Augmentation of Recipient Adaptive Alloimmunity by Donor Passenger Lymphocytes within the Transplant.

Chronic rejection of solid organ allografts remains the major cause of transplant failure. Donor-derived tissue-resident lymphocytes are transferred to the recipient during transplantation, but their impact on alloimmunity is unknown. Using mouse cardiac transplant models, we show that graft-versus-host recognition by passenger donor CD4 T cells markedly augments recipient cellular and humoral alloimmunity, resulting in more severe allograft vasculopathy and early graft failure. This augmentation is enhanced when donors were pre-sensitized to the recipient, is dependent upon avoidance of host NK cell recognition, and is partly due to provision of cognate help for allo-specific B cells from donor CD4 T cells recognizing B cell MHC class II in a peptide-degenerate manner. Passenger donor lymphocytes may therefore influence recipient alloimmune responses and represent a therapeutic target in solid organ transplantation.

Access to Transplantation and Transplant Outcome Measures (ATTOM): study protocol of a UK wide, in-depth, prospective cohort analysis.

INTRODUCTION: There is significant intercentre variability in access to renal transplantation in the UK due to poorly understood factors. The overarching aims of this study are to improve equity of access to kidney and kidney-pancreas transplantation across the UK and to optimise organ allocation to maximise the benefit and cost-effectiveness of transplantation. METHODS AND ANALYSIS: 6844 patients aged 18-75 years starting dialysis and/or receiving a transplant together with matched patients active on the transplant list from all 72 UK renal units were recruited between November 2011 and March 2013 and will be followed for at least 3 years. The outcomes of interest include patient survival, access to the transplant list, receipt of a transplant, patient-reported outcome measures (PROMs) including quality of life, treatment satisfaction, well-being and health status on different forms of renal replacement therapy. Sociodemographic and clinical data were prospectively collected from case notes and from interviews with patients and local clinical teams. Qualitative process exploration with clinical staff will help identify unit-specific factors that influence access to renal transplantation. A health economic analysis will explore costs and outcomes associated with alternative approaches to organ allocation. The study will deliver: (1) an understanding of patient and unit-specific factors influencing access to renal transplantation in the UK, informing potential changes to practices and policies to optimise outcomes and reduce intercentre variability; (2) a patient-survival probability model to standardise access to the renal transplant list and (3) an understanding of PROMs and health economic impact of kidney and kidney-pancreas transplantation to inform the development of a more sophisticated and fairer organ allocation algorithm. ETHICS AND DISSEMINATION: The protocol has been independently peer reviewed by National Institute for Health Research (NIHR) and approved by the East of England Research Ethics Committee. The results will be published in peer-reviewed journals and presented at conferences.

Diversity of the CD4 T Cell Alloresponse: The Short and the Long of It.

MHC alloantigen is recognized by two pathways: "directly," intact on donor cells, or "indirectly," as self-restricted allopeptide. The duration of each pathway, and its relative contribution to allograft vasculopathy, remain unclear. Using a murine model of chronic allograft rejection, we report that direct-pathway CD4 T cell alloresponses, as well as indirect-pathway responses against MHC class II alloantigen, are curtailed by rapid elimination of donor hematopoietic antigen-presenting cells. In contrast, persistent presentation of epitope resulted in continual division and less-profound contraction of the class I allopeptide-specific CD4 T cell population, with approximately 10,000-fold more cells persisting than following acute allograft rejection. This expanded population nevertheless displayed sub-optimal anamnestic responses and was unable to provide co-stimulation-independent help for generating alloantibody. Indirect-pathway CD4 T cell responses are heterogeneous. Appreciation that responses against particular alloantigens dominate at late time points will likely inform development of strategies aimed at improving transplant outcomes.

The mitochondria-targeted anti-oxidant MitoQ decreases ischemia-reperfusion injury in a murine syngeneic heart transplant model.

