Peripheral apoptosis and limited clonal deletion during physiologic murine B lymphocyte development.

Self-reactive and polyreactive B cells generated during B cell development are silenced by either apoptosis, clonal deletion, receptor editing or anergy to avoid autoimmunity. The specific contribution of apoptosis to normal B cell development and self-tolerance is incompletely understood. Here, we quantify self-reactivity, polyreactivity and apoptosis during physiologic B lymphocyte development. Self-reactivity and polyreactivity are most abundant in early immature B cells and diminish significantly during maturation within the bone marrow. Minimal apoptosis still occurs at this site, however B cell receptors cloned from apoptotic B cells show comparable self-reactivity to that of viable cells. Apoptosis increases dramatically only following immature B cells leaving the bone marrow sinusoids, but above 90% of cloned apoptotic transitional B cells are not self-reactive/polyreactive. Our data suggests that an apoptosis-independent mechanism, such as receptor editing, removes most self-reactive B cells in the bone marrow. Mechanistically, lack of survival signaling rather than clonal deletion appears to be the underpinning cause of apoptosis in most transitional B cells in the periphery.

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.

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.

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.

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.

Clonal Deletion of Tumor-Specific T Cells by Interferon-γ Confers Therapeutic Resistance to Combination Immune Checkpoint Blockade.

Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.

A TCR mechanotransduction signaling loop induces negative selection in the thymus.

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR-pMHC-CD8 trimolecular 'catch bonds', whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR-pMHC and pMHC-CD8 bimolecular 'slip bonds'. Catch bonds were not intrinsic to either the TCR-pMHC or the pMHC-CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC-CD8 interaction to the intracellular interaction of CD8 with TCR-CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans-cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus.

IL-7 receptor blockade blunts antigen-specific memory T cell responses and chronic inflammation in primates.

Targeting the expansion of pathogenic memory immune cells is a promising therapeutic strategy to prevent chronic autoimmune attacks. Here we investigate the therapeutic efficacy and mechanism of new anti-human IL-7Rα monoclonal antibodies (mAb) in non-human primates and show that, depending on the target epitope, a single injection of antagonistic anti-IL-7Rα mAbs induces a long-term control of skin inflammation despite repeated antigen challenges in presensitized monkeys. No modification in T cell numbers, phenotype, function or metabolism is observed in the peripheral blood or in response to polyclonal stimulation ex vivo. However, long-term in vivo hyporesponsiveness is associated with a significant decrease in the frequency of antigen-specific T cells producing IFN-γ upon antigen restimulation ex vivo. These findings indicate that chronic antigen-specific memory T cell responses can be controlled by anti-IL-7Rα mAbs, promoting and maintaining remission in T-cell mediated chronic inflammatory diseases.

Rebooting Human Immunology.

Recent progress in both conceptual and technological approaches to human immunology have rejuvenated a field that has long been in the shadow of the inbred mouse model. This is a healthy development both for the clinical relevance of immunology and for the fact that it is a way to gain access to the wealth of phenomenology in the many human diseases that involve the immune system. This is where we are likely to discover new immunological mechanisms and principals, especially those involving genetic heterogeneity or environmental influences that are difficult to model effectively in inbred mice. We also suggest that there are likely to be novel immunological mechanisms in long-lived, less fecund mammals such as human beings since they must remain healthy far longer than short-lived rodents in order for the species to survive.

T-cell egress from the thymus: Should I stay or should I go?

T-cells bearing the αßTCR play a vital role in defending the host against foreign pathogens and malignant transformation of self. Importantly, T-cells are required to remain tolerant to the host's own cells and tissues in order to prevent self-reactive responses that can lead to autoimmune disease. T-cells achieve the capacity for self/nonself discrimination by undergoing a highly selective and rigorous developmental program during their maturation in the thymus. This organ is unique in its ability to support a program of T-cell development that ensures the establishment of a functionally diverse αßTCR repertoire within the peripheral T-cell pool. The thymus achieves this by virtue of specialized stromal microenvironments that contain heterogeneous cell types, whose organization and function underpins their ability to educate, support, and screen different thymocyte subsets through various stages of development. These stages range from the entry of early T-cell progenitors into the thymus, through to the positive and negative selection of the αßTCR repertoire. The importance of the thymus medulla as a site for T-cell tolerance and the exit of newly generated T-cells into the periphery is well established. In this review, we summarize current knowledge on the developmental pathways that take place during αßT-cell development in the thymus. In addition, we focus on the mechanisms that regulate thymic egress and contribute to the seeding of peripheral tissues with newly selected self-tolerant αßT-cells.

Immune monitoring of transplant patients in transient mixed chimerism tolerance trials.

