Establishment of CD8+ T Cell Thymic Central Tolerance to Tissue-Restricted Antigen Requires PD-1.

Highly self-reactive T cells are censored from the repertoire by both central and peripheral tolerance mechanisms upon receipt of high-affinity TCR signals. Clonal deletion is considered a major driver of central tolerance; however, other mechanisms such as induction of regulatory T cells and functional impairment have been described. An understanding of the interplay between these different central tolerance mechanisms is still lacking. We previously showed that impaired clonal deletion to a model tissue-restricted Ag did not compromise tolerance. In this study, we determined that murine T cells that failed clonal deletion were rendered functionally impaired in the thymus. Programmed cell death protein 1 (PD-1) was induced in the thymus and was required to establish cell-intrinsic tolerance to tissue-restricted Ag in CD8+ thymocytes independently of clonal deletion. In bone marrow chimeras, tolerance was not observed in PD-L1-deficient recipients, but tolerance was largely maintained following adoptive transfer of tolerant thymocytes or T cells to PD-L1-deficient recipients. However, CRISPR-mediated ablation of PD-1 in tolerant T cells resulted in broken tolerance, suggesting different PD-1 signaling requirements for establishing versus maintaining tolerance. Finally, we showed that chronic exposure to high-affinity Ag supported the long-term maintenance of tolerance. Taken together, our study identifies a critical role for PD-1 in establishing central tolerance in autoreactive T cells that escape clonal deletion. It also sheds light on potential mechanisms of action of anti-PD-1 pathway immune checkpoint blockade and the development of immune-related adverse events.

Tackling cancer cell dormancy: Insights from immune models, and transplantation.

Disseminated non-dividing (dormant) cancer cells as well as those in equilibrium with the immune response remain the major challenge for successful treatment of cancer. The equilibrium between disseminated dormant cancer cells and the immune system is reminiscent of states that can occur during infection or allogeneic tissue and cell transplantation. We discuss here the major competing models of how the immune system achieves a self nonself discrimination (pathogen/danger patterns, quorum, and coinhibition/tuning models), and suggest that taking advantage of a combination of the proposed mechanisms in each model may lead to increased efficacy in tackling cancer cell dormancy.

Endothelial phosphoinositide 3-kinase-ß inactivation confers protection from immune-mediated vascular injury.

Heart transplant and recipient survival are limited by immune cell-mediated injury of the graft vasculature. We examined the role of the phosphoinositide 3-kinase-ß (PI3Kß) isoform in endothelial cells (EC) during coronary vascular immune injury and repair in mice. In minor histocompatibility-antigen mismatched allogeneic heart grafts, a robust immune response was mounted to each wild-type, PI3Kß inhibitor-treated, or endothelial-selective PI3Kß knockout (ECßKO) graft transplanted to wild-type recipients. However, microvascular EC loss and progressive occlusive vasculopathy only developed in control, but not PI3Kß-inactivated hearts. We observed a delay in inflammatory cell infiltration of the ECßKO grafts, particularly in the coronary arteries. Surprisingly, this was accompanied by an impaired display of proinflammatory chemokine and adhesion molecules by the ECßKO ECs. In vitro, tumor necrosis factor α-stimulated endothelial ICAM1 and VCAM1 expression was blocked by PI3Kß inhibition or RNA interference. Selective PI3Kß inhibition also blocked tumor necrosis factor α-stimulated degradation of inhibitor of nuclear factor kappa Bα and nuclear translocation of nuclear factor kappa B p65 in EC. These data identify PI3Kß as a therapeutic target to reduce vascular inflammation and injury.

On antigen-specific signals, immune class regulation and energetics: Report III from the workshops on foundational concepts of immune regulation.