BACKGROUND: Free radical production and mitochondrial dysfunction during cardiac graft reperfusion is a major factor in post-transplant ischemia-reperfusion (IR) injury, an important underlying cause of primary graft dysfunction. We therefore assessed the efficacy of the mitochondria-targeted anti-oxidant MitoQ in reducing IR injury in a murine heterotopic cardiac transplant model. METHODS: Hearts from C57BL/6 donor mice were flushed with storage solution alone, solution containing the anti-oxidant MitoQ, or solution containing the non-anti-oxidant decyltriphenylphosphonium control and exposed to short (30 minutes) or prolonged (4 hour) cold preservation before transplantation. Grafts were transplanted into C57BL/6 recipients and analyzed for mitochondrial reactive oxygen species production, oxidative damage, serum troponin, beating score, and inflammatory markers 120 minutes or 24 hours post-transplant. RESULTS: MitoQ was taken up by the heart during cold storage. Prolonged cold preservation of donor hearts before IR increased IR injury (troponin I, beating score) and mitochondrial reactive oxygen species, mitochondrial DNA damage, protein carbonyls, and pro-inflammatory cytokine release 24 hours after transplant. Administration of MitoQ to the donor heart in the storage solution protected against this IR injury by blocking graft oxidative damage and dampening the early pro-inflammatory response in the recipient. CONCLUSIONS: IR after heart transplantation results in mitochondrial oxidative damage that is potentiated by cold ischemia. Supplementing donor graft perfusion with the anti-oxidant MitoQ before transplantation should be studied further to reduce IR-related free radical production, the innate immune response to IR injury, and subsequent donor cardiac injury.

Avoiding immunological rejection in regenerative medicine.

One of the major goals of regenerative medicine is repair or replacement of diseased and damaged tissues by transfer of differentiated stem cells or stem cell-derived tissues. The possibility that these tissues will be destroyed by immunological rejection remains a challenge that can only be overcome through a better understanding of the nature and expression of potentially immunogenic molecules associated with cell replacement therapy and the mechanisms and pathways resulting in their immunologic rejection. This review draws on clinical experience of organ and tissue transplantation, and on transplantation immunology research to consider practical approaches for avoiding and overcoming the possibility of rejection of stem cell-derived tissues.

Impact of donor mismatches at individual HLA-A, -B, -C, -DR, and -DQ loci on the development of HLA-specific antibodies in patients listed for repeat renal transplantation.

We have analyzed the relationship between donor mismatches at each HLA locus and development of HLA locus-specific antibodies in patients listed for repeat transplantation. HLA antibody screening was undertaken using single-antigen beads in 131 kidney transplant recipients returning to the transplant waiting list following first graft failure. The number of HLA mismatches and the calculated reaction frequency of antibody reactivity against 10,000 consecutive deceased organ donors were determined for each HLA locus. Two-thirds of patients awaiting repeat transplantation were sensitized (calculated reaction frequency over 15%) and half were highly sensitized (calculated reaction frequency of 85% and greater). Antibody levels peaked after re-listing for repeat transplantation, were independent of graft nephrectomy and were associated with length of time on the waiting list (odds ratio 8.4) and with maintenance on dual immunosuppression (odds ratio 0.2). Sensitization was independently associated with increasing number of donor HLA mismatches (odds ratio 1.4). All mismatched HLA loci contributed to the development of HLA locus-specific antibodies (HLA-A: odds ratio 3.2, HLA-B: odds ratio 3.4, HLA-C: odds ratio 2.5, HLA-DRB1: odds ratio 3.5, HLA-DRB3/4/5: odds ratio 3.9, and HLA-DQ: odds ratio 3.0 (all significant)). Thus, the risk of allosensitization following failure of a first renal transplant increases incrementally with the number of mismatches at all HLA loci assessed. Maintenance of re-listed patients on dual immunosuppression was associated with a reduced risk of sensitization.

Functional characterization of late outgrowth endothelial progenitor cells in patients with end-stage renal failure.

Renal transplantation is potentially curative in renal failure, but long-term efficacy is limited by untreatable chronic rejection. Endothelial damage contributes to chronic rejection and is potentially repairable by circulating endothelial progenitor cells (EPC). The frequency and function of EPC are variably influenced by end-stage renal failure (ESRF). Here, we isolated and functionally characterized the late outgrowth EPC (LO-EPC) from ESRF patients to investigate their potential for endothelial repair. Patients with ESRF generated more LO-EPC colonies than healthy controls and had higher plasma levels of IL-1rα, IL-16, IL-6, MIF, VEGF, Prolactin, and PLGF. Patients' LO-EPC displayed normal endothelial cell morphology, increased secretion of PLGF, MCP-1, and IL-1ß, and normal network formation in vitro and in vivo. They demonstrated decreased adhesion to extracellular matrix. Integrin gene profiles and protein expression were comparable in patients and healthy volunteers. In some patients, mesenchymal stem cells (MSC) were co-isolated and could be differentiated into adipocytes and osteocytes in vitro. This is the first study to characterize LO-EPC from patients with ESRF. Their behavior in vitro reflects the presence of elevated trophic factors; their ability to proliferate in vitro and angiogenic function makes them candidates for prevention of chronic rejection. Their impaired adhesion and the presence of MSC are areas for potential therapeutic intervention.

