Long-term Graft Acceptance Research Articles

Long-term Tolerance to Islet Transplantation via Targeted Reduction of beta cell-specific T cells.

Activated beta-cell reactive CD4 + and CD8 + T effector cells undergo a profound DNA-damage response which is targetable by small molecule inhibitors of the p53 and cell cycle pathways that lead to apoptosis. The use of a combination of MDM2 and WEE1 inhibitors, which termed "p53 potentiation with checkpoint abrogation" (PPCA), conferred significant therapeutic efficacy in treating mouse models of new onset T1D. Specific targeting of these T effector cells by PPCA results in a loss of inflammatory T cell subsets, notably proliferation CD4 + Th0 and Th1 subsets and CD8 + T effector memory cells, as determined by single cell RNA-seq studies with the preservation of T regulatory cells. When autologous islet grafts are given to established diabetic NOD mice, a single course of PPCA results in long-term islet graft acceptance, restoration of normoglycemia and loss of beta cell specific CD4 + and CD8 + T cells. PPCA shows promise as a potential means of estimating islet graft tolerance in T1D recipients of islet graft transplantation.

CD94+ natural killer cells potentiate pulmonary ischaemia-reperfusion injury.

Pulmonary ischaemia-reperfusion injury (IRI) is a major contributor to poor lung transplant outcomes. We recently demonstrated a central role of airway-centred natural killer (NK) cells in mediating IRI; however, there are no existing effective therapies for directly targeting NK cells in humans. We hypothesised that a depleting anti-CD94 monoclonal antibody (mAb) would provide therapeutic benefit in mouse and human models of IRI based on high levels of KLRD1 (CD94) transcripts in bronchoalveolar lavage samples from lung transplant patients. We found that CD94 is highly expressed on mouse and human NK cells, with increased expression during IRI. Anti-mouse and anti-human mAbs against CD94 showed effective NK cell depletion in mouse and human models and blunted lung damage and airway epithelial killing, respectively. In two different allogeneic orthotopic lung transplant mouse models, anti-CD94 treatment during induction reduced early lung injury and chronic inflammation relative to control therapies. Anti-CD94 did not increase donor antigen-presenting cells that could alter long-term graft acceptance. Lung transplant induction regimens incorporating anti-CD94 treatment may safely improve early clinical outcomes.

Trained immunity is regulated by T cell-induced CD40-TRAF6 signaling

Trained immunity is characterized by histone modifications and metabolic changes in innate immune cells following exposure to inflammatory signals, leading to heightened responsiveness to secondary stimuli. Although our understanding of the molecular regulation of trained immunity has increased, the role of adaptive immune cells herein remains largely unknown. Here, we show that Tcells modulate trained immunity via cluster of differentiation 40-tissue necrosis factor receptor-associated factor 6 (CD40-TRAF6) signaling. CD40-TRAF6 inhibition modulates functional, transcriptomic, and metabolic reprogramming and modifies histone 3 lysine 4 trimethylation associated with trained immunity. Besides invitro studies, we reveal that single-nucleotide polymorphisms in the proximity of CD40 are linked to trained immunity responses invivo and that combining CD40-TRAF6 inhibition with cytotoxic T lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)-mediated co-stimulatory blockade induces long-term graft acceptance in a murine heart transplantation model. Combined, our results reveal that trained immunity is modulated by CD40-TRAF6 signaling between myeloid and adaptive immune cells and that this can be leveraged for therapeutic purposes.

Pregnancy dedifferentiates memory CD8+ T cells into hypofunctional cells with exhaustion-enriched programs.

Alloreactive memory, unlike naive, CD8+ T cells resist transplantation tolerance protocols and are a critical barrier to long-term graft acceptance in the clinic. We here show that semiallogeneic pregnancy successfully reprogrammed memory fetus/graft-specific CD8+ T cells (TFGS) toward hypofunction. Female C57BL/6 mice harboring memory CD8+ T cells generated by the rejection of BALB/c skin grafts and then mated with BALB/c males achieved rates of pregnancy comparable with naive controls. Postpartum CD8+ TFGS from skin-sensitized dams upregulated expression of T cell exhaustion (TEX) markers (Tox, Eomes, PD-1, TIGIT, and Lag3). Transcriptional analysis corroborated an enrichment of canonical TEX genes in postpartum memory TFGS and revealed a downregulation of a subset of memory-associated transcripts. Strikingly, pregnancy induced extensive epigenetic modifications of exhaustion- and memory-associated genes in memory TFGS, whereas minimal epigenetic modifications were observed in naive TFGS. Finally, postpartum memory TFGS durably expressed the exhaustion-enriched phenotype, and their susceptibility to transplantation tolerance was significantly restored compared with memory TFGS. These findings advance the concept of pregnancy as an epigenetic modulator inducing hypofunction in memory CD8+ T cells that has relevance not only for pregnancy and transplantation tolerance, but also for tumor immunity and chronic infections.

