Donor Human Leukocyte Antigen Research Articles

HLA DR Genome Editing with TALENs in Human iPSCs Produced Immune-Tolerant Dendritic Cells.

Although human induced pluripotent stem cells (iPSCs) can serve as a universal cell source for regenerative medicine, the use of iPSCs in clinical applications is limited by prohibitive costs and prolonged generation time. Moreover, allogeneic iPSC transplantation requires preclusion of mismatches between the donor and recipient human leukocyte antigen (HLA). We, therefore, generated universally compatible immune nonresponsive human iPSCs by gene editing. Transcription activator-like effector nucleases (TALENs) were designed for selective elimination of HLA DR expression. The engineered nucleases completely disrupted the expression of HLA DR on human dermal fibroblast cells (HDF) that did not express HLA DR even after stimulation with IFN-γ. Teratomas formed by HLA DR knockout iPSCs did not express HLA DR, and dendritic cells differentiated from HLA DR knockout iPSCs reduced CD4+ T cell activation. These engineered iPSCs might provide a novel translational approach to treat multiple recipients from a limited number of cell donors.

High-throughput sequencing defines donor and recipient HLA B-cell epitope frequencies for prospective matching in transplantation

Compatibility for human leukocyte antigen (HLA) genes between transplant donors and recipients improves graft survival but prospective matching is rarely performed due to the vast heterogeneity of this gene complex. To reduce complexity, we have combined next-generation sequencing and in silico mapping to determine transplant population frequencies and matching probabilities of 150 antibody-binding eplets across all 11 classical HLA genes in 2000 ethnically heterogeneous renal patients and donors. We show that eplets are more common and uniformly distributed between donors and recipients than the respective HLA isoforms. Simulations of targeted eplet matching shows that a high degree of overall compatibility, and perfect identity at the clinically important HLA class II loci, can be obtained within a patient waiting list of approximately 250 subjects. Internal epitope-based allocation is thus feasible for most major renal transplant programs, while regional or national sharing may be required for other solid organs.

HLA-A gene knockout using CRISPR/Cas9 system toward overcoming transplantation concerns

BackgroundThe treatment of many cancers and genetic diseases relies on novel engraftment approaches such as cell therapy and hematopoietic stem cell transplantation (HSCT). However, these methods are hindered by the alloreactive immune responses triggered by incompatible human leukocyte antigen (HLA) molecules. A successful HSCT procedure requires the eradication of donor and recipient HLA alloimmunization. Eliminating HLA-A gene expression using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9) could be a great approach to increase the possibility of a successful HSCT through extending pool of unrelated donors.ResultsOur dual gRNA approach introduced a large deletion in the HLA-A gene. Among 22 single-cloned cells, two clones (9.09%) and 11 clones (50%) received homozygous and heterozygous large deletions, respectively. Finally, the real-time PCR results also revealed that HLA-A gene expression was diminished significantly.ConclusionThe results suggested that CRISPR/Cas9 could be used as an efficient technique to introduce HLA-A gene knockout; thus, it can considerably lessen the burden of finding a fully matched donor by lowering the alleles required for a successful HSCT.

Factors that Influence Organ Donor Registration Among Asian American Physicians in Queens, New York.

Organ donation rates in the United States are lowest among Asians. Physicians are highly respected in Asian communities and may be influential in promoting donor registration, but little is known about their organ donor registration attitudes. We assessed associations between knowledge, attitudes, personal/professional experience, cultural/religious beliefs surrounding organ donation and donor registration status using multivariable logistic regression. We surveyed 121 Asian physicians in Queens, New York; 22% were registered donors. Registered donors were more likely to discuss donation wishes with their family (OR 9.47, 95% CI 2.60-34.51), know that donor human leukocyte antigen does not need to match organ recipients (OR 6.47, 95% CI 1.66-25.28), and have experience advising patients about organ donation (OR 5.35, 95% CI 1.50-19.02). Culturally tailored educational materials providing updated information to promote family discussion about organ donation could potentially increase Asian physicians' level of comfort and expertise in discussing organ donor registration with patients.