This review focuses on mechanistic studies performed in recipients of non-myeloablative bone marrow transplant regimens developed at Massachusetts General Hospital in HLA-identical and HLA-mismatched haploidentical combinations, initially as a platform for treatment of hematologic malignancies with immunotherapy in the form of donor leukocyte infusions, and later in combination with donor kidney transplantation for the induction of allograft tolerance. In patients with permanent mixed chimerism, central deletion may be a major mechanism of long-term tolerance. In patients in whom donor chimerism is only transient, the kidney itself plays a significant role in maintaining long-term tolerance. A high throughput sequencing approach to identifying and tracking a significant portion of the alloreactive T cell receptor repertoire has demonstrated biological significance in transplant patients and has been useful in pointing to clonal deletion as a long-term tolerance mechanism in recipients of HLA-mismatched combined kidney and bone marrow transplants with only transient chimerism.

Mode of Tolerance Induction and Requirement for Aire Are Governed by the Cell Types That Express Self-Antigen and Those That Present Antigen.

Aire controls the fate of autoreactive thymocytes (i.e., clonal deletion or development into regulatory T cells [Tregs]) through transcriptional control of the expression of tissue-restricted self-antigens (TRAs) from medullary thymic epithelial cells (mTECs) and bone marrow (BM)-derived cells. Although TRAs expressed by mTECs and BM-derived cells are suggested to complement each other to generate a full spectrum of TRAs, little is known about the relative contribution of TRAs from each component for establishment of self-tolerance. Furthermore, the precise role of Aire in specific types of Aire-expressing APCs remains elusive. We have approached these issues by generating two different types of transgenic mouse (Tg) model, which express a prefixed model self-antigen driven by the insulin promoter or the Aire promoter. In the insulin-promoter Tg model, mTECs alone were insufficient for clonal deletion, and BM-derived APCs were required for this action by utilizing Ag transferred from mTECs. In contrast, mTECs alone were able to induce Tregs, although at a much lower efficiency in the absence of BM-derived APCs. Importantly, lack of Aire in mTECs, but not in BM-derived APCs, impaired both clonal deletion and production of Tregs. In the Aire-promoter Tg model, both mTECs and BM-derived APCs could independently induce clonal deletion without Aire, and production of Tregs was impaired by the lack of Aire in mTECs, but not in BM-derived APCs. These results suggest that the fate of autoreactive thymocytes together with the requirement for Aire depend on the cell types that express self-antigens and the types of APCs involved in tolerance induction.

Tolerance is established in polyclonal CD4(+) T cells by distinct mechanisms, according to self-peptide expression patterns.

Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns.

Clonal Deletion Established via Invariant NKT Cell Activation and Costimulatory Blockade Requires In Vivo Expansion of Regulatory T Cells.

Recently, the immune-regulating potential of invariant natural killer T (iNKT) cells has attracted considerable attention. We previously reported that a combination treatment with a liposomal ligand for iNKT cells and an anti-CD154 antibody in a sublethally irradiated murine bone marrow transplant (BMT) model resulted in the establishment of mixed hematopoietic chimerism through in vivo expansion of regulatory T cells (Tregs). Herein, we show the lack of alloreactivity of CD8(+) T cells in chimeras and an early expansion of donor-derived dendritic cells (DCs) in the recipient thymi accompanied by a sequential reduction in the donor-reactive Vß-T cell receptor repertoire, suggesting a contribution of clonal deletion in this model. Since thymic expansion of donor DCs and the reduction in the donor-reactive T cell repertoire were precluded with Treg depletion, we presumed that Tregs should preform before the establishment of clonal deletion. In contrast, the mice thymectomized before BMT failed to increase the number of Tregs and to establish CD8(+) T cell tolerance, suggesting the presence of mutual dependence between the thymic donor-DCs and Tregs. These results provide new insights into the regulatory mechanisms that actively promote clonal deletion.

Thymic involution perturbs negative selection leading to autoreactive T cells that induce chronic inflammation.

Thymic involution and the subsequent amplified release of autoreactive T cells increase the susceptibility toward developing autoimmunity, but whether they induce chronic inflammation with advanced age remains unclear. The presence of chronic low-level proinflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality in virtually every chronic age-related disease. To determine how thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a Foxn1 conditional knockout mouse model that induces accelerated thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by a chronic inflammatory phenotype consisting of cellular infiltration into non-lymphoid tissues, increased TNF-α production, and elevated serum IL-6. Autoreactive T cell clones were detected in the periphery of Foxn1 conditional knockout mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related regulatory T cell accumulation in naturally aged mice, but not inflammatory infiltration. Taken together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a contributing source of chronic inflammation (inflammaging).

BIM Deficiency Protects NOD Mice From Diabetes by Diverting Thymocytes to Regulatory T Cells.

