T cell antigenicity and immunogenicity of allogeneic exosomes

Extracellular vesicles, including exosomes, are regularly released by allogeneic cells after transplantation. Recipient antigen-presenting cells (APCs) capture these vesicles and subsequently display donor MHC molecules on their surface. Recent evidence suggests that activation of alloreactive T cells by the so-called cross-dressed APCs plays an important role in initiating the alloresponse associated with allograft rejection. On the other hand, whether allogeneic exosomes can bind to T cells on their own and activate them remains unclear. In this study, we showed that allogeneic exosomes can bind to T cells but do not stimulate them in vitro unless they are cultured with APCs. On the other hand, allogeneic exosomes activate T cells in vivo and sensitize mice to alloantigens but only when delivered in an inflammatory environment.     

Adoptive Transfer of Tracer‐Alloreactive CD4+ T Cell Receptor Transgenic T Cells Alters the Endogenous Immune Response to an Allograft

T cell receptor transgenic (TCR‐Tg) T cells are often used as tracer populations of antigen‐specific responses to extrapolate findings to endogenous T cells. The extent to which TCR‐Tg T cells behave purely as tracer cells or modify the endogenous immune response is not clear. To test the impact of TCR‐Tg T cell transfer on endogenous alloimmunity, recipient mice were seeded with CD4⁺ or CD8⁺ TCR‐Tg or polyclonal T cells at the time of cardiac allograft transplantation. Only CD4⁺ TCR‐Tg T cells accelerated rejection and, unexpectedly, led to a dose‐dependent decrease in both transferred and endogenous T cells infiltrating the graft. In contrast, recipients of CD4⁺ TCR‐Tg T cells exhibited enhanced endogenous donor‐specific CD8⁺ T cell activation in the spleen and accelerated alloantibody production. Introduction of CD4⁺ TCR‐Tg T cells also perturbed the intragraft accumulation of innate cell populations. Transferred CD4⁺ TCR‐Tg T cells alter many aspects of endogenous alloimmunity, suggesting that caution should be used when interpreting experiments using these adoptively transferred cells because the overall nature of allograft rejection may be altered. These results also may have implications for adoptive CD4⁺ T cell immunotherapy in tumor and infectious clinical settings because cell infusion may have additional effects on natural immune responses.     

A Memory B Cell Crossmatch Assay for Quantification of Donor‐Specific Memory B Cells in the Peripheral Blood of HLA‐Immunized Individuals

Humoral responses against mismatched donor HLA are routinely measured as serum HLA antibodies, which are mainly produced by bone marrow–residing plasma cells. Individuals with a history of alloimmunization but lacking serum antibodies may harbor circulating dormant memory B cells, which may rapidly become plasma cells on antigen reencounter. Currently available methods to detect HLA‐specific memory B cells are scarce and insufficient in quantifying the complete donor‐specific memory B cell response due to their dependence on synthetic HLA molecules. We present a highly sensitive and specific tool for quantifying donor‐specific memory B cells in peripheral blood of individuals using cell lysates covering the complete HLA class I and class II repertoire of an individual. Using this enzyme‐linked immunospot (ELISpot) assay, we found a median frequency of 31 HLA class I and 89 HLA class II–specific memory B cells per million IgG‐producing cells directed at paternal HLA in peripheral blood samples from women (n = 22) with a history of pregnancy, using cell lysates from spouses. The donor‐specific memory B cell ELISpot can be used in HLA diagnostic laboratories as a cross‐match assay to quantify donor‐specific memory B cells in patients with a history of sensitizing events.     

Triple (GGTA1, CMAH,B2M) modified pigs expressing an SLA class Ilow phenotype—Effects on immune status and susceptibility to human immune responses

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9‐mediated deletion of β₂‐microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8⁺ T cell subset was observed in B2M^low pigs. Cells from B2M^low pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8⁺ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4⁺ T cells, natural killer cells) lysed targets from both SLA class I⁺ wildtype and SLA class I^low pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8⁺ T cells during the induction phase of an anti‐xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.     