This is a report from a one-week workshop held in Athens, Greece in July of 2022. The workshop aimed to identify emerging concepts relevant to the fundamentals of immune regulation and areas for future research. Theories of immune regulation emphasize the role of T cell help or co-stimulation (signal 2). The workshop participants considered how new data on the characteristics of agonist antigens, the role of the antigen receptor signals (signal 1) in driving fate decisions, the effect of energetics on immunity and a better understanding of class-control in the immune response, may impact theories of immune regulation. These ideas were discussed in the context of tumour immunology, autoimmunity, pregnancy and transplantation. Here we present the discussions as a narrative of different viewpoints to allow the reader to join the conversation. These discussions highlight the evolving understanding of the nature of specific antigen recognition and how both antigen-specific and non-specific mechanisms impact immune responses.

Metabolic Consequences of IgE- and Non-IgE-Mediated Mast Cell Degranulation.

Mast cells are important effector cells in the immune system and undergo activation (i.e., degranulation) by two major mechanisms: IgE-mediated and non-IgE-mediated mechanisms. Although IgE-mediated degranulation is well researched, the cellular mechanisms of non-IgE-mediated mast cell activation are poorly understood despite the potential to induce similar pathophysiological effects. To better understand non-IgE mast cell degranulation, we characterized and compared cellular metabolic shifts across several mechanisms of degranulation (allergen-induced [IgE-mediated], 20 nm of silver nanoparticle-mediated [non-IgE], and compound 48/80-mediated [non-IgE]) in murine bone marrow-derived mast cells. All treatments differentially impacted mitochondrial activity and glucose uptake, suggesting diverging metabolic pathways between IgE- and non-IgE-mediated degranulation. Non-IgE treatments depleted mast cells' glycolytic reserve, and compound 48/80 further inhibited the ability to maximize mitochondrial respiration. This cellular reprogramming may be indicative of a stress response with non-IgE treatments. Neither of these outcomes occurred with IgE-mediated degranulation, hinting at a separate programmed response. Fuel flexibility between the three primary mitochondrial nutrient sources was also eliminated in activated cells and this was most significant in non-IgE-mediated degranulation. Lastly, metabolomics analysis of bone marrow-derived mast cells following degranulation was used to compare general metabolite profiles related to energetic pathways. IgE-mediated degranulation upregulated metabolite concentrations for the TCA cycle and glycolysis compared with other treatments. In conclusion, mast cell metabolism varies significantly between IgE- and non-IgE-mediated degranulation suggesting novel cell regulatory mechanisms are potentially driving unexplored pathways of mast cell degranulation.

Anti-CD52 blocks EAE independent of PD-1 signals and promotes repopulation dominated by double-negative T cells and newly generated T and B cells.

Lymphocyte depletion using anti-CD52 antibody effectively reduces relapses of multiple sclerosis (MS). To begin to understand what mechanisms might control this outcome, we examined the effect of a murine-CD52-specific mAb on the depletion and repopulation of immune cells in mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We tested whether the tolerance-promoting receptor programmed cell death protein-1 (PD-1) is required for disease remission post anti-CD52, and found that PD-1-deficient mice with a more severe EAE were nevertheless effectively treated with anti-CD52. Anti-CD52 increased the proportions of newly generated T cells and double-negative (DN) T cells while reducing newly generated B cells; the latter effect being associated with a higher expression of CD52 by these cells. In the longer term, anti-CD52 caused substantial increases in the proportion of newly generated lymphocytes and DN T cells in mice with EAE. Thus, the rapid repopulation of lymphocytes from central lymphoid organs post anti-CD52 may limit further disease. Furthermore, these data identify DN T cells, a subset with immunoregulatory potential, as a significant hyperrepopulating subset following CD52-mediated depletion.

CD5 levels reveal distinct basal T-cell receptor signals in T cells from non-obese diabetic mice.

