to be limited to infected cells to avoid killing of healthy cells. To enable this, CD8+ T cells have T Cell Receptors (TCRs) which should discriminate between self and non-self through the recognition of antigenic peptides bound to Human Leukocyte Antigen class I (HLA-I) complexes-i.e., HLA-I immunopeptidomes-of patrolled cells. The majority of these antigenic peptides are produced by proteasomes through either peptide hydrolysis or peptide splicing. Proteasome-generated cis-spliced peptides derive from a given antigen, are immunogenic and frequently presented by HLA-I complexes. Theoretically, they also have a very large sequence variability, which might impinge upon our model of self/non-self discrimination and central and peripheral CD8+ T cell tolerance. Indeed, a large variety of cis-spliced epitopes might enlarge the pool of viral-human zwitter epitopes, i.e., peptides that may be generated with the exact same sequence from both self (human) and non-self (viral) antigens. Antigenic viral-human zwitter peptides may be recognized by CD8+ thymocytes and T cells, induce clonal deletion or other tolerance processes, thereby restraining CD8+ T cell response against viruses. To test this hypothesis, we computed in silico the theoretical frequency of zwitter non-spliced and cis-spliced epitope candidates derived from human proteome (self) and from the proteomes of a large pool of viruses (non-self). We considered their binding affinity to the representative HLA-A*02:01 complex, self-antigen expression in Medullary Thymic Epithelial cells (mTECs) and the relative frequency of non-spliced and cis-spliced peptides in HLA-I immunopeptidomes. Based on the present knowledge of proteasome-catalyzed peptide splicing and neglecting CD8+ TCR degeneracy, our study suggests that, despite their frequency, the portion of the cis-spliced peptides we investigated could only marginally impinge upon the variety of functional CD8+ cytotoxic T cells (CTLs) involved in anti-viral response.

Two terpene synthases in resistant Pinus massoniana contribute to defence against Bursaphelenchus xylophilus.

Pine wood nematode (PWN; Bursaphelenchus xylophilus), a destructive pest of Pinus massoniana, is causing a severe epidemic of pine wilt disease in China. When invaded by PWN, resistant P. massoniana secretes an abundance of oleoresin terpenoids as a defensive strategy. However, regulatory mechanisms of this defence in resistant P. massoniana have yet to be elucidated. Here, we characterized two terpene synthase genes, α-pinene synthase (PmTPS4) and longifolene synthase (PmTPS21), identified in resistant P. massoniana and investigate the contribution of these genes to the oleoresin defence strategy in resistant masson pines. Up-regulation of these two genes in the stem supported their involvement in terpene biosynthesis as part of the defence against PWN. Recombinant protein expression revealed catalytic activity for the two PmTPSs, with PmTPS4 primarily producing α-pinene, while PmTPS21 produced α-pinene and longifolene simultaneously. The major enzymatic products of the two terpene synthases had inhibitory effects on PWN in vitro. We demonstrated that PmTPS4 and PmTPS21 played positive roles in terpene-defence mechanisms against PWN infestation. The major products of these terpene synthases could directly inhibit the survival rate of PWN in vitro. We revealed that PmTPS21 was a novel bifunctional enzyme capable of simultaneous production of both monoterpene and sesquiterpene.

B7-CD28 co-stimulation modulates central tolerance via thymic clonal deletion and Treg generation through distinct mechanisms.

The molecular and cellular mechanisms mediating thymic central tolerance and prevention of autoimmunity are not fully understood. Here we show that B7-CD28 co-stimulation and B7 expression by specific antigen-presenting cell (APC) types are required for clonal deletion and for regulatory T (Treg) cell generation from endogenous tissue-restricted antigen (TRA)-specific thymocytes. While B7-CD28 interaction is required for both clonal deletion and Treg induction, these two processes differ in their CD28 signaling requirements and in their dependence on B7-expressing dendritic cells, B cells, and thymic epithelial cells. Meanwhile, defective thymic clonal deletion due to altered B7-CD28 signaling results in the accumulation of mature, peripheral TRA-specific T cells capable of mediating destructive autoimmunity. Our findings thus reveal a function of B7-CD28 co-stimulation in shaping the T cell repertoire and limiting autoimmunity through both thymic clonal deletion and Treg cell generation.

Cutting Edge: Intestinal Mucus Limits the Clonal Deletion of Developing T Cells Specific for an Oral Antigen.