Inflammation Causes Resistance to Anti‐CD20–Mediated B Cell Depletion

B cells play a central role in antibody‐mediated rejection and certain autoimmune diseases. However, B cell–targeted therapy such as anti‐CD20 B cell–depleting antibody (aCD20) has yielded mixed results in improving outcomes. In this study, we investigated whether an accelerated B cell reconstitution leading to aCD20 depletion resistance could account for these discrepancies. Using a transplantation model, we found that antigen‐independent inflammation, likely through toll‐like receptor (TLR) signaling, was sufficient to mitigate B cell depletion. Secondary lymphoid organs had a quicker recovery of B cells when compared to peripheral blood. Inflammation altered the pharmacokinetics (PK) and pharmacodynamics (PD) of aCD20 therapy by shortening drug half‐life and accelerating the reconstitution of the peripheral B cell pool by bone marrow–derived B cell precursors. IVIG (intravenous immunoglobulin) coadministration also shortened aCD20 drug half‐life and led to accelerated B cell recovery. Repeated aCD20 dosing restored B cell depletion and delayed allograft rejection, especially B cell–dependent, antibody‐independent allograft rejection. These data demonstrate the importance of further clinical studies of the PK/PD of monoclonal antibody treatment in inflammatory conditions. The data also highlight the disconnect between B cell depletion on peripheral blood compared to secondary lymphoid organs, the deleterious effect of IVIG when given with aCD20 and the relevance of redosing of aCD20 for effective B cell depletion in alloimmunity.     

Regulatory T Cell Infusion Can Enhance Memory T Cell and Alloantibody Responses in Lymphodepleted Nonhuman Primate Heart Allograft Recipients

The ability of regulatory T cells (Treg) to prolong allograft survival and promote transplant tolerance in lymphodepleted rodents is well established. Few studies, however, have addressed the therapeutic potential of adoptively transferred, CD4⁺CD25⁺CD127^?Foxp3⁺ (Treg) in clinically relevant large animal models. We infused ex vivo–expanded, functionally stable, nonselected Treg (up to a maximum cumulative dose of 1.87 billion cells) into antithymocyte globulin–lymphodepleted, MHC‐mismatched cynomolgus monkey heart graft recipients before homeostatic recovery of effector T cells. The monkeys also received tacrolimus, anti–interleukin‐6 receptor monoclonal antibodies and tapered rapamycin maintenance therapy. Treg administration in single or multiple doses during the early postsurgical period (up to 1 month posttransplantation), when host T cells were profoundly depleted, resulted in inferior graft function compared with controls. This was accompanied by increased incidences of effector memory T cells, enhanced interferon‐γ production by host CD8⁺ T cells, elevated levels of proinflammatory cytokines, and antidonor alloantibodies. The findings caution against infusion of Treg during the early posttransplantation period after lymphodepletion. Despite marked but transient increases in Treg relative to endogenous effector T cells and use of reputed “Treg‐friendly” agents, the host environment/immune effector mechanisms instigated under these conditions can perturb rather than favor the potential therapeutic efficacy of adoptively transferred Treg.     

Allospecific Memory B Cell Responses Are Dependent on Autophagy

Long‐lived, donor‐reactive memory B cells (Bmems) can produce alloantibodies that mediate transplant injury. Autophagy, an intrinsic mechanism of cell organelle/component recycling, is required for Bmem survival in infectious and model antigen systems, but whether autophagy affects alloreactive Bmem is unknown. We studied mice with an inducible yellow fluorescent protein (YFP) reporter expressed under the activation‐induced cytidine deaminase (AID) promoter active in B cells undergoing germinal center reactions. Up to 12 months after allogeneic sensitization, splenic YFP⁺ B cells were predominantly IgD^–IgM^–IgG⁺ and expressed CD73, CD80, and PD‐L2, consistent with Bmems. Labeled cells contained significantly more cells with autophagosomes and more autophagosomes per cell than unlabeled, naïve B cells. To test for a functional link, we quantified alloantibody formation in mice with B cells conditionally deficient in the requisite autophagy gene ATG7. These experiments revealed absent B cell ATG7 (1) prevented B cell autophagy, (2) inhibited secondary alloantibody responses without altering primary alloantibody formation, and (3) diminished frequencies of alloreactive Bmems. Pharmacological autophagy inhibition with 3‐methyladenine had similar effects on wild‐type mice. Together with new documentation of increased autophagosomes within human Bmems, our data indicate that targeting autophagy has potential for eliminating donor‐reactive Bmems in transplant recipients.     