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor-mismatched skin transplantation

Oral antigen exposure is a powerful, non-invasive route to induce immune tolerance to dietary antigens, and has been modestly successful at prolonging graft survival in rodent models of transplantation. To harness the mechanisms of oral tolerance for promoting long-term graft acceptance, we developed a mouse model where the antigen ovalbumin (OVA) was introduced orally prior to transplantation with skin grafts expressing OVA. Oral OVA treatment pre-transplantation promoted permanent graft acceptance and linked tolerance to skin grafts expressing OVA fused to the additional antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party allografts were spontaneously accepted. Oral OVA treatment promoted an anergic phenotype in OVA-reactive CD4+ and CD8+ conventional T cells (Tconvs) and expanded OVA-reactive Tregs pre-transplantation. However, skin graft acceptance following oral OVA resisted partial depletion of Tregs and blockade of PD-L1. Mechanistically, we revealed a role for the proximal gut draining lymph nodes (gdLNs) in mediating this effect, as an intestinal infection that drains to the proximal gdLNs prevented tolerance induction. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance.

Oral alloantigen exposure promotes donor-specific tolerance in a mouse model of minor mismatched skin transplantation

Abstract Oral tolerance is a powerful, non-invasive route to induce immune tolerance, yet it has been unsuccessful to promote graft acceptance in rodent models of transplantation. To this end, we developed a mouse model where the model antigen ovalbumin (OVA) was introduced orally, in a regimen designed to improve bioavailability, duodenal lymph node drainage and prolong duration of antigen exposure, prior to transplantation with skin grafts constitutively expressing membrane-bound OVA. We found that oral OVA treatment pre-transplantation promoted permanent graft acceptance as well as linked tolerance to skin grafts expressing OVA coupled to the additional model antigen 2W. Tolerance was donor-specific, as secondary donor-matched, but not third-party grafts were spontaneously accepted in the absence of additional immunosuppression. Treatment with this oral OVA regimen promoted an anergic phenotype in OVA-reactive CD8+ T cells, generation of iTregs, and dysfunction of OVA-reactive CD4+ T cells pre-transplantation. The tolerance induced by oral OVA was robust, as anti-CD25/anti-PD-L1 at the time of transplantation failed to prevent skin graft acceptance. Mechanistically, we revealed a critical role for the proximal gut draining lymph nodes (gLNs) in mediating this effect, as an intestinal infection that drains to the proximal gLNs prevented the induction of transplantation tolerance. Our study extends previous work applying oral antigen exposure to transplantation and serves as proof of concept that the systemic immune mechanisms supporting oral tolerance are sufficient to promote long-term graft acceptance. Regimens that improve bioavailability and duration of oral alloantigen administration may hold promise in the clinic. This work was supported by a NIAID grant to ASC and MLA (P01AI-97113). Peter Wang was supported by undergraduate research grants from the Katen Scholars Program and the Dean's Fund for Undergraduate Research.

CXCR4 blockade reduces the severity of murine heart allograft rejection by plasmacytoid dendritic cell-mediated immune regulation

Allograft-specific regulatory T cells (Treg cells) are crucial for long-term graft acceptance after transplantation. Although adoptive Treg cell transfer has been proposed, major challenges include graft-specificity and stability. Thus, there is an unmet need for the direct induction of graft-specific Treg cells. We hypothesized a synergism of the immunotolerogenic effects of rapamycin (mTOR inhibition) and plerixafor (CXCR4 antagonist) for Treg cell induction. Thus, we performed fully-mismatched heart transplantations and found combination treatment to result in prolonged allograft survival. Moreover, fibrosis and myocyte lesions were reduced. Although less CD3+ T cell infiltrated, higher Treg cell numbers were observed. Noteworthy, this was accompanied by a plerixafor-dependent plasmacytoid dendritic cells-(pDCs)-mobilization. Furthermore, in vivo pDC-depletion abrogated the plerixafor-mediated Treg cell number increase and reduced allograft survival. Our pharmacological approach allowed to increase Treg cell numbers due to pDC-mediated immune regulation. Therefore pDCs can be an attractive immunotherapeutic target in addition to plerixafor treatment.

