Donor HLA Antigens Research Articles

Islet transplantation outcomes in type 1 diabetes and transplantation of HLA-DQ8/DR4: results of a single-centre retrospective cohort in Canada

In solid organ transplantation, HLA matching between donor and recipient is associated with superior outcomes. In islet transplantation, an intervention for Type 1 diabetes, HLA matching between donor and recipient is not performed as part of allocation. Susceptibility to Type 1 diabetes is associated with the presence of certain HLA types. This study was conducted to determine the impact of these susceptibility antigens on isletallograft survival. This is a single-centre retrospective cohort study. This cohort of transplant recipients (n=268) received islets from 661 donor pancreases between March 11th, 1999 and August 29th, 2018at the University of Alberta Hospital (Edmonton, AB, Canada). The frequency of the Type 1 diabetes susceptibility HLA antigens (HLA-A24, -B39, -DQ8, -DQ2 and-DQ2-DQA1∗05) in recipients and donors were determined. Recipient and donor HLA antigens were examined in relation to time to first C-peptide negative status/graft failure or last observation point. Taking into account multiple transplants per patient, we fitted a Gaussian frailty survival analysis model with baseline hazard function stratified by transplant number, adjusted for cumulative islet dose and other confounders. Across all transplants recipients of donors positive for HLA-DQ8 had significantly better graft survival (adjusted HRs 0.33 95% CI 0.17-0.66; p=0.002). At first transplant only, donors positive for HLA-DQ2-DQA1∗05 had inferior graft survival (adjusted HR 1.96 95% CI 1.10-3.46); p=0.02), although this was not significant in the frailty analysis taking multiple transplants into account (adjusted HR 1.46 95% CI 0.77-2.78; p=0.25). Other HLA antigens were not associated with graft survival after adjustment for confounders. Our findings suggest islet transplantation from HLA-DQ8 donors is associated with superior graft outcomes. A donor positive for HLA-DQ2-DQA1∗05at first transplant was associated with inferior graft survival but not when taking into account multiple transplants per recipient. The relevance of HLA-antigens on organ allocation needs further evaluation and inclusion in islet transplant registries and additional observational and interventional studies to evaluate the role of HLA-DQ8 in islet graft survival are required. None.

No Association Between Early Donor Specific Antibody and Subsequent Allograft Function at 3 Years Post-Pediatric Heart Transplantation. First Results of a Prospective Multi-Institutional Study

<h3>Purpose</h3> We have shown that sensitized and non-sensitized pediatric heart transplant (PHTx) recipients had similar 1 year patient and graft survival (CTOTC-04 study). In this follow-up study (CTOTC-09), we assess the impact of ‘preformed' (at Tx) and first year newly detected donor specific antibody (ndDSA) on allograft function at 3 years. <h3>Methods</h3> Consecutive children listed for Tx at 9 centers were prospectively enrolled. Management of sensitization, immunosuppression and rejection surveillance were standardized. The primary endpoint was PCWP at 3 years. Secondary endpoints included other hemodynamics, ejection fraction (EF), BNP, graft and patient survival and rejection. DSA was defined by single antigen Luminex testing with ≥1 antibody specific towards donor HLA antigens with MFI>1000. <h3>Results</h3> Of 407 enrolled, 370 achieved Tx (mean 8.7 years). Sensitization status at Tx: non-sensitized (n=169, 46%), sensitized/no DSA (n= 120, 32%), sensitized/DSA (n=76, 21%); 5 (1%) unknown. Subjects with and without early DSA (preformed and/or first year ndDSA) had similar PCWP at 3 years (table). There were no significant differences between the 2 groups for: other hemodynamics (PA, RA, RVEDP, LVEDP; all P > 0.08); cardiac index (P= 0.315); EF (P=0.682); and BNP (P=0.900). Subanalysis of the 3 sensitization groups at Tx stratified by presence/absence of first year ndDSA showed no significant impact of ndDSA on PCWP, EF or BNP (all P > 0.1). Graft and patient survival did not differ between groups, though freedom from all acute rejection types was inferior in subjects with DSA at Tx. <h3>Conclusion</h3> Sensitization status at Tx and development of first year ndDSA are not associated with worse allograft function or inferior survival at 3 years. Ongoing follow-up, including assessment of CAD, is in progress. This information is critical for defining Tx strategies and the long-term impact of DSA on graft outcomes.