Contrasting effects of B cell depletion on CD4+ and CD8+ memory T cell responses generated after transplantation

Alloreactive memory T cells play a key role in transplantation by accelerating allograft rejection and preventing tolerance induction. Some studies using µMT mice, which are constitutionally devoid of B cells, showed that B cells were required for the generation of memory T cells after allotransplantation. However, whether B cell depletion in normal adult mice has the same effect on memory responses by CD4⁺ and CD8⁺ T cells activated after transplantation has not been thoroughly investigated. In this study, we tested the effect of anti‐CD20 antibody‐mediated B cell depletion on CD4⁺ and CD8⁺ memory T cell alloresponses after skin transplantation in wild‐type mice. We found that B cell depletion prevented the development of memory alloresponses by CD4⁺ T cells but enhanced that of CD8⁺ memory T cells. Next, we tested the influence of B cell depletion on hematopoietic chimerism. In OT‐II CD4⁺ anti‐OVA TCR transgenic mice sensitized to ovalbumin antigen, B cell depletion also impaired allospecific memory T cell responses and thereby enhanced donor hematopoietic chimerism and T cell deletion after bone marrow transplantation. This study underscores the complexity of the relationships between B and T cells in the generation and reactivation of different memory T cell subsets after transplantation.     

Antigen‐dependent interactions between regulatory B cells and T cells at the T:B border inhibit subsequent T cell interactions with DCs

IL‐10+ regulatory B cells (Bregs) inhibit immune responses in various settings. While Bregs appear to inhibit inflammatory cytokine expression by CD4+ T cells and innate immune cells, their reported impact on CD8+ T cells is contradictory. Moreover, it remains unclear which effects of Bregs are direct versus indirect. Finally, the subanatomical localization of Breg suppressive function and the nature of their intercellular interactions remain unknown. Using novel tamoxifen‐inducible B cell–specific IL‐10 knockout mice, we found that Bregs inhibit CD8+ T cell proliferation and inhibit inflammatory cytokine expression by both CD4+ and CD8+ T cells. Sort‐purified Bregs from IL‐10‐reporter mice were adoptively transferred into wild‐type hosts and examined by live‐cell imaging. Bregs localized to the T:B border, specifically entered the T cell zone, and made more frequent and longer contacts with both CD4+ and CD8+ T cells than did non‐Bregs. These Breg:T cell interactions were antigen‐specific and reduced subsequent T:DC contacts. Thus, Bregs inhibit T cells through direct cognate interactions that subsequently reduce DC:T cell interactions.     

CD4+CD28null T cells are not alloreactive unless stimulated by interleukin‐15

Proinflammatory, cytotoxic CD4⁺CD28^null T cells can be substantially expanded in patients with end‐stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4⁺CD28^null T cells were stimulated with HLA‐mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28⁺ T cells. CD4⁺CD28^null T cells contained alloreactive (CD137⁺) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)‐15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL‐15 increased the frequency of proliferating alloreactive CD4⁺CD28^null T cells to 30.5% without inducing CD28 expression (P < .05). After allogeneic stimulation together with IL‐15 and IL‐21, frequency of degranulating CD107a⁺CD4⁺CD28^null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon‐γ and tumor necrosis factor‐α increased by the combination of IL‐15 and IL‐21 (P < .001 and P < .05, respectively). Finally, CD4⁺CD28^null T cells did not show significant suppression. Thus, CD4⁺CD28^null T cells represent a population with absent alloreactivity unless IL‐15 is present.     

Implications of Resident Memory T Cells for Transplantation

Recent studies have established resident memory T cells (T_RM) as the dominant memory lymphocyte population surveying most nonlymphoid tissues. Unlike other memory T cell lineages, T_RM do not recirculate through blood and are permanently confined to their tissue of residence. T_RM orchestrate local immune responses and have been shown to accelerate local pathogen control in many experimental infection models. Here we briefly summarize recent advances in T_RM differentiation, maintenance, and their protective function. While little is known, we have speculated on the potential implications of T_RM for transplantation biology. Areas of emphasis include the role of passenger T_RM in controlling latent viral recrudescence in donor organs, donor T_RM as a source of graft‐versus‐host disease, the ability of T_RM to potently induce inflammation through sensing and alarm functions, and differentiation of host T cells into T_RM in response to local cues inside the allograft. Further investigation of T_RM in the context of transplantation might identify therapeutic targets to prolong graft survival.     