Peripheral CD19+CD24highCD38high B-regulatory cells in lung transplant recipients

BackgroundThe role of CD19+CD24highCD38high B-regulatory cells in solid-organ Transplant (Tx) in acceptance are still scarce. In previous studies on kidney transplant recipients may suggest a protective role of this cell subtype in graft tolerance and the existence of a cross talk between B-and T-regulatory clones. In lung transplantation, the role of B-regulatory cells has never been investigated. In a murine tracheal transplantation model, this subset seems able to prevent tracheal obliteration when in combination with rapamycin. Aim of this study is to analyze peripheral CD19+CD24highCD38high B-reg cells counts in a cohort of lung recipients, their association with several clinical and pharmacological variables and their possible association with T regulatory cell. MethodsFrom Jan 2009 to Dec 2014, 117 lung Tx recipients were submitted to an immunological follow up I-FU(median: 108.7 months (6.7–310.5)). Immunological follow up consisted of a complete blood peripheral immuno-phenotype, inclusive of CD19+CD24highCD38high B-cells (globally 1106 determinations). We tested the association between B-reg and relevant variables by linear or regression models for repeated measures, adjusting for time from Tx. ResultsAmong all variables analyzed at multivariate analysis: chronic rejection (OR − 0.19, p = .039), use of Mycophenolate (OR − 0.38, p < .001) and the presence of a concomitant pulmonary infection of S. aureus (OR 0.66, p = .002) and A. fumigatus (OR 0.50, p = .009) were significantly associated to B-reg cell.No significant correlation between CD19+CD24highCD38high B-reg cells and T-reg cells counts was found in our cohort. ConclusionsOur present data highlight, for the first time, that this cell subset might participate in long-term lung graft acceptance mechanisms.

Blood CD9+ B cell, a biomarker of bronchiolitis obliterans syndrome after lung transplantation.

Bronchiolitis obliterans syndrome is the main limitation for long-term survival after lung transplantation. Some specific B cell populations are associated with long-term graft acceptance. We aimed to monitor the B cell profile during early development of bronchiolitis obliterans syndrome after lung transplantation. The B cell longitudinal profile was analyzed in peripheral blood mononuclear cells from patients with bronchiolitis obliterans syndrome and patients who remained stable over 3years of follow-up. CD24hi CD38hi transitional B cells were increased in stable patients only, and reached a peak 24months after transplantation, whereas they remained unchanged in patients who developed a bronchiolitis obliterans syndrome. These CD24hi CD38hi transitional B cells specifically secrete IL-10 and express CD9. Thus, patients with a total CD9+ B cell frequency below 6.6% displayed significantly higher incidence of bronchiolitis obliterans syndrome (AUC=0.836, PPV=0.75, NPV=1). These data are the first to associate IL-10-secreting CD24hi CD38hi transitional B cells expressing CD9 with better allograft outcome in lung transplant recipients. CD9-expressing B cells appear as a contributor to a favorable environment essential for the maintenance of long-term stable graft function and as a new predictive biomarker of bronchiolitis obliterans syndrome-free survival.

Effect of Timing and Complement Receptor Antagonism on Intragraft Recruitment and Protolerogenic Effects of Mesenchymal Stromal Cells in Murine Kidney Transplantation.