Hypoproliferative human neural progenitor cell xenografts survived extendedly in the brain of immunocompetent rats

BackgroundThere is a huge controversy about whether xenograft or allograft in the “immune-privileged” brain needs immunosuppression. In animal studies, the prevailing sophisticated use of immunosuppression or immunodeficient animal is detrimental for the recipients, which results in a short lifespan of animals, confounds functional behavioral readout of the graft benefits, and discourages long-term follow-up.MethodsNeuron-restricted neural progenitor cells (NPCs) were derived from human embryonic stem cells (ESCs, including H1, its gene-modified cell lines for better visualization, and HN4), propagated for different passages, and then transplanted into the brain of immunocompetent rats without immunosuppressants. The graft survivals, their cell fates, and HLA expression levels were examined over time (up to 4 months after transplantation). We compared the survival capability of NPCs from different passages and in different transplantation sites (intra-parenchyma vs. para- and intra-cerebroventricle). The host responses to the grafts were also investigated.ResultsOur results show that human ESC-derived neuron-restricted NPCs survive extendedly in adult rat brain parenchyma with no need of immunosuppression whereas a late-onset graft rejection seems inevitable. Both donor HLA antigens and host MHC-II expression level remain relatively low with little change over time and cannot predict the late-onset rejection. The intra-/para-cerebroventricular human grafts are more vulnerable to the immune attack than the intrastriatal counterparts. Prevention of graft hyperplasia by using hypoproliferative late passaged human NPCs further significantly extends the graft survival time. Our new data also shows that a subpopulation of host microglia upregulate MHC-II expression in response to the human graft, but fail to present the human antigen to the host immune system, suggestive of the immune-isolation role of the blood–brain barrier (BBB).ConclusionsThe present study confirms the “immune privilege” of the brain parenchyma and, more importantly, unveils that choosing hypoproliferative NPCs for transplantation can benefit graft outcome in terms of both lower tumor-genic risk and the prolonged survival time without immunosuppression.

Early Antibody-Mediated Rejection in Non-Pre-Sensitized Heart Transplant Recipient

Introduction Antibody-mediated rejection (AMR) is an important cause of allograft dysfunction. It usually occurs after the first month of heart transplant (HT), but an early presentation is possible. We present an unusual case of a female HT recipient who had early AMR with no evidence of pre-sensitization to the donor. Case Report A woman, 45 years old, idiopathic dilated cardiomyopathy, two kids, with previous negative panel reactive antibody (PRA) was submitted to HT with success. The prospective crossmatch was negative and she did not receive blood cells during the surgery. The donor was a woman, 47 years old, who had a hemorrhagic stroke. After the procedure, she needed low doses of vasoactive drugs, received immunosuppression therapy according to institutional protocol and maintained normal ventricular function. On the 5th day, she developed biventricular graft dysfunction (left ventricular ejection fraction of 40%) requiring inotropes. The PRA revealed a donor specific antibody (DSA) not previously detected and the result of endomyocardial biopsy (EMB) confirmed acute AMR (pAMR2). Patient was successfully treated with methylprednisolone, plasmapheresis, immunoglobulin and rituximab. Biventricular function recovered; no DSA was detected and new EMB showed no rejection findings. Summary AMR results from recipient antibodies against donor-HLA antigens, causing complement activation, inflammation and injury to the allograft endothelium. In general, it occurs in the first two months after HT due to a rise in DSA by the contact with donor-HLA antigens. Early AMR, that occurs in the first week, is related to pre-sensitization to donor HLA antigens and associated with graft dysfunction. In this case report, no previous antibody had been detected. Two possibilities could explain such findings: the presence of non-HLA antibodies or the presence of memory B cells with levels of DSA too low to be detected on PRA. This case highlights the importance of monitoring antibodies in HT recipients with early allograft dysfunction even with previous negative PRA.