Allo‐HLA Cross‐Reactivities of Cytomegalovirus‐, Influenza‐, and Varicella Zoster Virus–Specific Memory T Cells Are Shared by Different Healthy Individuals

Virus‐specific T cells can recognize allogeneic HLA (allo‐HLA) through TCR cross‐reactivity. The allospecificity often differs by individual (private cross‐reactivity) but also can be shared by multiple individuals (public cross‐reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross‐reactivities of human virus‐specific CD8⁺ T cells directed against allo‐HLA by assessing their reactivity in mixed‐lymphocyte reactions. Further characterization was done by studying TCR usage with primer‐based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with ⁵¹chromium‐release assays. We identified three novel public allo‐HLA cross‐reactivities of human virus‐specific CD8⁺ T cells. CMV B35/IPS CD8⁺ T cells cross‐reacted with HLA‐B51 and/or HLA‐B58/B57 (23% of tetramer‐positive individuals), FLU A2/GIL (influenza IMP[58‐66] HLA‐A*02:01/GILGFVFTL) CD8⁺ T cells with HLA‐B38 (90% of tetramer‐positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593‐601] HLA‐A*02:01/ALWALPHAA) CD8⁺ T cells with HLA‐B55 (two unrelated individuals). Cross‐reactivity was tested against different cell types including endothelial and epithelial cells. All cross‐reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo‐HLA cross‐reactivity of virus‐specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.     

Selective CD28 Blockade Results in Superior Inhibition of Donor‐Specific T Follicular Helper Cell and Antibody Responses Relative to CTLA4‐Ig

Donor‐specific antibodies (DSAs) are a barrier to improved long‐term outcomes after kidney transplantation. Costimulation blockade with CTLA4‐Ig has shown promise as a potential therapeutic strategy to control DSAs. T follicular helper (Tfh) cells, a subset of CD4⁺ T cells required for optimal antibody production, are reliant on the CD28 costimulatory pathway. We have previously shown that selective CD28 blockade leads to superior allograft survival through improved control of CD8⁺ T cells relative to CTLA4‐Ig, but the impact of CD28‐specific blockade on CD4⁺ Tfh cells is unknown. Thus, we identified and characterized donor‐reactive Tfh cells in a murine skin transplant model and then used this model to evaluate the impact of selective CD28 blockade with an anti‐CD28 domain antibody (dAb) on the donor‐specific Tfh cell–mediated immune response. We observed that the anti‐CD28 dAb led to superior inhibition of donor‐reactive CXCR5⁺PD‐1^high Tfh cells, CD95⁺GL7⁺ germinal center B cells and DSA formation compared with CTLA4‐Ig. Interestingly, donor‐reactive Tfh cells differentially upregulated CTLA4 expression, suggesting an important role for CTLA4 in mediating the superior inhibition observed with the anti‐CD28 dAb. Therefore, selective CD28 blockade as a novel approach to control Tfh cell responses and prevent DSA after kidney transplantation warrants further study.     

Cytomegalovirus‐Responsive CD8+ T Cells Expand After Solid Organ Transplantation in the Absence of CMV Disease

Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid organ transplant recipients. Approximately 60% of adults are CMV seropositive, indicating previous exposure. Following resolution of the primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8⁺ T cell responses to CMV polypeptides immediate‐early‐1 and pp65 were analyzed in 16 CMV‐seropositive kidney and heart transplant recipients longitudinally pretransplantation and posttransplantation. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis, and CMV viral load monitoring, with approximately half receiving T cell–depleting induction therapy. The frequency of CMV‐responsive CD8⁺ T cells, defined by the production of effector molecules in response to CMV peptides, increased during the course of 1 year posttransplantation. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV‐responsive T cell population posttransplantation.     

B cell–derived IL‐1β and IL‐6 drive T cell reconstitution following lymphoablation

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4⁺ and CD8⁺ T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell–derived IL‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.     