Mesenchymal stromal cells (MSCs) have protolerogenic effects in renal transplantation, but they induce long-term regulatory T cells (Treg)-dependent graft acceptance only when infused before transplantation. When given posttransplant, MSCs home to the graft where they promote engraftment syndrome and do not induce Treg. Unfortunately, pretransplant MSC administration is unfeasible in deceased-donor kidney transplantation. To make MSCs a therapeutic option also for deceased organ recipients, we tested whether MSC infusion at the time of transplant (day 0) or posttransplant (day 2) together with inhibition of complement receptors prevents engraftment syndrome and allows their homing to secondary lymphoid organs for promoting tolerance. We analyzed intragraft and splenic MSC localization, graft survival, and alloimmune response in mice recipients of kidney allografts and syngeneic MSCs given on day 0 or on posttransplant day 2. C3a receptor (C3aR) or C5a receptor (C5aR) antagonists were administered to mice in combination with the cells or were used together to treat MSCs before infusion. Syngeneic MSCs given at day 0 homed to the spleen increased Treg numbers and induced long-term graft acceptance. Posttransplant MSC infusion, combined with a short course of C3aR or C5aR antagonist or administration of MSCs pretreated with C3aR and C5aR antagonists, prevented intragraft recruitment of MSCs and graft inflammation, inhibited antidonor T-cell reactivity, but failed to induce Treg, resulting in mild prolongation of graft survival. These data support testing the safety/efficacy profile of administering MSCs on the day of transplant in deceased-donor transplant recipients and indicate that complement is crucial for MSC recruitment into the kidney allograft.

Relationship Between Mixed Chimerism and Acceptance of HLA-matched and -Mismatched Kidney Transplants after Withdrawal of Immunosuppressive Drugs

Classical studies in cattle and rodents showed that mixed chimerism established in fetuses and neonates was permanent, and was linked to immune tolerance and the acceptance of organ grafts from the hematopoietic cell donors without need for immunosuppressive (IS) drugs. The goal of the current clinical study was to determine whether establishment of persistent mixed chimerism for at least 1 year in HLA-matched and -mismatched recipients of combined kidney and hematopoietic cell transplants from living donors followed the preclinical paradigm and allowed for permanent chimerism and acceptance of transplant kidneys in the absence of IS drugs. Recipients were given a 10-day post-transplant conditioning regimen of total lymphoid irradiation and rabbit anti-thymocyte globulin. Of 29 HLA-matched patients, 24 developed mixed chimerism for at least 1 yr and were completely withdrawn from IS drugs from mo 6 through 18. There was no subsequent rejection in 23 of the 24, with up to 12 yrs of follow-up. Mixed chimerism persisted after drug withdrawal in 10, as in the preclinical studies. However, 14 had a pattern that differed from the paradigm and accepted the organ grafts after drug withdrawal despite loss of chimerism during the 2nd yr. Pts with long-term graft acceptance off drug had specific unresponsiveness to donor allo-antigens in the MLR even in the absence of stable mixed chimerism. There were 2 graft losses among the 29 during the 12-yr observation period due to return of underlying kidney disease. Biopsies obtained from mixed chimeric pts just before discontinuation of IS drugs showed no rejection. Of 22 HLA haplotype-mismatched patients, 18 have been followed for more than 1 yr, and 9 developed mixed chimerism that persisted at least 1 yr. MMF was withdrawn from the chimeric pts such that they were maintained on tacrolimus (TAC) monotherapy at the end of the 1st yr, and biopsies showed no rejection at this time. However, withdrawal of TAC in 6 during the 2nd yr resulted in loss of mixed chimerism with evidence of mild rejection in 3; they returned to IS drugs. Mixed chimerism in these HLA-mismatched pts did not follow the “classical” paradigm - it was unstable and dependent on IS drugs. The remaining persistent chimeras have been maintained on TAC monotherapy. There has been no graft loss or chronic rejection in the 22 pts, with up to 7 yrs of follow-up. Neither severe or chronic infection, nor graft versus host disease, has been observed in the HLA-matched and -mismatched pts. In conclusion, persistent mixed chimerism protected against rejection in both HLA-matched and -mismatched patients. Whereas long-term acceptance of kidney transplants off IS drugs was observed even after loss of chimerism in HLA-matched pts, protection against rejection was associated with persistence of mixed chimerism in HLA-mismatched pts. The latter outcome in HLA-mismatched pts is desirable if chimerism can be maintained on low-dose TAC monotherapy.

Successful Treatment of T Cell-Mediated Acute Rejection with Delayed CTLA4-Ig in Mice.

