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 Vβ‐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.     

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

The mixed chimerism approach achieves donor‐specific tolerance in organ transplantation, but clinical use is inhibited by the toxicities of current bone marrow (BM) transplantation (BMT) protocols. Blocking the CD40:CD154 pathway with anti‐CD154 monoclonal antibodies (mAbs) is exceptionally potent in inducing mixed chimerism, but these mAbs are clinically not available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that CD4 or CD8 activation through an intact direct or CD4 T cell activation through the indirect pathway is sufficient to trigger BM rejection despite CTLA4Ig treatment. In the absence of CD4 T cells, CD8 T cell activation via the direct pathway, in contrast, leads to a state of split tolerance. Interruption of the CD40 signals in both the direct and indirect pathway of allorecognition or lack of recipient CD154 is required for the induction of chimerism and tolerance. We developed a novel BMT protocol that induces mixed chimerism and donor‐specific tolerance to fully mismatched cardiac allografts relying on CD28 costimulation blockade and mTOR inhibition without targeting the CD40 pathway. Notably, MHC‐mismatched/minor antigen‐matched skin grafts survive indefinitely whereas fully mismatched grafts are rejected, suggesting that non‐MHC antigens cause graft rejection and split tolerance.     

Regulatory B cells require antigen recognition for effective allograft tolerance induction

Through multiple mechanisms, regulatory B cells (Breg) have been shown to play an important role in the development of allograft tolerance. However, a careful understanding of the role of antigen‐specificity in Breg‐mediated allograft tolerance has remained elusive. In experimental models of islet and cardiac transplantation, it has been established that Bregs can be induced in vivo by anti‐CD45RB ± anti‐TIM1antibody treatment, resulting in prolonged, Breg‐dependent allograft tolerance. The importance of Breg antigen recognition has been suggested but not confirmed through adoptive transfer experiments, using tolerant WT C57BL/6 animals challenged with either BALB/c or C3H grafts. However, the importance of receptor‐specificity has not been formally tested. Here, we utilize the novel ovalbumin‐specific B cell receptor transnuclear (OBI) mice in multiple primary tolerance and adoptive transfer experiments to establish that Breg‐dependent allograft tolerance relies on antigen recognition by B cells. Additionally, we identify that this Breg‐dependent tolerance relies on the function of transforming growth factor‐β. Together, these experiments mark important progress toward understanding how best to improve Breg‐mediated allograft tolerance.     

Neutrophil derived CSF1 induces macrophage polarization and promotes transplantation tolerance

The colony‐stimulating factor 1 (CSF1) regulates the differentiation and function of tissue macrophages and determines the outcome of the immune response. The molecular mechanisms behind CSF1‐mediated macrophage development remain to be elucidated. Here we demonstrate that neutrophil‐derived CSF1 controls macrophage polarization and proliferation, which is necessary for the induction of tolerance. Inhibiting neutrophil production of CSF1 or preventing macrophage proliferation, using targeted nanoparticles loaded with the cell cycle inhibitor simvastatin, abrogates the induction of tolerance. These results provide new mechanistic insights into the developmental requirements of tolerogenic macrophages and identify CSF1 producing neutrophils as critical regulators of the immunological response.     

Maintaining T cell tolerance of alloantigens: Lessons from animal studies

Achieving host immune tolerance of allogeneic transplants represents the ultimate challenge in clinical transplantation. It has become clear that different cells and mechanisms participate in acquisition versus maintenance of allograft tolerance. Indeed, manipulations which prevent tolerance induction often fail to abrogate tolerance once it has been established. Hence, elucidation of the immunological mechanisms underlying maintenance of T cell tolerance to alloantigens is essential for the development of novel interventions that preserve a robust and long lasting state of allograft tolerance that relies on T cell deletion in addition to intra‐graft suppression of inflammatory immune responses. In this review, we discuss some essential elements of the mechanisms involved in the maintenance of naturally occurring or experimentally induced allograft tolerance, including the newly described role of antigen cross‐dressing mediated by extracellular vesicles.     

Tracking of TCR‐Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long‐lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25⁺ regulatory T cells (Tregs), blocking programmed death ligand‐1 (PD‐L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD‐1/PD‐L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.     

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.     

PD‐1 expression on CD8+ T cells regulates their differentiation within lung allografts and is critical for tolerance induction

Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8⁺ T cells are considered a barrier to immunosuppression‐mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8⁺ T lymphocytes. Here we demonstrate that costimulation blockade‐mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD‐1) expression on CD8⁺ T cells. In the absence of PD‐1 expression, CD8⁺ T cells form prolonged interactions with graft‐infiltrating CD11c⁺ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.     

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.     

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse‐1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2‐photon microscopy, we analyzed the effects of DNAse‐1 on NETs in mouse orthotopic lung allografts damaged by ischemia‐reperfusion injury. Although DNAse‐1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4⁺ T cells and donor‐derived antigen presenting cells. DNAse‐1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen‐specific CD4⁺ T cell proliferation through activating toll‐like receptor (TLR) — Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil‐specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse‐1 administration to wild‐type recipients promotes the recognition of allo‐ and self‐antigens and prevents immunosuppression‐mediated lung allograft acceptance through a MyD88‐dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.     