Copresentation of intact and processed MHC alloantigen by recipient dendritic cells enables delivery of linked help to alloreactive CD8 T cells by indirect-pathway CD4 T cells.

In transplantation, direct-pathway CD8 T cells that recognize alloantigen on donor cells require CD4 help for activation and cytolytic function. The ability of indirect-pathway CD4 T cells to provide this help remains unexplained, because a fundamental requirement for epitope linkage is seemingly broken. The simultaneous presentation, by host dendritic cells (DCs), of both intact MHC class I alloantigen and processed alloantigen would deliver linked help, but has not been demonstrated definitively. In this study, we report that following in vitro coculture with BALB/c DCs, small numbers (~1.5%) of C57BL/6 (B6) DCs presented acquired H-2(d) alloantigen both as processed allopeptide and as unprocessed Ag. This represented class I alloantigen provides a conformational epitope for direct-pathway allorecognition, because B6 DCs isolated from cocultures and transferred to naive B6 mice provoked cytotoxic CD8 T cell alloimmunity. Crucially, this response was dependent upon simultaneous presentation of class II-restricted allopeptide, because despite acquiring similar amounts of H-2(d) alloantigen upon coculture, MHC class II-deficient B6 DCs failed to elicit cytotoxic alloimmunity. The relevance of this pathway to solid-organ transplantation was then confirmed by the demonstration that CD8 T cell cytotoxicity was provoked in secondary recipients by transfer of DCs purified from wild-type, but not from MHC class II-deficient, C57BL/6 recipients of BALB/c heart transplants. These experiments demonstrate that representation of conformationally intact MHC alloantigen by recipient APC can induce cytotoxic alloimmunity, but simultaneous copresentation of processed allopeptide is essential, presumably because this facilitates linked recognition by indirect-pathway CD4 Th cells.

Allorecognition pathways in transplant rejection and tolerance.

With the advent of cellular therapies, it has become clear that the success of future therapies in prolonging allograft survival will require an intimate understanding of the allorecognition pathways and effector mechanisms that are responsible for chronic rejection and late graft loss.Here, we consider current understanding of T-cell allorecognition pathways and discuss the most likely mechanisms by which these pathways collaborate with other effector mechanisms to cause allograft rejection. We also consider how this knowledge may inform development of future strategies to prevent allograft rejection.Although both direct and indirect pathway CD4 T cells appear active immediately after transplantation, it has emerged that indirect pathway CD4 T cells are likely to be the dominant alloreactive T-cell population late after transplantation. Their ability to provide help for generating long-lived alloantibody is likely one of the main mechanisms responsible for the progression of allograft vasculopathy and chronic rejection.Recent work has suggested that regulatory T cells may be an effective cellular therapy in transplantation. Given the above, adoptive therapy with CD4 regulatory T cells with indirect allospecificity is a rational first choice in attempting to attenuate the development and progression of chronic rejection; those with additional properties that enable inhibition of germinal center alloantibody responses hold particular appeal.

Regulation of allograft survival by inhibitory FcγRIIb signaling.

Fcγ receptors (FcγR) provide important immunoregulation. Targeting inhibitory FcγRIIb may therefore prolong allograft survival, but its role in transplantation has not been addressed. FcγRIIb signaling was examined in murine models of acute or chronic cardiac allograft rejection by transplanting recipients that either lacked FcγRIIb expression (FcγRIIb(-/-)) or overexpressed FcγRIIb on B cells (B cell transgenic [BTG]). Acute heart allograft rejection occurred at the same tempo in FcγRIIb(-/-) C57BL/6 (B6) recipients as wild type recipients, with similar IgG alloantibody responses. In contrast, chronic rejection of MHC class II-mismatched bm12 cardiac allografts was accelerated in FcγRIIb(-/-) mice, with development of more severe transplant arteriopathy and markedly augmented effector autoantibody production. Autoantibody production was inhibited and rejection was delayed in BTG recipients. Similarly, whereas MHC class I-mismatched B6.K(d) hearts survived indefinitely and remained disease free in B6 mice, much stronger alloantibody responses and progressive graft arteriopathy developed in FcγRIIb(-/-) recipients. Notably, FcγRIIb-mediated inhibition of B6.K(d) heart graft rejection was abrogated by increasing T cell help through transfer of additional H2.K(d)-specific CD4 T cells. Thus, inhibitory FcγRIIb signaling regulates chronic but not acute rejection, most likely because the supra-optimal helper CD4 T cell response in acute rejection overcomes FcγRIIb-mediated inhibition of the effector B cell population. Immunomodulation of FcγRIIb in clinical transplantation may hold potential for inhibiting progression of transplant arteriopathy and prolonging transplant survival.