Type 1 diabetes in non-obese diabetic (NOD) mice occurs when autoreactive T cells eliminate insulin producing pancreatic ß cells. While extensively studied in T-cell receptor (TCR) transgenic mice, the contribution of alterations in thymic selection to the polyclonal T-cell pool in NOD mice is not yet resolved. The magnitude of signals downstream of TCR engagement with self-peptide directs the development of a functional T-cell pool, in part by ensuring tolerance to self. TCR interactions with self-peptide are also necessary for T-cell homeostasis in the peripheral lymphoid organs. To identify differences in TCR signal strength that accompany thymic selection and peripheral T-cell maintenance, we compared CD5 levels, a marker of basal TCR signal strength, on immature and mature T cells from autoimmune diabetes-prone NOD and -resistant B6 mice. The data suggest that there is no preferential selection of NOD thymocytes that perceive stronger TCR signals from self-peptide engagement. Instead, NOD mice have an MHC-dependent increase in CD4+ thymocytes and mature T cells that express lower levels of CD5. In contrast, T cell-intrinsic mechanisms lead to higher levels of CD5 on peripheral CD8+ T cells from NOD relative to B6 mice, suggesting that peripheral CD8+ T cells with higher basal TCR signals may have survival advantages in NOD mice. These differences in the T-cell pool in NOD mice may contribute to the development or progression of autoimmune diabetes.

The historical postulate is not the basis of self-nonself discrimination: A response to Bretscher's proposal.

Peter Bretscher was the first to envision that the problem of self-nonself discrimination in the adaptive immune system could be solved by positing that antigen inactivates single lymphocytes, whereas antigen-mediated lymphocyte cooperation is required to stimulate their activation. These ideas led to a two-signal model for lymphocyte activation: an antigen-specific signal that when generated alone results in tolerance and the combination of two (or more) antigen-specific signals resulting in lymphocyte activation and immunity. This 'quorum model' is consistent with the concept known as the historical postulate that posits that the early life timing of antigen exposure is the key to self-tolerance. Bretscher proposes that the historical postulate is 'the basis, at level of the system, for self-nonself discrimination' and contends that the Danger model violates this postulate. Herein I argue that the data do not support putting the historical postulate alone at the top of the hierarchy of concepts underlying self-nonself discrimination. The location of antigen is at least as important because it determines whether central tolerance will be engaged. Location of antigen together with timing of antigen exposure are major factors determining whether quorum, the basis for self-nonself discrimination, is achieved.

Trisomy 21 impairs PGE2 production in dermal fibroblasts.

The triplication of human chromosome 21 results in Down syndrome (DS), the most common genetic form of intellectual disability. This aneuploid condition also results in an enhanced risk of a spectrum of comorbid conditions, such as leukemia, early onset Alzheimer's disease, and diabetes. Individuals with DS also display an increased incidence of wound healing complications and resistance to solid tumor development. Due to this unique phenotype and the involvement of eicosanoids in key comorbidities like poor healing and tumor development, we hypothesized that cells from DS individuals would display altered eicosanoid production. Using age- and sex-matched dermal fibroblasts we interrogated this hypothesis. Briefly, assessment of over 90 metabolites derived from cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome p450 systems revealed a possible deficiency in the COX system. Basal gene expression and Western blotting experiments showed significantly decreased gene expression of COX1 and 2, and COX2 protein abundance in DS fibroblasts compared to euploid controls. Further, using two different stressors, scratch wound or LPS, we found that DS fibroblasts could not upregulate COX2 abundance and prostaglandin E2 production. Together, these findings show that dermal fibroblasts from DS individuals have a deficient COX2 response, which may contribute to wound healing complications and tumor resistance in DS.

Desensitization using imlifidase and EndoS enables chimerism induction in allosensitized recipient mice.