Clinical observations that kidney transplant recipients receiving belatacept who experienced T cell-mediated acute rejection can be successfully treated and subsequently maintained on belatacept-based immunosuppression suggest that belatacept is able to control memory T cells. We recently reported that treatment with CTLA4-Ig from day 6 posttransplantation successfully rescues allografts from acute rejection in a BALB/c to C57BL/6 heart transplant model, in part, by abolishing B cell germinal centers and reducing alloantibody titers. Here, we show that CTLA4-Ig is additionally able to inhibit established T cell responses independently of B cells. CTLA4-Ig inhibited the in vivo cytolytic activity of donor-specific CD8+ T cells, and the production of IFNγ by graft-infiltrating T cells. Delayed CTLA4-Ig treatment did not reduce the numbers of graft-infiltrating T cells nor prevented the accumulation of antigen-experienced donor-specific memory T cells in the spleen. Nevertheless, delayed CTLA4-Ig treatment successfully maintained long-term graft acceptance in the majority of recipients that had experienced a rejection crisis, and enabled the acceptance of secondary BALB/c heart grafts transplanted 30 days after the first transplantation. In summary, we conclude that delayed CTLA4-Ig treatment is able to partially halt ongoing T cell-mediated acute rejection. These findings extend the functional efficacy of CTLA4-Ig therapy to effector T cells and provide an explanation for why CTLA4-Ig-based immunosuppression in the clinic successfully maintains long-term graft survival after T cell-mediated rejection.

Intra-graft injection of tacrolimus promotes survival of vascularized composite allotransplantation

BackgroundImmunosuppressive therapies derived from solid organ transplantation are effective in promoting survival of vascularized composite allotransplantation (VCA), but they cause serious side effects that are difficult to justify for this non–life-saving procedure. Unlike solid organ transplantation, hand and face transplants offer the possibility of site-specific immunosuppression for reducing systemic exposure while increasing intra-graft concentrations of the drug. Therefore, in this study, we tested whether a single intra-graft injection tacrolimus could promote VCA survival. MethodsBrown Norway-to-Lewis hind limb transplantations were performed, and animals were left untreated (group I), treated with a daily injection of 1-mg/kg tacrolimus for 21 days (group 2) or injected with 7-mg tacrolimus directly into the transplanted limb on day 1 (group III). Graft rejection was monitored, and animals were sacrificed at grade 3 rejection or 200 days after transplantation. ResultsIntra-graft injection of tacrolimus significantly prolonged allograft survival as compared to untreated animals or animals treated with systemic tacrolimus. Half of the intra-graft–treated rats rejected their graft on average at day 70.5. Interestingly, the other half remained rejection-free for more than 200 days without signs of kidney or liver toxicity. In these animals, tacrolimus was detected in the VCA skin but not in the blood until day 200. Long-term survival was not linked to induction of donor-specific tolerance but to a higher level of lymphocyte chimerism. ConclusionsIntra-graft delivery of tacrolimus may promote VCA survival by increasing tissue drug availability and promoting the establishment of transient chimerism and thus long-term graft acceptance.

Increased CD40 Ligation and Reduced BCR Signalling Leads to Higher IL-10 Production in B Cells From Tolerant Kidney Transplant Patients.

An increased percentage of peripheral transitional B cells producing IL-10 has been observed in patients tolerant to kidney allografts. In healthy volunteers, the balance between the CD40 and B-cell receptor (BCR) signalling modulated IL-10 production by B cells, with stimulation via the BCR decreasing CD40-mediated IL-10 production. In this study, we evaluate whether in tolerant kidney transplant patients, the increased IL-10 production by B cells was due to an altered CD40 and/or BCR signalling. B cells obtained from a new cohort of tolerant renal transplant recipients and those from age- and sex-matched healthy volunteers were activated via CD40 and BCR, either alone or in combination. In tolerant patients, we observed higher percentages of B cells producing IL-10 after CD40 ligation and higher expression of CD40L on activated T cells compared with healthy controls. Furthermore, B cells from tolerant recipients had reduced extracellular signal-regulated kinase signalling after BCR-mediated activation compared with healthy controls. In keeping with this, combining BCR signalling with CD40 ligation did not reduce IL-10 secretion as was observed in healthy control transitional B cells. Altogether, our data suggest that the altered response of B cells in tolerant recipients may contribute to long-term stable graft acceptance.