Post-transplant de novo non donor-specific HLA antibodies are not associated with poor graft outcome in non-sensitized pediatric recipients of kidney transplantation

While de novo donor-specific HLA antibodies (dnDSAs) have a detrimental impact on kidney graft outcome, the clinical significance of de novo non donor-specific antibodies (dnNDSAs) is more controversial. We retrospectively evaluated for Ab development and characteristics of dnNDSAs serially collected post-transplant sera and, when available, graft biopsy eluates, from 144 non-sensitized, primary pediatric kidney recipients, consecutively transplanted at a single center between 2003 and 2017, using HLA class I and class II single-antigen flow-bead assays (SAB). The results were compared with clinical-pathologic data from HLA antibody negative and HLA dnDSA-positive patients.Forty-five out of 144 patients developed dnNDSAs (31%). Among the dnNDSA-positive patients, 86% displayed one or more class I/II antibodies recognizing antigens included in the CREG/shared epitope groups that also comprise the mismatched donor HLA antigens. Despite potential pathogenicity, as suggested by their occasional presence within the graft, dnNDSAs displayed significantly lower MFI, and limited complement binding and graft homing properties, when compared with dnDSAs. In parallel, the graft survival probability was significantly lower in patients with dnDSA than in those with dnNDSA or without HLA antibodies (p < 0.005). Indeed, the dnNDSA-positive patients remaining dnDSA-negative throughout the posttransplant period did not develop clinical antibody mediated rejection and graft loss, and maintained good graft function at a median follow-up of 9 years. The biological characteristics of dnNDSAs may account for the low graft damaging capability when compared to dnDSAs.

Successful Clearance of Donor Specific Anti-HLA Antibodies after Desensitization with Rituximab, Velcade and Plasma-Exchange Followed By a Haploidentical Stem Cell Transplantion with Post-Transplantation Cyclophosphamide

Background: T cell - replete haploidentical stem cell transplantation (haploSCT) is increasingly performed and has expanded donor pool and transplant option for many patients (pt). However, the presence of recipient antibodies against donor HLA antigens (DSA) have been reported to be associated with engraftment failure and may limit the access to transplantation as the only lifesafing treatment modality. Guidelines for detection and treatment of DSA have been recently published but no standardized desensitization schedule exists so far. We report here the results of our institutional desensitization procedure initially developed for highly immunized patients with severe sickle cell disease undergoing haploSCT and then further adapted to all pt with DSA undergoing a mismatched allogeneic SCT. Methods: From March 2014 to July 2019, 20 pt had detectable DSA and did perform desensitization before undergoing a haploSCT. The DSA level was determined by using the LUMINEX technique (One Lamda, Inc). In case of positivity, the single antigen test was done to identify each class I and II HLA antibody-specificity. The values were expressed in mean fluorescence intensity (MFI). DSA were considered positive if the MFI value was ≥ 1000. The desensitization treatment included Rituximab 375 mg/m2, Velcade 1.3 mg/m2 and Plasma-Exchange followed by intravenous polyvalent immunoglobulins (Figure). DSA level controls were done routinely during desensitization, before starting the conditioning regimen and the day before graft injection. Pt who did not decrease DSA levels were not considered for haploSCT. Results: Median age was 61 years (range, 22-73). Diagnosis was acute myeloid leukemia in 6 pt, myelodysplastic / myeloproliferative syndrome in 10 pt, chronic lymphocytic leukemia in 1 pt and severe sickle cell disease in 3 pt. All donors were first-degree family members. Half of the pt were women [donor-recipient sex match: F/F=4; M/F=6; F/M=5; M/M=5pt]. The median MFI value before desensitization was 4700 (range, 1000-16000). Most pt (17) had DSAs against HLA class I antigens. Seventeen pt successfully decreased DSA levels to a median MFI value of 500 (range, 500-1200) and could proceed to haploSCT. They all engrafted and no DSA rebound was observed. One pt relapsed during the desensitization procedure and 2 pt with high DSAs above 10000 did not respond with remaining MFI of 16000 and 9500, respectively, and did not undergo haploSCT. One pt with CMML experienced primary graft failure for relapse and one pt with CLL did not engraft for unknown cause. Conclusions: The presence of DSA under 10000 should not be a barrier to transplantation. Our report shows that the hereby described desensitization schedule effectively cleared DSA allowing engraftment after haploSCT. Further studies are needed to determine the role of specificity and strength of DSA in order to better predict the likelihood of successful desensitization. Disclosures Harbi: Sanofi: Honoraria. Blaise:Jazz Pharmaceuticals: Honoraria.

Deceased donors as nondirected donors in kidney paired donation.