Unlinked memory helper responses promote long-lasting humoral alloimmunity.

Essential help for long-lived alloantibody responses is theoretically provided only by CD4 T cells that recognize target alloantigen, processed and presented by the allospecific B cell. We demonstrate that in an alloresponse to multiple MHC disparities, cognate help for class-switched alloantibody may also be provided by CD4 T cells specific for a second "helper" alloantigen. This response was much shorter-lived than when help was provided conventionally, by Th cell recognition of target alloantigen. Nevertheless, long-lasting humoral alloimmunity developed when T cell memory against the helper alloantigen was first generated. Costimulatory blockade abrogated alloantibody produced through naive Th cell recognition of target alloantigen but, crucially, blockade was ineffective when help was provided by memory responses to the accessory helper alloantigen. These results suggest that memory Th cell responses against previously encountered graft alloantigen may be the dominant mechanism for providing help to generate new specificities of alloantibody in transplant patients receiving immunosuppression.

Generating an iPSC bank for HLA-matched tissue transplantation based on known donor and recipient HLA types.

The likelihood for immunological rejection of Human Leukocyte Antigens (HLA)-mismatched induced pluripotent stem cells (iPSCs) limits their therapeutic potential. Here we show how a tissue bank from 150 selected homozygous HLA-typed volunteers could match 93% of the UK population with a minimal requirement for immunosuppression. Our model provides a practical approach for using existing HLA-typed samples to generate an iPSC stem cell bank that circumvents prospective typing of a large number of individuals.

Germinal center alloantibody responses are mediated exclusively by indirect-pathway CD4 T follicular helper cells.

The durable alloantibody responses that develop in organ transplant patients indicate long-lived plasma cell output from T-dependent germinal centers (GCs), but which of the two pathways of CD4 T cell allorecognition is responsible for generating allospecific T follicular helper cells remains unclear. This was addressed by reconstituting T cell-deficient mice with monoclonal populations of TCR-transgenic CD4 T cells that recognized alloantigen only as conformationally intact protein (direct pathway) or only as self-restricted allopeptide (indirect pathway) and then assessing the alloantibody response to a heart graft. Recipients reconstituted with indirect-pathway CD4 T cells developed long-lasting IgG alloantibody responses, with splenic GCs and allospecific bone marrow plasma cells readily detectable 50 d after heart transplantation. Differentiation of the transferred CD4 T cells into T follicular helper cells was confirmed by follicular localization and by acquisition of signature phenotype. In contrast, IgG alloantibody was not detectable in recipient mice reconstituted with direct-pathway CD4 T cells. Neither prolongation of the response by preventing NK cell killing of donor dendritic cells nor prior immunization to develop CD4 T cell memory altered the inability of the direct pathway to provide allospecific B cell help. CD4 T cell help for GC alloantibody responses is provided exclusively via the indirect-allorecognition pathway.

Late outgrowth endothelial progenitor cells engineered for improved survival and maintenance of function in transplant-related injury.

Chronic allograft vasculopathy (CAV) is a major cause of organ transplant failure that responds poorly to treatment. Endothelial activation, dysfunction and apoptosis contribute to CAV, whereas strategies for protecting endothelium and maximizing endothelial repair may diminish it. Late outgrowth endothelial progenitor cells (LO-EPC) can home to areas of injury and integrate into damaged vessels, implying a role in vascular repair; however, in an allograft, LO-EPC would be exposed to the hazardous microenvironment associated with transplant-related ischaemia reperfusion (I/R) injury and persistent inflammation. We evaluated the in vitro effect of I/R injury and the proinflammatory cytokine tumour necrosis factor (TNF)-α on LO-EPC phenotype and function. We show that LO-EPC are intrinsically more tolerant than mature EC to I/R injury induced apoptosis, maintaining their proliferative, migratory and network formation capacity. Under inflammatory conditions, LO-EPC were activated and released higher levels of inflammatory cytokines, upregulated adhesion molecule expression, and were more susceptible to apoptosis. Lentiviral vector-mediated overexpression of the protective gene A20 in LO-EPC maintained their angiogenic phenotype and function, and protected them against TNF-α-mediated apoptosis, reducing ICAM-1 expression and inflammatory cytokine secretion. Administration of ex vivo modified LO-EPC overexpressing A20 might effect vascular repair of damaged allografts and protect from CAV.

Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses.

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody-mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin-ß-receptor (LTßR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal-center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTßR-Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTßR-Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.

Immunological considerations for embryonic and induced pluripotent stem cell banking.

Recent advances in stem cell technology have generated enthusiasm for their potential to study and treat a diverse range of human disease. Pluripotent human stem cells for therapeutic use may, in principle, be obtained from two sources: embryonic stem cells (hESCs), which are capable of extensive self-renewal and expansion and have the potential to differentiate into any somatic tissue, and induced pluripotent stem cells (iPSCs), which are derived from differentiated tissue such as adult skin fibroblasts and appear to have the same properties and potential, but their generation is not dependent upon a source of embryos. The likelihood that clinical transplantation of hESC- or iPSC-derived tissues from an unrelated (allogeneic) donor that express foreign human leucocyte antigens (HLA) may undergo immunological rejection requires the formulation of strategies to attenuate the host immune response to transplanted tissue. In clinical practice, individualized iPSC tissue derived from the intended recipient offers the possibility of personalized stem cell therapy in which graft rejection would not occur, but the logistics of achieving this on a large scale are problematic owing to relatively inefficient reprogramming techniques and high costs. The creation of stem cell banks comprising HLA-typed hESCs and iPSCs is a strategy that is proposed to overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. Estimates have shown that a stem cell bank containing around 10 highly selected cell lines with conserved homozygous HLA haplotypes would provide matched tissue for the majority of the UK population. These simulations have practical, financial, political and ethical implications for the establishment and design of stem cell banks incorporating cell lines with HLA types that are compatible with different ethnic populations throughout the world.

Donor CD4 T cells contribute to cardiac allograft vasculopathy by providing help for autoantibody production.

BACKGROUND: The development of autoantibody after heart transplantation is increasingly associated with poor graft outcome, but what triggers its development and whether it has a direct causative role in graft rejection is not clear. Here, we study the development of antinuclear autoantibody in an established mouse model of heart allograft vasculopathy. METHODS AND RESULTS: Humoral vascular changes, including endothelial complement staining, were present in bm12 heart grafts, explanted 50 days after transplantation. Alloantibody was not detectable, but long-lasting autoantibody responses developed in C57BL/6 recipients from the third week after transplantation. No autoantibody was generated if donor CD4 T cells were depleted before heart graft retrieval or in recipients that lacked B-cell major histocompatibility complex class II expression, indicating that humoral autoimmunity is a consequence of donor CD4 T-cell allorecognition of the major histocompatibility complex class II complex on recipient autoreactive B cells. An effector role for autoantibody in graft rejection was confirmed by abrogation of humoral vascular rejection, and attenuation of vasculopathy, in B-cell deficient recipients and by development of vascular obliteration and accelerated rejection in recipients primed for autoantibody before transplantation. CONCLUSIONS: Passenger CD4 T cells within heart transplants can contribute to allograft vasculopathy by providing help to recipient B cells for autoantibody generation.

Indirect allorecognition: not simple but effective.

Donor dendritic cells are potent but short-lived stimulators of early transplant rejection, and recipient antigen presenting cells presenting donor major histocompatibility complex peptides sustain immunoreactivity and contribute to chronic rejection. We briefly consider how CD4 T cells that recognize allopeptide can provide help for effector and regulatory responses and highlight the implications for promoting graft survival.

Lymphoid tissue formation in allografts: innocent until proven guilty.

Tertiary lymphoid organs (TLOs) are lymphoid-like structures commonly found at sites of chronic inflammation. Commonly associated with autoimmune responses, they have recently been described within organ allografts. Although TLOs are similar structurally to secondary lymphoid organs, their function remains largely unknown. In this review, we discuss how immune responses within TLOs may influence graft survival.