Biopsy-proven acute cellular rejection as an efficacy endpoint of randomized trials in liver transplantation: a systematic review and critical appraisal.

Biopsy-proven acute cellular rejection (ACR) is the primary efficacy endpoint in most randomized trials evaluating immunosuppression in liver transplantation. However, ACR is not a major cause of graft loss, and a certain grade of immune activation may be even beneficial for long-term graft acceptance. Validated criteria to select candidates for liver biopsy are lacking, and routine clinical practice relies on liver tests, which are inaccurate markers of ACR. Indeed, both the agreement among clinicians to select candidates for liver biopsy and the correlation between the clinical suspicion of ACR and histological findings are poor. In randomized trials evaluating immunosuppression protocols, this concern grows exponentially due to the open-label and multicenter nature of most studies. Therefore, biopsy-proven ACR is a suboptimal efficacy endpoint given its limited impact on prognosis and the heterogeneous diagnosis, which may increase the risk of bias. Chronic rejection and/or graft loss would be more appropriate endpoints, but would certainly require larger studies with prolonged surveillances. An objective method to select candidates for liver biopsy is therefore urgently needed, and only severe episodes of histological ACR should be considered as potentially harmful. Emerging surrogate markers of ACR and antibody-mediated rejection require further investigation to determine their clinical role.

Combining autologous dendritic cell therapy with CD3 antibodies promotes regulatory T cells and permanent islet allograft acceptance.

Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation.

Establishment of Transplantation Tolerance via Minimal Conditioning in Aged Recipients

Mixed hematopoietic chimerism is a powerful means of generating donor-specific tolerance, allowing long-term graft acceptance without lifelong dependence on immunosuppressive drugs. To avoid the need for whole body irradiation and associated side effects, we utilized a radiation-free minimal conditioning regime to induce long-term tolerance across major histocompatibility barriers. We found that low-dose busulfan, in combination with host T cell depletion and short-term sirolimus-based immunosuppression, facilitated efficient donor engraftment. Tolerance was achieved when mice were transplanted with whole or T cell-depleted bone marrow, or purified progenitor cells. Tolerance induction was associated with an expansion in regulatory T cells and was not abrogated in the absence of a thymus, suggesting a dominant or compensatory peripheral mode of tolerance. Importantly, we were able to generate durable chimerism and tolerance to donor skin grafts in both young and aged mice, despite age-related thymic atrophy and immune senescence. Clinically, this is especially relevant as the majority of transplant recipients are older patients whose immune recovery might be dangerously slow and would benefit from radiation-free minimal conditioning regimes that allow efficient donor engraftment without fully ablating the recipient immune system.

Vascular endothelium as a target of immune response in renal transplant rejection.

This review of clinical and experimental studies aims at analyzing the interplay between graft endothelium and host immune system in renal transplantation, and how it affects the survival of the graft. Graft endothelium is indeed the first barrier between self and non-self that is encountered by host lymphocytes upon reperfusion of vascularized solid transplants. Endothelial cells (EC) express all the major sets of antigens (Ag) that elicit host immune response, and therefore represent a preferential target in organ rejection. Some of the Ag expressed by EC are target of the antibody-mediated response, such as the AB0 blood group system, the human leukocyte antigens (HLA), and MHC class I related chain A antigens (MICA) systems, and the endothelial cell-restricted Ag; for each of these systems, the mechanisms of interaction and damage of both preformed and de novo donor-specific antibodies are reviewed along with their impact on renal graft survival. Moreover, the rejection process can force injured EC to expose cryptic self-Ag, toward which an autoimmune response mounts, overlapping to the allo-immune response in the damaging of the graft. Not only are EC a passive target of the host immune response but also an active player in lymphocyte activation; therefore, their interaction with allogenic T-cells is analyzed on the basis of experimental in vitro and in vivo studies, according to the patterns of expression of the HLA class I and II and the co-stimulatory molecules specific for cytotoxic and helper T-cells. Finally, as the response that follows transplantation has proven to be not necessarily destructive, the factors that foster graft endothelium functioning in spite of rejection, and how they could be therapeutically harnessed to promote long-term graft acceptance, are described: accommodation that is resistance of EC to donor-specific antibodies, and endothelial cell ability to induce Foxp3+ regulatory T-cells, that are crucial mediators of tolerance.

