CD8 T-cell recognition of acquired alloantigen promotes acute allograft rejection.

Adaptive CD8 T-cell immunity is the principal arm of the cellular alloimmune response, but its development requires help. This can be provided by CD4 T cells that recognize alloantigen "indirectly," as self-restricted allopeptide, but this process remains unexplained, because the target epitopes for CD4 and CD8 T-cell recognition are "unlinked" on different cells (recipient and donor antigen presenting cells (APCs), respectively). Here, we test the hypothesis that the presentation of intact and processed MHC class I alloantigen by recipient dendritic cells (DCs) (the "semidirect" pathway) allows linked help to be delivered by indirect-pathway CD4 T cells for generating destructive cytotoxic CD8 T-cell alloresponses. We show that CD8 T-cell-mediated rejection of murine heart allografts that lack hematopoietic APCs requires host secondary lymphoid tissue (SLT). SLT is necessary because within it, recipient dendritic cells can acquire MHC from graft parenchymal cells and simultaneously present it as intact protein to alloreactive CD8 T cells and as processed peptide alloantigen for recognition by indirect-pathway CD4 T cells. This enables delivery of essential help for generating cytotoxic CD8 T-cell responses that cause rapid allograft rejection. In demonstrating the functional relevance of the semidirect pathway to transplant rejection, our findings provide a solution to a long-standing conundrum as to why SLT is required for CD8 T-cell allorecognition of graft parenchymal cells and suggest a mechanism by which indirect-pathway CD4 T cells provide help for generating effector cytotoxic CD8 T-cell alloresponses at late time points after transplantation.

Targeting indirect pathway CD4 T-cell alloresponses in the prevention of chronic transplant rejection.

BACKGROUND: Uniquely, alloantigen is recognised by two pathways: as intact antigen on the surface of donor antigen-presenting cells (direct) and as self-restricted processed allopeptide (indirect). The indirect pathway is believed to be longlasting, and is generally considered to be a single entity. Here we address how indirect responses against different alloantigens differ in their strength and longevity, and how this knowledge could be used to direct immunoregulatory therapy with antigen-specific regulatory T cells (Tregs). METHODS: A murine model of cardiac transplantation was used (bm12.Kd.IE to C57BL/6). Indirect CD4 T-cell allorecognition of mismatched donor MHC class I and II, and of H-Y minor histocompatibility antigen was assessed by quantifying proliferation of adoptively transferred monoclonal T-cell receptor transgenic T cells (TCR75, Tea, Mar). Antigen presentation by dendritic cells and B cells was assessed by selective depletion with diphtheria toxin or depleting anti-CD20 monoclonal antibody. Tregs were generated by in-vitro culture. FINDINGS: Indirect pathway responses were heterogeneous. Whereas the indirect response against class I alloantigen was longlasting and persistently strong, the response against class II alloantigen decayed within 2 weeks. Leucocyte depletion studies confirmed that this difference was due to rapid destruction of MHC class II expressing donor B cells and dendritic cells in the recipient, whereas anti-class I responses were generated by continual processing of graft parenchymal cells; recognition of donor haemopoietic fraction was not required. Notably, transfer of MHC class I specific Tregs at transplant or 3 weeks later abrogated germinal centre alloantibody responses and blocked development of allograft vasculopathy, whereas class II specific Tregs were ineffective when transferred at the late timepoint. INTERPRETATION: Although indirect allorecognition is considered to be a single entity, our results show that it consists of a number of responses that vary in duration and strength according to target alloantigen. The ability of class I allopeptide specific Tregs, but not class II specific Tregs, to prevent rejection when transferred at a late timepoint suggests that antigen-specific targeting of dominant and longlasting pathways might be particularly effective at preventing chronic rejection. FUNDING: Wellcome Trust Clinical Research Training Fellowship.

Recipient natural killer cell allorecognition of passenger donor lymphocytes and its effect on adaptive alloimmunity after transplantation.