T cell exhaustion is associated with antigen abundance and promotes transplant acceptance

Exhaustion of T cells limits their ability to clear chronic infections or eradicate tumors. Here, in the context of transplant, we investigated whether T cell exhaustion occurs and has a role in determining transplant outcome. A peptide/MHC tetramer‐based approach was used to track exhausted CD8⁺ T cells in a male‐to‐female skin transplant model. Transplant of large whole‐tail skins, but not small tail skins (0.8 cm × 0.8 cm), led to exhaustion of anti‐male tetramer⁺ CD8⁺ T cells and subsequently the acceptance of skin grafts. To study CD4⁺ T cell exhaustion, we used the TCR‐transgenic B6 TEa cells that recognize a major transplant antigen I‐Eα from Balb/c mice. TEa cells were adoptively transferred either into B6 recipients that received Balb/c donor skins or into CB6F1 mice that contained an excessive amount of I‐Eα antigen. Adoptively transferred TEa cells in skin‐graft recipients were not exhausted. By contrast, virtually all adoptively transferred TEa cells were exhausted in CB6F1 mice. Those exhausted TEa cells lost ability to reject Balb/c skins upon further transfer into lymphopenic B6.Rag1^?/? mice. Hence, T cell exhaustion develops in the presence of abundant antigen and promotes transplant acceptance. These findings are essential for better understanding the nature of transplant tolerance.     

Combined Anti‐CD154/CTLA4Ig Costimulation Blockade‐Based Therapy Induces Donor‐Specific Tolerance to Vascularized Osteomyocutaneous Allografts

Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor‐derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2^d) mice were transplanted into C57BL/6 (H2^b) recipients. Immunosuppression consisted of 1 mg anti‐CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0–7, then every other day for 3 weeks). Long‐term allograft survival, donor‐specific tolerance and donor–recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long‐term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor‐specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor‐specific tolerance was further assessed in vivo by secondary donor‐specific skin graft survival and third‐party graft rejection. A significant increase of Foxp3⁺ regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti‐CD154/CTLA4Ig costimulation blockade‐based therapy induces donor‐specific tolerance in a stringent murine alloOMC transplant model.     

Erosion of Transplantation Tolerance After Infection

Recent clinical studies suggest that operational allograft tolerance can be persistent, but long‐term surviving allografts can be rejected in a subset of patients, sometimes after episodes of infection. In this study, we examined the impact of Listeria monocytogenes (Lm) infection on the quality of tolerance in a mouse model of heart allograft transplantation. Lm infection induced full rejection in 40% of tolerant recipients, with the remaining experiencing a rejection crisis or no palpable change in their allografts. In the surviving allografts on day 8 postinfection, graft‐infiltrating cell numbers increased and exhibited a loss in the tolerance gene signature. By day 30 postinfection, the tolerance signature was broadly restored, but with a discernible reduction in the expression of a subset of 234 genes that marked tolerance and was down‐regulated at day 8 post‐Lm infection. We further demonstrated that the tolerant state after Lm infection was functionally eroded, as rejection of the long‐term surviving graft was induced with anti‐PD‐L1 whereas the same treatment had no effect in noninfected tolerant mice. Collectively, these observations demonstrate that tolerance, even if initially robust, exists as a continuum that can be eroded following bystander immune responses that accompany certain infections.     

Myeloid‐derived suppressor cells expand after transplantation and their augmentation increases graft survival

Myeloid‐derived suppressor cells (MDSCs) expand in an inflammatory microenvironment such as cancer and autoimmunity. To study if transplantation induces MDSCs and these cells regulate allograft survival, C57BL/6 donor hearts were transplanted into BALB/c recipients and endogenous MDSCs were characterized. The effects of adoptive transfer of transplant (tx), tumor (tm), and granulocyte‐colony stimulating factor (g‐csf)–expanded MDSCs or depletion of MDSC were assessed. MDSCs expanded after transplantation (1.7‐4.6‐fold) in the absence of immunosuppression, homed to allografts, and suppressed proliferation of CD4 T cells in vitro. Tx‐MDSCs differed phenotypically from tm‐MDSCs and g‐csf‐MDSCs. Among various surface markers, Rae‐1 expression was notably low and TGF‐β receptor II was high in tx‐MDSCs when compared to tm‐MDSCs and g‐csf‐MDSCs. Adoptive transfer of these three MDSCs led to differential graft survival: control (6 days), tx‐MDSCs (7.5 days), tm‐MDSCs (9.5 days), and g‐csf‐MDSCs (19.5 days). In combination with anti‐CD154 mAb, MDSCs synergistically extended graft survival from 40 days (anti‐CD154 alone) to 86 days with tm‐MDSCs and 132 days with g‐csf‐MDSCs. Early MDSC depletion (day 0 or 20), however, abrogated graft survival, but late depletion (day 25) did not. In conclusion, MDSCs expanded following transplantation, migrated to cardiac allografts, prolonged graft survival, and were synergistic with anti‐CD154 mAb.