Mixed hematopoietic chimerism induction as a way to foster tolerance to donor organs in recipients who have been sensitized to donor antigens is challenging. Donor-specific antibodies (DSA) are a dominant barrier toward successful donor bone marrow engraftment. Although desensitization methods are routinely used in recipients with allosensitization for allogeneic bone marrow transplantation, engraftment is frequently unsuccessful. To overcome the barrier of prior sensitization we tested enzymatic desensitization of donor-specific IgG using imlifidase and endoglycosidase of Streptococcus pyogenes (EndoS), which both partially block the function of DSA in mice, as a novel approach to improve murine bone marrow engraftment in primed hosts. We found that EndoS was capable of inhibiting antibody-mediated killing of donor cells in vivo. Furthermore, the effect of EndoS depended on the titer of DSA and the genetic background of the recipients. In combination with imlifidase, EndoS improved the survival of donor bone marrow cells. Together with cyclophosphamide, bortezomib, T cell depletion, and nonlethal irradiation, imlifidase in combination with EndoS allowed allogeneic bone marrow engraftment in sensitized recipients. We conclude that enzymatic inactivation of DSA, using the combination of imlifidase and EndoS, can be used for inducing donor hematopoietic chimerism in allosensitized recipient mice in combination with other desensitization strategies.

Significant association between tumor mutational burden and immune-related adverse events during immune checkpoint inhibition therapies.

More than 2000 immuno-oncology agents are being tested or are in use as a result of the cancer immunotherapy revolution. Manipulation of co-inhibitory receptors has achieved tumor eradication in a minority of patients, but widespread immune-related adverse events (irAEs) compromised tolerance to healthy self-tissues in the majority. We have proposed that a major mechanism of irAEs is similar to a graft-versus-malignancy effect of graft-versus-host disease. To verify our hypothesis, we retrieved post-marketing data of adverse events from the U.S. Food and Drug Administration Adverse Event Reporting System. A significant positive correlation was revealed in 7677 patients between the reporting odds ratio of irAEs during immune checkpoint inhibitor therapy and the corresponding tumor mutational burden across 19 cancer types. These results can be interpreted to mean that the ICI drugs unleashed T cells against "altered-self," self, and tumors resulting in better overall survival.

On T cell development, T cell signals, T cell specificity and sensitivity, and the autoimmunity facilitated by lymphopenia.

We propose a framework to explain how T cells achieve specificity and sensitivity, how the affinity of the TcR peptide/MHC interaction controls positive and negative thymic selection and mature T cell survival, and whether antigen-dependent activation and inactivation takes place. Two distinct types of signalling can lead to mature T cell multiplication. One requires the TcR to recognize with a certain affinity an antigen-derived peptide, an agonist peptide, bound to an MHC molecule. The other, the tonic signal, leads to naïve T cell survival and modest proliferation if the T cell successfully competes for endogenous, self-peptide/MHC ligands, involving lower affinity TCR/ligand interactions. Many suggest lymphopenia contributes to autoimmunity by increasing the strength of TcR-tonic signalling, and so activation of anti-self T cells. We suggest T cell activation requires antigen-mediated cooperation between T cells. Increased tonic signalling under lymphopenic conditions facilitates T cell proliferation and so antigen-dependent cooperation and activation of anti-self T cells.

The case for allele-specific recognition by the TCR.

There is a sharp difference in how one views TCR structure-function-behaviour dependent on whether its recognition of major histocompatibility complex-encoded restriction elements (R) is germline selected or somatically generated. The generally accepted or Standard model is built on the assumption that recognition of R is by the V regions of the αß TCR, which is not driven by allele specificity, whereas the competing model posits that recognition of R is allele-specific. The establishing of allele-specific recognition of R by the TCR would rule out the Standard model and clear the road to a consideration of a competing construct, the Tritope model. Here, the case for allele-specific recognition (germline selected) is detailed making it obvious that the Standard model is untenable.

Exploiting autoimmunity unleashed by low-dose immune checkpoint blockade to treat advanced cancer.