BACKGROUND: Memory T cells are known to reside in peripheral non-lymphoid tissue, but how their presence within solid organ allografts affects transplant outcomes is not known. We have previously described how graft-versus-host (GVH) allorecognition by passenger CD4 T cells within MHC class II-mismatched bm12 heart grafts provokes antinuclear humoral autoimmunity in C57BL/6 recipient mice. Here we aimed to examine how such GVH recognition affects the alloresponse to allografts with greater mismatching. METHODS: A MHC class I and II mismatched murine model of cardiac transplantation was developed (bm12.Kd.IE to C57BL/6). After transplantation, cellular and humoral responses against mismatched antigens were measured with ELISPOT and ELISA, and the effect of GVH recognition assessed by depletion of donor CD4 T cells before graft procurement. Antinuclear autoantibody development was assessedwith HeP-2 indirect immunofluorescence. The role of recipient natural killer (NK) cells was examined by depletion with anti-NK1.1 antibody. FINDINGS: Bm12.Kd.IE heart grafts provoked strong germinal centre alloantibody and autoantibody responses in C57BL/6 recipients and developed allograft vasculopathy. By contrast, heart grafts from CD4 T-cell-depleted donors developed only minimal vasculopathy, and the alloantibody responses were weaker, without observable autoantibody. Bm12.Kd.IE CD4 T cells survived long term when transferred to RAG hosts suggesting that avoidance of killing by host NK cells might be essential for autoantibody development. In support, in a model of alloantibody-mediated vasculopathy, depletion of NK cells from a C57BL/6 recipient of a BALB/c heart graft resulted in the development of autoantibody, amplification of the alloantibody response, and rapid allograft rejection. This amplification was abrogated by depletion of donor CD4 T cells. INTERPRETATION: Although host adaptive immunity is expected to bring about destruction of passenger lymphocytes within heart allografts, this process occurs too slowly to prevent GVH-mediated augmentation of the alloresponse to the graft. Rather, rapid killing of donor lymphocytes by host alloreactive NK cells is essential. Passenger CD4 lymphocytes might therefore contribute to chronic rejection in recipients receiving an allograft that does not prompt innate NK cell recognition. FUNDING: Wellcome Trust Clinical Research Training Fellowship.

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.

Stem cell medicine encounters the immune system.

Recent progress in deriving human embryonic stem (hES) cells and defining their capacity to differentiate has inspired hope that they could become a source of replacement cells for damaged or diseased tissues. We review the immunological barriers to transplanting hES cells and consider several potential solutions, including stem-cell banking, modification of the immunogenicity of donor cells and induction of tolerance to the graft. We evaluate the probable efficacy of these approaches with a view to facilitating the use of hES cells in clinical practice.

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.

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.

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.

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.

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.

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.

TL1A both promotes and protects from renal inflammation and injury.

Death receptor 3 (DR3), a member of the TNF receptor (TNFR) superfamily, is induced in human renal tubular epithelial cells (TEC) in response to injury. This study examined the expression and actions of TL1A, the principal ligand for DR3. In histologically normal tissue from biopsy or nephrectomy specimens of renal allografts, TL1A mRNA and protein were expressed in vascular endothelial cells but not in TEC. In specimens of acute or antibody-mediated allograft rejection, vascular endothelial cells and infiltrating leukocytes expressed increased TL1A mRNA and protein, but TEC expressed TL1A protein without mRNA, consistent with uptake of exogenous ligand. Addition of TL1A to organ cultures of human or mouse kidney caused activation of NF-kappaB, expression of TNFR2, activation of caspase-3, and apoptosis in TEC. Inhibition of NF-kappaB activation increased TL1A-mediated caspase-3 activation and apoptosis of TEC, but it did not reduce the induction of TNFR2. In organ culture of DR3-deficient mouse kidneys, addition of TL1A induced TNFR2 but did not activate NF-kappaB and did not increase apoptosis of TEC. These data suggest that TL1A may contribute to renal inflammation and injury through DR3-mediated activation of NF-kappaB and caspase-3, respectively, but that an unidentified receptor may mediate the NF-kappaB-independent induction of TNFR2 in TEC.

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.

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.

Pathways of helper CD4 T cell allorecognition in generating alloantibody and CD8 T cell alloimmunity.