Because regulatory T-cell (Treg) development can be induced by the same agonist self-antigens that induce negative selection, perturbation of apoptosis will affect both negative selection and Treg development. But how the processes of thymocyte deletion versus Treg differentiation bifurcate and their relative importance for tolerance have not been studied in spontaneous organ-specific autoimmune disease. We addressed these questions by removing a critical mediator of thymocyte deletion, BIM, in the NOD mouse model of autoimmune diabetes. Despite substantial defects in the deletion of autoreactive thymocytes, BIM-deficient NOD (NODBim(-/-)) mice developed less insulitis and were protected from diabetes. BIM deficiency did not impair effector T-cell function; however, NODBim(-/-) mice had increased numbers of Tregs, including those specific for proinsulin, in the thymus and peripheral lymphoid tissues. Increased levels of Nur77, CD5, GITR, and phosphorylated IκB-α in thymocytes from NODBim(-/-) mice suggest that autoreactive cells receiving strong T-cell receptor signals that would normally delete them escape apoptosis and are diverted into the Treg pathway. Paradoxically, in the NOD model, reduced thymic deletion ameliorates autoimmune diabetes by increasing Tregs. Thus, modulating apoptosis may be one of the ways to increase antigen-specific Tregs and prevent autoimmune disease.

Differential roles for Bim and Nur77 in thymocyte clonal deletion induced by ubiquitous self-antigen.

Negative selection, primarily mediated through clonal deletion of self-reactive thymocytes, is critical for establishing self-tolerance and preventing autoimmunity. Recent studies suggest that the molecular mechanisms of negative selection differ depending on the thymic compartment and developmental stage at which thymocytes are deleted. Using the physiological HY(cd4) TCR transgenic model of negative selection against ubiquitous self-antigen, we previously found that one of the principal mediators implicated in clonal deletion, Bim, is required for caspase-3 activation but is ultimately dispensable for negative selection. On the basis of these data, we hypothesized that Nur77, another molecule thought to be a key mediator of clonal deletion, could be responsible for Bim-independent deletion. Despite comparable Nur77 induction in thymocytes during negative selection, Bim deficiency resulted in an accumulation of high-affinity-signaled thymocytes as well as impairment in caspase-mediated and caspase-independent cell death. Although these data suggested that Bim may be required for Nur77-mediated cell death, we found that transgenic Nur77 expression was sufficient to induce apoptosis independently of Bim. However, transgenic Nur77-induced apoptosis was significantly inhibited in the context of TCR signaling, suggesting that endogenous Nur77 could be similarly regulated during negative selection. Although Nur77 deficiency alone did not alter positive or negative selection, combined deficiency in Bim and Nur77 impaired clonal deletion efficiency and significantly increased positive selection efficiency. Collectively, these data shed light on the different roles for Bim and Nur77 during ubiquitous Ag-mediated clonal deletion and highlight potential differences from their reported roles in tissue-restricted Ag-mediated clonal deletion.

Surrogate light chain expression beyond the pre-B cell stage promotes tolerance in a dose-dependent fashion.

While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.

The self-obsession of T cells: how TCR signaling thresholds affect fate 'decisions' and effector function.

Self-reactivity was once seen as a potential characteristic of T cells that was eliminated by clonal selection to protect the host from autoimmune pathology. It is now understood that the T cell repertoire is in fact broadly self-reactive, even self-centered. The strength with which a T cell reacts to self ligands and the environmental context in which this reaction occurs influence almost every aspect of T cell biology, from development to differentiation to effector function. Here we highlight recent advances and discoveries that relate to T cell self-reactivity, with a particular emphasis on T cell antigen receptor (TCR) signaling thresholds.

Elevated T cell receptor signaling identifies a thymic precursor to the TCRαß(+)CD4(-)CD8ß(-) intraepithelial lymphocyte lineage.

The origin and developmental pathway of intestinal T cell receptor αß(+) CD4(-)CD8ß(-) intraepithelial lymphocytes (unconventional iIELs), a major population of innate-like resident cytolytic T cells, have remained elusive. By cloning and expressing several TCRs isolated from unconventional iIELs, we identified immature CD4(lo)CD8(lo)(DP(lo))CD69(hi)PD-1(hi) thymocytes as the earliest postsignaling precursors for these cells. Although these precursors displayed multiple signs of elevated TCR signaling, a sizeable fraction of them escaped deletion to selectively engage in unconventional iIEL differentiation. Conversely, TCRs cloned from DP(lo)CD69(hi)PD-1(hi) thymocytes, a population enriched in autoreactive thymocytes, selectively gave rise to unconventional iIELs upon transgenic expression. Thus, the unconventional iIEL precursor overlaps with the DP(lo) population undergoing negative selection, indicating that, concomitant with the downregulation of both CD4 and CD8 coreceptors, a balance between apoptosis and survival signals results in outcomes as divergent as clonal deletion and differentiation to the unconventional iIEL lineage.