As a result of the cancer immunotherapy revolution, more than 2000 immuno-oncology agents are currently being tested or are in use to improve responses. Not unexpectedly, the 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo for their development of cancer therapy by the blockade of co-inhibitory signals. Unfortunately, manipulation of the co-inhibitory receptors has also resulted in a safety issue: widespread iatrogenic immune-related adverse events (irAEs). Autoimmunity is emerging as the nemesis of immunotherapy. Originally, it was assumed that CTLA-4 blockade selectively targets T cells relevant to the antitumour immune response. However, an uncontrolled pan T cell activation was induced compromising tolerance to healthy self-tissues. The irAEs are very similar to that of a chronic graft-versus-host-disease (GVHD) reaction following allogeneic bone marrow transplantation (BMT). We hypothesized that ipilimumab induced a graft-versus-malignancy (GVM) effect, which eradicated metastatic melanoma in a minority of patients, but also involved an auto-GVHD reaction that resulted in widespread autoimmunity in the majority. Therefore, we argued for a profound theoretical point against the consensus of experts. The task is not to desperately put the genie back in the bottle by immune-suppressive treatments, but instead to harness the autoimmune forces. In this way, the same goal could be achieved by an antibody as by the adoptive transfer of alloreactive donor lymphocytes, but without severe GVHD. The proof-of-principle of a low-dose-combination immune checkpoint therapy, consisting only of approved drugs and treatments, was demonstrated in 111 stage IV cancer patients.

Two Strikes and You're Out? The Pathogenic Interplay of Coinhibitor Deficiency and Lymphopenia-Induced Proliferation.

Lymphopenia-induced proliferation (LIP) occurs when resources for T cell survival in a host are in excess. LIP has been associated with the development of inflammatory disease in situations where an additional disease-predisposing cofactor is present during LIP. This has led to the view of LIP-driven autoimmunity as a two hit model; however, not all cofactors have equal ability to precipitate autoimmunity and we have recently shown that in some circumstances, such as the absence of the coinhibitory molecule PD-1, additional hits are required. Herein we review factors controlling LIP, including coinhibitory molecules and other attenuators of TCR signaling, with a focus on their contribution to LIP-driven autoimmunity. Rather than viewing LIP-associated autoimmunity as an n-hit model, we suggest a more quantitative view of lymphopenia with respect to the factors that promote LIP as a tool to predict autoimmune potential and to inform tumor immunotherapy approaches.

Best practices for enhancing surgical research: a perspective from the Canadian Association of Chairs of Surgical Research

Summary: The Canadian Association of Chairs of Surgical Research was created in 2014, with representation from every departmental surgical research committee across Canada, to establish Canadian surgical research as a beacon for health care innovation and to propose solutions for the daily challenges facing surgeon-researchers. Our key mandate has been to identify challenges for surgeons and scientists performing research to prevent further erosion of this vital area of activity that benefits patients, health care service providers and Canadian society. This article outlines the findings of a nationwide survey sent to all members of departments of surgery across Canada, seeking input on current threats and potential solutions. The results suggest that surgical research in Canada is experiencing a decline in funding and an increase in challenges affecting research productivity of academic surgeons, such as pressures to be clinically active, unpredictable surgical schedules, growing administrative demands, and increasing complexity of patient populations. Although surgeons are productive in their research endeavours, institutional changes and sharing of best practices are needed to ensure sustainable growth of research programs.

Application of central immunologic concepts to cancer: helping T cells and B cells become intolerant of tumors.

CD4-mediated T-cell help in the activation of CD8(+) T cells and B cells, through linked-recognition of antigenic determinants, is a long-standing concept foundational to our understanding of immunity (presence of help) versus tolerance (lack of help). Surprisingly, this function of CD4(+) T cells has not been extensively examined as a means to overcome immune tolerance of the self-antigens made by tumor cells. Hesitation to employ this powerful mechanism may be due to the potential to cause unwanted autoimmune pathology. In this issue of the European Journal of Immunology, Snook et al. [Eur. J. Immunol. 2014. 44: 1956-1966] identify a state of split tolerance, showing that CD4(+) T cells specific for a number of tumor-associated self-antigens are robustly tolerant, while their CD8(+) T-cell and B-cell counterparts are far less tolerant. Furthermore, the authors demonstrate that provision of linked foreign helper epitopes, such as influenza hemagglutinin, substantially enhances both CD8(+) T-cell and B-cell responses to tumor self-antigens without causing any overt autoimmune pathology. These findings provide a strong rationale to employ foreign helper epitopes in cancer vaccines and highlight the need to fully explore therapeutic strategies that are based on well-established immunologic concepts.