A layer of mucus functions to segregate contents of the intestinal lumen from the intestinal epithelium. The MUC2 mucin is the primary constituent of intestinal mucus and plays critical protective roles against luminal microbes and other noxious agents. In this study, we investigated whether MUC2 helps maintain CD8 T cell tolerance toward intestinal luminal Ags by gavaging wild-type and Muc2-/- mice with a model Ag and monitoring immune responses posttreatment. We report that orally delivered OVA rapidly disseminates through the blood of Muc2-/- (but not control) mice and causes immune activation of Ag-specific CD8 T cells at both local and distal sites. Further, the administration of oral OVA to Muc2-/- mice led to its presentation by thymic dendritic cells and the deletion of Ag-specific thymocytes. Collectively, our findings suggest that intestinal mucus helps limit the shaping of the TCR repertoire of developing thymocytes by intestinal luminal Ags.

B cells as antigen-presenting cells in transplantation rejection and tolerance.

Transplantation of fully allogeneic organs into immunocompetent recipients invariably elicits T cell and B cell responses that lead to the production of donor-specific antibodies (DSA). When immunosuppression is inadequate donor-specific T cell and B cell responses escape, leading to T cell-mediated rejection (TCMR), antibody mediated (ABMR) rejection, or mixed rejection (MR) exhibiting features of both TCMR and ABMR. Current literature suggests that ABMR is a major cause of late graft loss, and that new therapies to curtail the donor-specific humoral response are necessary. The majority of research into B cell responses elicited by allogeneic allografts in both preclinical models and clinical studies, has focused on the function of B cells as antibody-secreting cells and the pathogenic effects of DSA as mediators of ABMR. However, it has long been recognized that the DSA response to allografts is T cell-dependent, and that B cells engage in cognate interactions with T cells that provide "help" and promote B cell differentiation into antibody-secreting cells (ASCs). This review focusses the function of B cells as antigen-presenting cells (APCs) to T cells in lymphoid organs, how they may be critical APCs to T cell in the allograft, and the functional consequences of these interactions.

Regulatory T Cell Development.

Foxp3-expressing CD4+ regulatory T (Treg) cells play key roles in the prevention of autoimmunity and the maintenance of immune homeostasis and represent a major barrier to the induction of robust antitumor immune responses. Thus, a clear understanding of the mechanisms coordinating Treg cell differentiation is crucial for understanding numerous facets of health and disease and for developing approaches to modulate Treg cells for clinical benefit. Here, we discuss current knowledge of the signals that coordinate Treg cell development, the antigen-presenting cell types that direct Treg cell selection, and the nature of endogenous Treg cell ligands, focusing on evidence from studies in mice. We also highlight recent advances in this area and identify key unanswered questions.

Treg Therapies Revisited: Tolerance Beyond Deletion.

Induction of immune tolerance is the Holy Grail in transplantation medicine and autoimmunity. Currently, patients are required to use immunosuppressive drugs for the rest of their lives, resulting in unwanted side effects and complication from global suppression of the immune response. It is well established that regulatory T cells (Tregs) are critical for the maintenance of immune tolerance towards self-antigens by several mechanisms of immune regulation, in parallel with intrathymic deletion of self-reactive T cells during ontogeny. Therefore, approaches for increasing Treg numbers or function in vivo could provide an all-purpose solution for tolerance induction. Currently, most state-of-the-art therapeutics for treating autoimmune diseases or preventing allograft rejection work either by general immunosuppression or blocking inflammatory reactions and are non-specific. Hence, these approaches cannot provide satisfactory long-term results, let alone a cure. However, in animal models the therapeutic potential of Treg expansion for inducing effective tolerance has now been demonstrated in various models of autoimmunity and allogeneic transplantation. Here, we focus on therapies for increasing the size of the Treg pool by expanding endogenous Treg numbers in vivo or by adoptive transfer of Tregs. In particular, we discuss IL-2 based approaches (low dose IL-2, IL-2 complexes) for inducing Treg expansion in vivo as well as cell-based approaches (polyclonal, antigen specific, or cell engineered) for adoptive Treg therapy. We also mention new questions arising from the first clinical studies on Treg therapy in the fields of transplantation and autoimmunity.

Deletion of donor-reactive T cell clones after human liver transplant.

We recently developed a high throughput T cell receptor ß chain (TCRß) sequencing-based approach to identifying and tracking donor-reactive T cells. To address the role of clonal deletion in liver allograft tolerance, we applied this method in samples from a recent randomized study, ITN030ST, in which immunosuppression withdrawal was attempted within 2 years of liver transplantation. We identified donor-reactive T cell clones via TCRß sequencing following a pre-transplant mixed lymphocyte reaction and tracked these clones in the circulation following transplantation in 3 tolerant and 5 non-tolerant subjects. All subjects showed a downward trend and significant reductions in donor-reactive TCRß sequences were detected post-transplant in 6 of 8 subjects, including 2 tolerant and 4 non-tolerant recipients. Reductions in donor-reactive TCRß sequences were greater than those of all other TCRß sequences, including 3rd party-reactive sequences, in all 8 subjects, demonstrating an impact of the liver allograft after accounting for repertoire turnover. Although limited by patient number and heterogeneity, our results suggest that partial deletion of donor-reactive T cell clones may be a consequence of liver transplantation and does not correlate with success or failure of early immunosuppression withdrawal. These observations underscore the organ- and/or protocol-specific nature of tolerance mechanisms in humans.