Rapamycin Interferes With Postdepletion Regulatory T Cell Homeostasis and Enhances DSA Formation Corrected by CTLA4‐Ig

Previously, we demonstrated that alemtuzumab induction with rapamycin as sole maintenance therapy is associated with an increased incidence of humoral rejection in human kidney transplant patients. To investigate the role of rapamycin in posttransplant humoral responses after T cell depletion, fully MHC mismatched hearts were transplanted into hCD52Tg mice, followed by alemtuzumab treatment with or without a short course of rapamycin. While untreated hCD52Tg recipients acutely rejected B6 hearts (n = 12), hCD52Tg recipients treated with alemtuzumab alone or in conjunction with rapamycin showed a lack of acute rejection (MST > 100). However, additional rapamycin showed a reduced beating quality over time and increased incidence of vasculopathy. Furthermore, rapamycin supplementation showed an increased serum donor‐specific antibodies (DSA) level compared to alemtuzumab alone at postoperation days 50 and 100. Surprisingly, additional rapamycin treatment significantly reduced CD4⁺CD25⁺FoxP3⁺ T reg cell numbers during treatment. On the contrary, ICOS⁺PD‐1⁺CD4 follicular helper T cells in the lymph nodes were significantly increased. Interestingly, CTLA4‐Ig supplementation in conjunction with rapamycin corrected rapamycin‐induced accelerated posttransplant humoral response by directly modulating Tfh cells but not Treg cells. This suggests that rapamycin after T cell depletion could affect Treg cells leading to an increase of Tfh cells and DSA production that can be reversed by CTLA4‐Ig.     

Uremia‐Associated Premature Aging of T Cells Does Not Predict Infectious Complications After Renal Transplantation

Patients with end‐stage renal disease have prematurely aged T cell systems. We tested whether T cell aging parameters were associated with the risk of infections after renal transplantation (RTx). We studied 188 patients over 1 year. Peripheral T cells were analyzed before and at 3 and 6 mo after RTx for frequency of recent thymic emigrants, relative telomere length and differentiation status. These parameters were related to the occurrence of opportunistic and serious infections. Overall, 84 patients developed an infection. In this group, 50 developed an opportunistic infection and 53 developed a serious infection. T cell aging parameters assessed before RTx were not associated with infection risk. The memory T cells showed a decrease within the first 3 mo in both groups (p < 0.001). The CD4⁺ memory T cells increased between 3 and 6 mo within the infection group (p = 0.015). The number of CD8⁺ memory T cells increased in both groups (p < 0.001) but reached baseline levels only in the infection group. In the infection group, the CD8⁺CD28^null T cell percentage increased between 3 and 6 mo (p = 0.024), tending to be higher than at baseline (p = 0.061). These differences in post‐RTx dynamics resulted from infections. Parameters of uremia‐associated premature aging of peripheral T cells do not predict posttransplant infections.     

Disruption of Transplant Tolerance by an “Incognito” Form of CD8 T Cell–Dependent Memory

Several approaches successfully achieve allograft tolerance in preclinical models but are challenging to translate into clinical practice. Many clinically relevant factors can attenuate allograft tolerance induction, including intrinsic genetic resistance, peritransplant infection, inflammation, and preexisting antidonor immunity. The prevailing view for immune memory as a tolerance barrier is that the host harbors memory cells that spontaneously cross‐react to donor MHC antigens. Such preexisting “heterologous” memory cells have direct reactivity to donor cells and resist most tolerance regimens. In this study, we developed a model system to determine if an alternative form of immune memory could also block tolerance. We posited that host memory T cells could potentially respond to donor‐derived non‐MHC antigens, such as latent viral antigens or autoantigens, to which the host is immune. Results show that immunity to a model nonself antigen, ovalbumin (OVA), can dramatically disrupt tolerance despite undetectable initial reactivity to donor MHC antigens. Importantly, this blockade of tolerance was CD8⁺ T cell–dependent and required linked antigen presentation of alloantigens with the test OVA antigen. As such, this pathway represents an unapparent, or “incognito,” form of immunity that is sufficient to prevent tolerance and that can be an unforeseen additional immune barrier to clinical transplant tolerance.