PD-1 is not required for natural or peripherally induced regulatory T cells: Severe autoimmunity despite normal production of regulatory T cells.

The expression of the coinhibitor PD-1 on T cells is important for the establishment of immune homeostasis. We previously found that PD-1 is particularly critical for the control of self-tolerance during lymphopenia-induced proliferation of recent thymic emigrants (RTEs). Previous studies suggested that PD-1 modulates the generation of Treg cells, particularly peripherally induced Treg (pTreg) cells, and controls Th17 cells. However, these conclusions were derived indirectly from studies on the ligand PD-L1, and not PD-1 itself. Herein we directly tested whether T-cell PD-1 expression was needed for Treg cell generation and examined if a paucity of Treg cells or enhanced Th17 cells could explain the severe lymphopenia-potentiated autoimmunity caused by PD-1 KO RTEs. Employing the murine FoxP3(EGFP) reporter system to simultaneously monitor conversion of WT and PD-1 KO T cells to pTreg cells in the same animal, we found that PD-1 deficiency did not inhibit pTreg cell generation or lead to Th17-cell-mediated autoimmunity. Surprisingly, pTreg cell numbers were increased in PD-1 KO versus WT cell populations. Furthermore, we noted an increased conversion to pTreg cells by RTEs. Our data suggest that the primary role for PD-1 is to restrain T-cell activation/proliferation to self-Ags rather than promote generation of Treg cells.

T cells generated in the absence of a thoracic thymus fail to establish homeostasis.

Cervical thymus mimics the thoracic thymus in supporting T-cell development and exists in a subset of mice and humans. Importantly, it remains unknown whether the cervical thymus can generate T cells that are self-tolerant in the complete absence of signals from the thoracic thymus. Using a fetal liver reconstitution model in thoracic thymectomized RAG(-/-) mice, we found that T cells could be generated without contribution from the thoracic thymus. However, these mice had decreased T cells, increased proportions of effector memory T cells and Treg phenotype cells, increased serum IgG1/2b, and increased frequency of T cells expressing IFN-γ, IL-17 or IL-10. Half of the mice that received a thoracic thymectomy and fetal liver cells, unlike sham surgery controls, developed substantial morbidity with age. Disease was associated with lymphopenia-driven activation rather than inherent defects in the cervical thymus, as both thoracic and cervical thymocytes could generate disease in lymphopenic recipients. Administration of the homeostatic cytokine IL-7 caused a rapid, transient increase in T-cell numbers and reduced the time to disease onset. Together the data suggests that the cervical thymus can function in the complete absence of the thoracic thymus; however, the T cells generated do not establish homeostasis.

Control of in vivo collateral damage generated by T cell immunity.

An ongoing dilemma faced during an immune response is generating an effective, often proinflammatory response to eliminate pathogens and/or infected cells while also minimizing collateral damage to adjacent noninfected tissues. The factors limiting bystander cell injury during an Ag-specific immune response in vivo are largely unknown. In this study, using an in vivo model of islet transplants in TCR transgenic mice, we show that both CD4 and CD8 T cells do have the capacity to inflict adjacent tissue damage and that this injury is greatly enhanced in sensitized hosts. CD4 T cell-mediated killing of specific and bystander cells occurred via different mechanisms. Unlike specific target cell killing, CD4-mediated bystander injury required tissue Fas expression and was inhibited with anti-IFN-γ Ab treatment in vivo. Moreover, bystander cell injury was not entirely nonspecific but rather required, in naive recipients, that the MHC allele expressed by the bystanders was self. Importantly, the coinhibitor programmed death-1 plays an important role in restraining bystander cell injury mediated either by defined TCR transgenic T cells or by polyclonal T cell populations. Thus, the differential requirements for specific versus bystander cell injury suggest that there are opportunities for inhibiting immune pathology without compromising Ag-specific immunity in vivo.