Current Evidence on the Clinical Relevance of Donor-specific Antibodies in Paediatric Liver Transplantation.

The clinical impact of donor-specific antibodies (DSA) occurring before or after liver transplantation (LT) against donor-human leucocyte antigen (HLA) on graft outcome is still unclear. We aim to present the current consensus based on recent paediatric LT case series. Compared to kidney transplantation, the liver seems to be less susceptible to antibody-mediated graft damage, which is likely due to protective Kupffer cell activity. The incidence of DSA after liver transplantation is higher in children than in adults. DSA directed against HLA class II molecules, mainly DQ, occur more often. The presence of such anti-class II DSA (DQ/DR), especially of the complement-binding IgG3 subclass, may be associated with endothelial injury, T-cell-mediated rejection (TCMR), inflammation, and fibrosis. Regular DSA-posttransplant monitoring cannot as yet be recommended in routine practice but may be useful in selected cases.

Donor-Specific Human Leukocyte Antigen Antibody Formation After Allograft Glenoid Reconstruction Occurs But Does Not Impact Clinicoradiographic Outcomes

Background: Recurrent shoulder instability is a prevalent condition, with glenoid bone loss as a common cause. Arthroscopic repair using distal tibial allografts provides long-lasting treatment by restoring glenoid surface area and presumably avoids risks of sensitization against donor human leukocyte antigen (HLA). Two case studies have challenged this assumption, suggesting that small bone allografts are able to induce host adaptive immune responses to donor HLA. The incidence of small bone allograft HLA sensitization and its effects on resorption and patient outcomes are unclear. Purpose/Hypothesis: The purpose was to assess the rate of sensitization against donor HLA after distal tibial allograft procedures for shoulder instability due to glenoid bone loss and to find whether HLA sensitization negatively affects patient-reported and radiographic outcomes. We hypothesized that sensitized patients would have worse radiographic and self-reported outcomes compared with nonsensitized patients. Study Design: Cohort study; Level of evidence, 3. Methods: A total of 71 patients with a mean age of 28.85 years (range, 13.58-61.31 years) were enrolled, with 58 patients submitting sufficient pre- and postoperative blood samples for HLA antibody testing. In patients who developed HLA antibodies postoperatively, donor HLA typing was used to confirm donor-specific sensitization. Pre- and postoperative computerized tomography scans (0.9 ± 0.8 years follow-up) were used to grade resorption based on the modified Zhu resorption grade classification (ie, grade 0 = no resorption; grade 1 = less than 25% resorption; grade 2 = between 25% and 50% resorption; and grade 3 = larger than 50% resorption). The Western Ontario Shoulder Instability Index outcome scores were obtained preoperatively and at regular postoperative appointments. Resorption and outcome data were compared between sensitized and nonsensitized patients using the Fisher exact test, independent 2-tailed Student t tests, and the Wilcoxon rank-sum test to determine the effect of HLA sensitization on radiographic and patient-reported outcomes. Results: A total of 7 (12.1%) patients with sufficient HLA samples were sensitized against donor HLA postoperatively. Sensitized patients did not have significantly higher rates of resorption (21.9% vs 14.3%, 21.9% vs 28.6%, 43.8% vs 28.6%, and 12.5% vs 28.6% for respective resorption grades 0-3; P = .67; α = .05). Self-reported outcomes were not statistically significant between sensitized and nonsensitized patients (24.9 ± 27.61 vs 40.16 ± 18.99; P = .37; α = .05) and did not differ significantly based on resorption grade (47.4 ± 0.0 vs 55.2 ± 18.8, 30.4 ± 15.8 vs 39.9 ± 20.9, 41.2 ± 0.0 vs 39.1 ± 13.1, and -24.9 ± 0 vs 24.4 ± 19.6 for resorption grades 0-3; P > .05; α = .05). Conclusion: Sensitization against donor HLA after small bone graft allografting was not previously considered but has been brought to light as a possibility. Aside from potential complications for future organ transplants, HLA sensitization does not introduce a risk for adverse outcomes or higher grades of resorption compared with nonsensitized patients after small bone allografting for shoulder instability.