As proof of concept, we simulate a revised kidney allocation system that includes deceased donor (DD) kidneys as chain-initiating kidneys (DD-CIK) in a kidney paired donation pool (KPDP), and estimate potential increases in number of transplants. We consider chains of length 2 in which the DD-CIK gives to a candidate in the KPDP, and that candidate's incompatible donor donates to theDD waitlist. In simulations, we vary initial pool size, arrival rates of candidate/donor pairs and (living) nondirected donors (NDDs), and delay time from entry to the KPDP until a candidate is eligible to receive a DD-CIK. Using data on candidate/donor pairs and NDDs from the Alliance for Paired Kidney Donation, and the actual DDs from the Scientific Registry of Transplant Recipients (SRTR) data, simulations extend over 2years. With an initial pool of 400, respective candidate and NDD arrival rates of 2 per day and 3 per month, and delay times for access to DD-CIK of 6months or less, including DD-CIKs increases the number of transplants by at least 447 over 2years, and greatly reduces waiting times of KPDP candidates. Potential effects on waitlist candidates are discussed as are policy and ethical issues.

First-in-Human Trial of Bcma-CD19 Compound CAR with Remarkable Donor-Specific Antibody Reduction

Ma: iCAR Bio Therapeutics Ltd: Employment. Wada:iCell Gene Therapeutics LLC: Employment. Ma:iCell Gene Therapeutics LLC: Consultancy, Equity Ownership, Research Funding; iCAR Bio Therapeutics Ltd: Consultancy, Equity Ownership, Research Funding.

Anticorps non-HLA et transplantation d’organe : une vision critique

In solid organ transplantation, the deleterious effect of antibodies directed against donor HLA antigens, whether preformed or de novo, is well established. Anti-HLA antibodies have been associated not only with the risk of antibody-mediated rejection but also with late graft dysfunction and are now considered to be the leading cause of allograft loss after renal transplantation. In addition to HLA antibodies, the possible involvement of non-HLA antibodies targeting donor endothelial cells has long been the subject of intense research. The purpose of this review is to discuss current knowledge and remaining issues related to the involvement of non-HLA antibodies in solid organ transplantation. More specifically, the clinical data underlying the hypothesis of the role of non-HLA antibodies will be discussed, as well as the different techniques for antibody detection, their clinical relevance and their antigenic targets.

Virtual Crossmatch: Reality of Perception

Results of 773 actual flow crossmatches (aFXMs) and virtual flow crossmatches (vFXMs) performed for living and deceased donor kidney transplantation in our center were analyzed retrospectively and evaluated for their concordance. Prediction of vFXMs was based on antibody identification using single antigen bead assay and locally established mean fluorescence intensity cutoff point compared with donor HLA antigens. The vast majority of aFXMs were in concordance with vFXMs with an overall concordance of 97%. Twenty-three predicted to be negative showed positive aFXMs; 12 of them had 0% calculated panel-reactive antibody, and 11 were found in patients with multiple non−donor-specific HLA antibodies. Three predicted positive vFXMs yielded negative aFXMs; 2 of them had allele-specific antibodies. ConclusionsvFXMs based on precise characterization of antibody specificities detected by single antigen bead assay using our cutoff point accurately predicted FXMs in the majority of patients and can be used safely to allocate kidney offers without performing physical crossmatches in selected patients.

Cardiac transplantation across preformed HLA-antibody barriers

Cardiac transplantation across preformed HLA-antibody barriers

OR10 Data-driven modeling of flow cytometric crossmatch: Enhanced virtual crossmatching

Aim Increasingly HLA laboratories are using virtual crossmatching to predict recipient and donor compatibility using HLA antibody data and donor HLA type. However, virtual crossmatch interpretation is based on HLA experience and expertise of individual transplant centers. The purpose of this study is to develop data-driven algorithms (DDA) that predict flow cytometric crossmatch (FXM) outcomes using HLA antibody mean fluorescent intensity (MFI) data and donor HLA typing. These methods are independent of human intuition or experience and may provide insights that are otherwise imperceptible. Methods Two data sets consisting of 222 and 109 FXM with single antigen bead data for both HLA class I and II antibodies were used. Single antigen bead data was compiled against donor HLA antigens using the OneLambda assay and Luminex. The first DDA found the optimal MFI threshold using summation of mean fluorescent intensity data for class I and/or II that predicted either T cell or B cell mean channel shifts (MCS) above FXM cutoff. The second DDA applied a least-squares regression model to the HLA locus-specific data (second data set) to predict the actual T or B cell MCS. Results The threshold method yielded between 84.9% and 91.1% accuracy when using class I donor specific antibody (DSA) data to predict T cell outcome, and class I and II DSA data to predict B cell outcome. Optimal MFI thresholds of 3450 and 7560 were found for T and B cell prediction. For quality assurance, prediction of T cell MCS was attempted using class II data, resulting in 61.9% accuracy. The least-squares model increased accuracy to 93.6% and 97.2% for T and B cell MCS, respectively. Class I DSA influenced T and B cell MCS more than class II. The relative importance of an individual HLA locus on T cell prediction was found to be HLA-B > -A > -C. In the least-squares fitting, B64 had a large positive effect while A34 had a large negative effect on T cell MCS. Conclusions Utilizing DDA can expand accuracy of biologic systems beyond human experience and expertise. We showed how DDA can improve HLA crossmatch by generating high-level accuracy T and B cell FXM outcomes. Further improvements to the algorithm will incorporate HLA antigen expression and HLA antibody avidity.