CD8 Intrinsic Defects in Myd88-/- Recipients Determine the Effective Control of Effector T Cell Accumulation in Kidney Allografts.

Previous reports indicate that MyD88 plays distinct roles in transplantation by regulating DC maturation and T cell priming. The Purpose of this study is to determine whether Myd88 signaling in T cells is required in kidney allograft rejection in mice. Methods: Orthotopic kidney transplants were performed between MHC fully mismatched mice, in which B6 kidneys were transplanted into binephrectomized wild type (WT) or MyD88-/- BALB/c mice (allografts). To determine whether Myd88 expression in T cells is required for accumulation of effector T cells in the kidney allografts, we used a dual adoptive transfer approaches in which 1:1 mixed naïve CD8 T cells from both Myd88-/- and WT mice were infused into BALB/c RAG1-/- mice. Kidney grafts were examined by histology and phenotypes of cellular infiltrates were identified by immunohistochemistry and flow cytometry between POD7 and 14. T cell function was assessed by a mixed lymphocyte reaction. Results: Recipient MyD88 is not required for the initiation of acute allograft rejection featured by cellular infiltration and tubulitis, however, recipient MyD88 deficiency results in spontaneous clearance of the rejection response and subsequent restoration of renal graft function, leading to long-term allograft survival compared to MyD88 sufficient (WT) recipients. The graft protection was associated with a reduced intragraft accumulation of CD11b-Gr-1+ cells and activated CD8+ T cells in the MyD88-/- mice. T cells, specifically CD8 T cells, from the MyD88-/- recipients failed to mount a donor specific recall response as indicated by diminished proliferation and cytokine production upon donor antigen re-stimulation in vitro. Interestingly, exogenous IL-6 restored the proliferation of T cells from MyD88-/- recipients to that of T cells from WT recipients. Furthermore, adoptively transferred MyD88-/- CD8 T cells in RAG1-/- recipients exhibited a diminished expression of chemokine receptors, specifically CCR4 and CXCR3. This was accompanied by an impaired ability to accumulate in the kidney allografts, even in a MyD88 sufficient environment. Conclusion: This study provides a novel mechanism linking the lack of intrinsic MyD88 signaling in T cells to compromised effector T cell expansion and accumulation in the kidney allografts, thereby leading to the spontaneous resolution of initial cellular infiltration of kidney allografts and long-term graft acceptance.

Myeloid Progenitors Can Induce Tolerance in the Context of Autologous HSC Transplantation.

i. Purpose. We have published that tolerance for skin graft transplantation can be consistently induced with Myeloid Progenitor Cells (MP) without long-term high-level donor-derived engraftment. Importantly, MP are in clinical trials to test their ability to lower infections in bone marrow transplantation, making them clinically available. MP can be given either two months prior to or at the time of organ transplantation for tolerance induction. Using MP from B10;B6-Rag2-/-Il2rg-/- mice (devoid of T, B or NK cells) we have established that donor-matched lymphoid cells are not required. Here we test whether tolerance is preserved when MP are combined with host-autologous HSC which would reduce HCT-related complications. ii. Methods. In our model mice (typically BALB/c) were irradiated (8.5Gy) and reconstituted with 500 BALB/c or 4,000 FVB HSC and 100,000 third party MP. MP-matched (or mismatched) skin grafts are placed on the same day and their fate is followed long-term.Figure: No Caption available.iii. Results. Specific tolerance induction in this model can be achieved with autologous HSC (which can rapidly restore the hematopoietic system) and skin graft-matched MP. Tolerance induced by 100,000 skin graft-matched MP is slightly lower (p=0.02, logrank) when combined with 500 host autologous HSC (solid black line) than it is with 4,000 allogeneic HSC which are not matched to the skin graft (solid grey line): At the dose tested approx. 80% of the grafts (35/43) show long-term skin graft acceptance without further immunosuppression. Grafts not matched to host, HSC or MP are all rejected (dashed lines). Ongoing experiments test whether increasing the MP dose will further improve outcome. iv. Conclusion. Organ matched MP can induce tolerance in combination with autologous HSC significantly increasing the potential clinical applicability of this approach to tolerance induction and importantly, indicating that reduced intensity preconditioning regimens should be feasible.