Follicular regulatory T cells control humoral and allergic immunity by restraining early B cell responses.

Follicular regulatory T (TFR) cells have specialized roles in modulating follicular helper T (TFH) cell activation of B cells. However, the precise role of TFR cells in controlling antibody responses to foreign antigens and autoantigens in vivo is still unclear due to a lack of specific tools. A TFR cell-deleter mouse was developed that selectively deletes TFR cells, facilitating temporal studies. TFR cells were found to regulate early, but not late, germinal center (GC) responses to control antigen-specific antibody and B cell memory. Deletion of TFR cells also resulted in increased self-reactive immunoglobulin (Ig) G and IgE. The increased IgE levels led us to interrogate the role of TFR cells in house dust mite models. TFR cells were found to control TFH13 cell-induced IgE. In vivo, loss of TFR cells increased house-dust-mite-specific IgE and lung inflammation. Thus, TFR cells control IgG and IgE responses to vaccines, allergens and autoantigens, and exert critical immunoregulatory functions before GC formation.

Inventories of naive and tolerant mouse CD4 T cell repertoires reveal a hierarchy of deleted and diverted T cell receptors.

Deletion or Treg cell differentiation are alternative fates of autoreactive MHCII-restricted thymocytes. How these different modes of tolerance determine the size and composition of polyclonal cohorts of autoreactive T cells with shared specificity is poorly understood. We addressed how tolerance to a naturally expressed autoantigen of the central nervous system shapes the CD4 T cell repertoire. Specific cells in the tolerant peripheral repertoire either were Foxp3+ or displayed anergy hallmarks and, surprisingly, were at least as frequent as in the nontolerant repertoire. Despite this apparent lack of deletional tolerance, repertoire inventories uncovered that some T cell receptors (TCRs) were lost from the CD4 T cell pool, whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance. Importantly, the contribution of different diverter TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. This reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal permissive antigen reactivity.

Autoreactive T-Lymphocytes in Inflammatory Skin Diseases.

The presence of one or several autoantigen(s) and a response by the adaptive immune system are the key criteria to classify a pathology as an autoimmune disease. The list of entities fulfilling this criterion is currently growing in the light of recent advancements in the pathogenetic understanding of a number of important dermatoses. The role of autoreactive T-lymphocytes differs amongst these pathologies. While they are directly involved as effector cells attacking and sometimes killing their respective target in some diseases (e.g., vitiligo), they provide help to B-lymphocytes, which in turn produce the pathogenic autoreactive antibodies in others (pemphigus and pemphigoid). Atopic dermatits is a chimera in this regard, as there is evidence for both functions. Psoriasis is an example for an entity where autoantigens were finally identified, suggesting that at least a subgroup of patients should be classified as suffering from a true autoimmune rather than autoinflammatory condition. Identification of resident memory T-lymphocytes (TRM) helped to understand why certain diseases relapse at the same site after seemingly effective therapy. Therefore, the in-depth characterization of autoreactive T-lyphocytes goes way beyond an academic exercise and opens the door toward improved therapies yielding durable responses. TRM are particularly suitable targets in this regard, and the clinical efficacy of some established and emerging therapeutic strategies such as the inhibition of Janus Kinase 3 or interleukin 15 may rely on their capacity to prevent TRM differentiation and maintenance. Research in this field brings us closer to the ultimate goal in the management of autoimmunity at large, namely resetting the immune system in order to restore the state of tolerance.

Pulling RANK on Cancer: Blocking Aire-Mediated Central Tolerance to Enhance Immunotherapy.

A major breakthrough in cancer treatment occurred with the development of strategies that overcome T-cell tolerance toward tumor cells. These approaches enhance antitumor immunity by overcoming mechanisms that are normally in place to prevent autoimmunity but simultaneously prevent rejection of tumor cells. Although tolerance mechanisms that restrict antitumor immunity take place both in the thymus and periphery, only immunotherapies that target peripheral tolerance mechanisms occurring outside of the thymus are currently available. We review here recent gains in our understanding of how thymic tolerance mediated by the autoimmune regulator (Aire) impedes antitumor immunity. It is now clear that transient depletion of Aire-expressing cells in the thymus can be achieved with RANKL blockade. Finally, we discuss key findings that support the repurposing of anti-RANKL as a cancer immunotherapy with a unique mechanism of action.