HLA Donor Specific Antibody and HLA Antigen Bias are Independent Risk Factors for Chronic Lung Allograft Dysfunction

Immune responses to both HLA donor specific antibody (DSA) as well as lung associated self antigens have been implicated in chronic lung allograft dysfunction (CLAD). Furthermore, several studies have suggested that a bias in the HLA-DR antigens may promote a T-cell driven response to self-antigens. The goal of this study was two-fold; firstly we investigated the association of de novo HLA DSA with bronchiolitis obliterans syndrome (BOS) as well as restrictive allograft syndrome (RAS). Secondly, we investigated the HLA genotypes of both recipients and donors to determine if there was any antigen bias associated with either form of CLAD.

P233 On the performance characteristics of luminex single antigen bead (LSAB) assay mean fluorescence intensity cutpoint in predicting flow cytometry crossmatch (FCXM) results

Aim LSAB mean fluorescence intensity (MFI) is often used to predict physical FCXM results, and LSAB MFI cutpoints are used to list unacceptable antigens. We therefore determined the accuracy of LSAB assay MFI cutpoint used to score the LSAB assay as positive or negative in foretelling FCXM results. Methods One hundred and forty-six pre-transplant sera from 146 patients with end stage renal disease were tested against potential kidney donor’s T cells and B cells using a 3-color flow cytometry clinical protocol; the same 146 sera were tested using LSAB assay and the cumulative MFI directed at donor HLA class I and/or II antigens were determined. A T cell FCXM with a median channel shift (MCS) ≥ 40 with the patients serum vs. negative control serum was classified as a positive test; a B cell FCXM with MCS ≥ 50 with the patients serum vs. negative control serum was classified as a positive test. In the LSAB assay, MFI > 2000 directed at donor HLA antigens was scored as positive DSA. These cutpoints are our laboratory’s cutpoints for both ASHI proficiency testing and for clinical use. Results Statistical analysis demonstrated a significant association between LSAB results and FXCM results (Table 1). LSAB assay results predicted T Cell FCXM results with a sensitivity of 70% and a specificity of 100% (Fischers Exact Test, P 0.0001, Positive Predictive Value (PPV = 100%, Negative Predictive Value (NPV) = 91%). LSAB assay results also predicted B Cell FCXM results with a sensitivity of 86% and a specificity of 72% (Fischers Exact Test, P 0.0001, PPV = 62%, NPV = 91%) (Table 1). In a subset of 31 patients, both donor FCXM and auto FCXM results were available and 17 of 31 donor positive FCXM were also auto B cell FCXM positive (17/17) and auto T cell FCXM positive (1/17). Conclusions LSAB assay results are significantly associated with FCXM results. Auto B cell FCXM and high resolution HLA typing should further refine the relationship between these two high sensitive assays. Download high-res image (138KB) Download full-size image