BACKGROUND: The relative contributions of the "direct" and "indirect" pathways of CD4 T cell allorecognition in providing help for generating effective humoral and CD8 T cell alloimmunity remain unclear. Here, the generation of alloantibody and cytotoxic CD8 T cell responses to a vascularized allograft were examined in a murine adoptive-transfer model in which help could only be provided by transferred CD4 T cells recognizing alloantigen exclusively through the direct pathway. METHODS: Rejection kinetics and the development of alloantibody and cytotoxic CD8 T cell responses to MHC-mismatched H-2d heart grafts were compared when CD4 T cell help was present (wild-type H-2d recipients), or absent (CD4 T cell deficient, MHC class II-/- H-2b recipients [B6CII-/-]), or available only through the direct pathway (B6CII-/- mice reconstituted with wild-type CD4 T cells). RESULTS: BALB/c allografts were rejected by B6 mice rapidly (median survival time [MST] 7 days) with strong CD8 T cell effector and alloantibody responses, but were rejected by B6CII-/- mice more slowly (MST 23 days), with markedly reduced CD8 T cell responses and no detectable alloantibody. CD4 T cell reconstitution of B6CII-/- recipients accelerated heart graft rejection to near that of wild-type recipients (MST 13 days), with complete restoration of cytotoxic CD8 T cell responses but without detectable IgM or IgG alloantibody. CONCLUSIONS: Different pathways of helper T cell allorecognition are responsible for generating humoral and CD8 T cell alloimmunity. CD4 T cell help provided exclusively through the direct pathway generates strong cytotoxic CD8 T cell responses that effect rapid heart graft rejection.

Alloantibody-associated chronic rejection of MHC class I-disparate heart grafts is modulated by intravenous soluble donor alloantigen.

Increasing evidence suggests that there may be a causal relationship between the development of donor-specific alloantibodies and chronic allograft vasculopathy (CAV). PVG.RT1(u) rat heart allografts spontaneously undergo chronic rejection when transplanted into unmodified PVG.R8 congenic recipients that differ only at the classical MHC class I RT1.A locus. Here we show that development of vasculopathy in this experimental model is associated with production of a strong anti-A(u) antibody response. Perioperative intravenous administration of recombinant soluble RT1.A(u) heavy chain that is sequence identical to donor MHC class I, or chimaeric A(u/a) (donor/recipient) protein had a variable effect resulting generally in either sensitisation and accelerated rejection, or abrogation of alloantibody and attenuation of chronic rejection. These findings highlight the potential for soluble donor MHC class I alloantigen given at the time of heart transplantation to influence alloantibody production and graft outcome.

Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching.

BACKGROUND: Human embryonic stem (hES) cells are a promising source for transplantation to replace diseased or damaged tissue, but their differentiated progeny express human leucocyte antigens (HLAs) that will probably cause graft rejection. The creation of a bank of HLA-typed hES cells, from which a best match could be selected, would help reduce the likelihood of graft rejection. We investigated how many hES cell lines would be needed to make matching possible in most cases. METHODS: The number of hES cell lines needed to achieve varying degrees of HLA match was estimated by use of, as a surrogate for hES-cell donor embryos, blood group and HLA types on a series of 10,000 consecutive UK cadaveric organ donors. The degree of blood group compatibility and HLA matching for a recipient population consisting of 6577 patients registered on the UK kidney transplant waiting list was determined, assuming all donor hES cell lines could provide a transplant for an unlimited number of recipients. FINDINGS: A bank of 150 consecutive donors provided a full match at HLA-A, HLA-B, and HLA-DR for a minority of recipients (<20%); a beneficial match (defined as one HLA-A or one HLA-B mismatch only) or better for 37.9% (range 27.9-47.5); and an HLA-DR match or better for 84.9% (77.5-90.0). Extending the number of donors beyond 150 conferred only a very gradual incremental benefit with respect to HLA matching. A panel of only ten donors homozygous for common HLA types selected from 10,000 donors provided a complete HLA-A, HLA-B and HLA-DR match for 37.7% of recipients, and a beneficial match for 67.4%. INTERPRETATION: Approximately 150 consecutive blood group compatible donors, 100 consecutive blood group O donors, or ten highly selected homozygous donors could provide the maximum practical benefit for HLA matching. The findings from these simulations have practical, political, and ethical implications for the establishment of hES-cell banks.

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.

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.