P085 Lung re-transplant with repeated mismatch HLA antigens

Avoidance of previous donor mismatched HLA antigens is often considered for lung re-transplant due to concerns of potential HLA sensitization and memory responses. However, this approach may result in the exclusion of a large number of potential donors from consideration. Recently, we successfully performed a re-transplant with a history of multiple repeated mismatches. A 30+ year old male with a past medical history of cystic fibrosis underwent two previous bilateral lung transplants in August 2004 and in November 2010. Both allografts failed due to CLAD/BOS that developed after respiratory viral infections. The patient also subsequently developed detectable class I and II HLA antibodies with a cPRA > 88% in May 2016. Among these antibodies, strong anti-A1, B7 and DQ2 were first donor HLA specific. The second donor shared an A3-B18-DR4 haplotype with the first donor but not the recipient. No HLA antibodies specific to this A3-B18-DR4 haplotype antigens or to any other mismatched HLA antigens of the second donor were detected. A third lung transplant became urgently necessary in October 2016. Adding his previous donors’ mismatched HLA antigens to the unacceptable HLA list resulted in a cPRA > 96%. Therefore, a personalized listing strategy was developed based on discussions between the HLA lab and the transplant program. A deceased donor became available in November 2016. The virtual crossmatch revealed that the donor had no unacceptable HLA antigens based on HLA antibody detection. However, this donor carried the A3-B18-DR4 haplotype which was a repeated mismatch haplotype of the previous two donors. Based on a risk and benefit assessment, this donor was accepted as the source of lungs for the third transplant. The patient received 5 plasmapheresis sessions post-transplant to prevent antibody mediated rejection. Frequent post-transplant HLA antibody monitoring has not detected development of donor specific antibodies to the third donor and the new allograft functions well. Lung re-transplant can be performed with a cross of a repeated HLA mismatch provided that the recipient has no detectable HLA antibodies specific to the current donor. Our preliminary findings obtained from our study conducted on UNOS lung transplant data supports this conclusion. An assessment of risk and benefit of crossing the repeated mismatch HLA antigens should be done on a case-by-case basis.

The mechanism of humoral immune response to allogeneic organ transplantation

The problem of antibody-mediated rejection of donor organ remains extremely relevant. The main targets of the antibodies are mainly donor HLA-antigens (Human Leucocyte Antigens), expressed, in particular, by the cells of graft vascular endothelium. This review describes the mechanisms of the development of humoral alloimmunity which are based on B-cell recognition of epitopes of donor HLA-molecules and affinity maturation of B-cell receptors in the germinal centers of peripheral lymphatic system. Monitoring of epitope load and cross-reactivity indicators to evaluate HLA-compatibility of donor and recipient plays an important role in the prevention of allograft humoral rejection.

HLA Amino Acid Polymorphisms and Kidney Allograft Survival.

BackgroundThe association of HLA mismatching with kidney allograft survival has been well established. We examined whether amino acid (AA) mismatches (MMs) at the antigen recognition site of HLA molecules represent independent and incremental risk factors for kidney graft failure (GF) beyond those MMs assessed at the antigenic (2-digit) specificity.MethodsData on 240 024 kidney transplants performed between 1987 and 2009 were obtained from the Scientific Registry of Transplant Recipients. We imputed HLA-A, -B, and -DRB1 alleles and corresponding AA polymorphisms from antigenic specificity through the application of statistical and population genetics inferences. GF risk was evaluated using Cox proportional-hazards regression models adjusted for covariates including patient and donor risk factors and HLA antigen MMs.ResultsWe show that estimated AA MMs at particular positions in the peptide-binding pockets of HLA-DRB1 molecule account for a significant incremental risk that was independent of the well-known association of HLA antigen MMs with graft survival. A statistically significant linear relationship between the estimated number of AA MMs and risk of GF was observed for HLA-DRB1 in deceased donor and living donor transplants. This relationship was strongest during the first 12 months after transplantation (hazard ratio, 1.30 per 15 DRB1 AA MM; P < 0.0001).ConclusionsThis study shows that independent of the well-known association of HLA antigen (2-digit specificity) MMs with kidney graft survival, estimated AA MMs at peptide-binding sites of the HLA-DRB1 molecule account for an important incremental risk of GF.

A Modified Protocol with Improved Detection Rate for Mis-Matched Donor HLA from Low Quantities of DNA in Urine Samples from Kidney Graft Recipients.

BackgroundUrine from kidney transplant recipient has proven to be a viable source for donor DNA. However, an optimized protocol would be required to determine mis-matched donor HLA specificities in view of the scarcity of DNA obtained in some cases.MethodsIn this study, fresh early morning urine specimens were obtained from 155 kidney transplant recipients with known donor HLA phenotype. DNA was extracted and typing of HLA-A, B and DRB1 loci by polymerase chain reaction-specific sequence primers was performed using tailor-made condition according to the concentration of extracted DNA.ResultsHLA typing of DNA extracted from urine revealed both recipient and donor HLA phenotypes, allowing the deduction of the unknown donor HLA and hence the degree of HLA mis-match. By adopting the modified procedures, mis-matched donor HLA phenotypes were successfully deduced in all of 35 tested urine samples at DNA quantities spanning the range of 620–24,000 ng.ConclusionsThis urine-based method offers a promising and reliable non-invasive means for the identification of mis-matched donor HLA antigens in kidney transplant recipients with unknown donor HLA phenotype or otherwise inadequate donor information.

Polyomavirus Reactivation and Immune Responses to Kidney-Specific Self-Antigens in Transplantation.

Humoral immune responses against donor antigens are important determinants of long-term transplant outcomes. Reactivation of the polyomavirus BK has been associated with de novo antibodies against mismatched donor HLA antigens in kidney transplantation. The effect of polyomavirus reactivation (BK viremia or JC viruria) on antibodies to kidney-specific self-antigens is unknown. We previously reported excellent 5-year outcomes after minimization of immunosuppression for BK viremia and after no intervention for JC viruria. Here, we report the 10-year results of this trial (n=193) along with a nested case-control study (n=40) to explore associations between polyomavirus reactivation and immune responses to the self-antigens fibronectin (FN) and collagen type-IV (Col-IV). Consistent with 5-year findings, subjects taking tacrolimus, compared with those taking cyclosporin, had less acute rejection (11% versus 22%, P=0.05) and graft loss (9% versus 22%, P=0.01) along with better transplant function (eGFR 65±19 versus 50±24 ml/min per 1.73 m2, P<0.001) at 10 years. Subjects undergoing immunosuppression reduction for BK viremia had 10-year outcomes similar to those without viremia. In the case-control study, antibodies to FN/Col-IV were more prevalent during year 1 in subjects with polyomavirus reactivation than in those without reactivation (48% versus 11%, P=0.04). Subjects with antibodies to FN/Col-IV had more acute rejection than did those without these antibodies (38% versus 8%, P=0.02). These data demonstrate the long-term safety and effectiveness of minimizing immunosuppression to treat BK viremia. Furthermore, these results indicate that polyomavirus reactivation associates with immune responses to kidney-specific self-antigens that may increase the risk for acute rejection through unclear mechanisms.

OR49 Detecting chimerism following lung transplant with graft vs. host disease

Graft-versus-host disease (GVHD) is a major post-transplant complication, but is rarely found in solid organ transplant. A 50 year old male presented with skin rash and GI symptoms consistent with GVHD two months after a lung transplant, and skin and gastrointestinal biopsies were performed that confirmed the diagnosis. Subsequently, we evaluated a peripheral blood sample (PBL) from the patient for the percentage T cells and B cells expressing donor HLA antigens. A previously described method was used to determine the donor’s typing by SSP (Hahn, 2000). In order to determine the relative amount of donor cells that were present in the recipient, crossmatches were carried out using cells acquired from PBL and biopsy samples of the recipient’s colon. Cells from the colon biopsy were then isolated using a tissue grinder often utilized for spleen lymphocyte isolation. After washing, the cells were re-suspended in McCoy’s 5A Medium and used untreated for a flow crossmatch. During flow crossmatch the cells were incubated with serum (negative control, positive control, the patient’s own serum, and donor specific anti-HLA A1 serum), washed, and later stained with CD3 and CD19 for T-cells and B-cells respectively. The HLA-A1 antigen was solely donor specific, therefore, if there were any HLA-A1 positive cells present in the PBL or biopsy, the donor T-cells/B-cells would deviate from the negative control. This method allows for measurement of live cells that are donor derived, which can be assessed before and after treatment to determine efficacy. The crossmatch performed before treatment displayed a deviation in the T-cells when incubated with anti-HLA-A1 serum that was then measured as 5% of the total T-cells gated. Therefore, 5% of the CD3+ T cell population in the colon was of donor origin. After treatment with solumedrol, another colon biopsy was performed and the crossmatch showed 0% of the CD3+ T-cells were donor derived. As a result, the crossmatch showed the treatment was working and the patient’s symptoms had resolved. A follow-up PBL crossmatch 355 days post-transplant was performed and found 22% donor HLA-A1 expressing CD3+ T cells suggesting the transplanted lung can act as a reservoir for donor T cells or a semi-indirect recognition pathway may present donor HLA on recipient T cells.