Analysis of HLA haplotype and clinical factors during hematopoietic stem cell transplantation

BackgroundThe human leukocyte antigen (HLA) haplotype of the recipient in hematopoietic stem cell transplantation (HSCT) is a key factor in its success or failure. We analyzed the relationship between HLA haplotype frequency and associated clinical factors in HSCT patients. MethodsPatients who underwent allogeneic HSCT between 2000 and 2019 at our institution were enrolled in this study. The HSCT composition was 77 bone marrow transplantations (BMT), 38 peripheral blood stem cell transplantations (PBSCT), and 36 cord blood transplantations (CBT). Patients were classified into three groups according to their donor HLA haplotype frequency in the Japan Population: group A, top 1–10 haplotypes; group B, top 11–100 haplotypes; and group C, haplotype 101-. We then compared various items including clinical biomarkers with the HLA haplotype frequency. ResultsA significant negative correlation was identified between older persons and length of survival. There are also significant correlations between survival and levels of immunoglobulin G, D-dimer, and C-reactive protein, as well as the platelet-large cell ratio before transplantation. A total of 96, 30, and 25 patients were classified into groups A, B, and C, respectively. The HSCT match rate was significantly higher in group A patients than in those of groups B and C. In contrast, the death rate, D-dimer level, and length of time for engraftment were significantly higher in group B and C patients than in those of group A. ConclusionAn assessment of transplant-related complications is important in improving the performance of HSCT. The present data suggest that a special therapeutic strategy is necessary for HSCT using low-frequency HLA haplotypes.

Circulating Donor Heart Exosome Profiling Enables Noninvasive Detection of Antibody-mediated Rejection.

Endomyocardial biopsy remains the gold standard for distinguishing types of immunologic injury-acute versus antibody-mediated rejection (AMR). Exosomes are tissue-specific extracellular microvesicles released by many cell types, including transplanted heart. Circulating transplant heart exosomes express donor-specific human leukocyte antigen (HLA) I molecules. As AMR is mediated by antibodies to donor HLAs, we proposed that complement deposition that occurs with AMR at tissue level would also occur on circulating donor heart exosomes. Plasma exosomes in 4 patients were isolated by column chromatography and ultracentrifugation. Donor heart exosomes were purified using anti-donor HLA I antibody beads and complement C4d protein expression was assessed in this subset as marker for AMR. Three patients had no rejection episodes. Circulating donor heart exosomes showed troponin protein and mRNA expression at all follow-up time points. One patient developed AMR on day 14 endomyocardial biopsy that was treated with rituximab, IVIG/plasmapheresis. Time-specific detection of C4d protein was seen in donor heart exosome subset in this patient, which resolved with treatment. C4d was not seen in other 3 patients' donor exosomes. Anti-donor HLA I specificity enables characterization of circulating donor heart exosomes in the clinical setting. Further characterization may open the window to noninvasively diagnose rejection type, such as AMR.

Individual HLAs influence immunological events in allogeneic stem cell transplantation from HLA-identical sibling donors.

In allogeneic hematopoietic stem cell transplantation (allo-HSCT), the effects of patient and donor human leukocyte antigen (HLA) matching status on graft-versus-host disease (GVHD) have been extensively elucidated, but the effects of specific HLAs on acute GVHD remain unclear. Using data from a Japanese registry, we retrospectively analyzed 4392 patients with leukemia or myelodysplastic syndrome who received transplants from HLA-identical sibling donors to investigate the effects of HLAs on acute GVHD. From unbiased searches of HLA-A, -B, and -DR, HLA-B60 was significantly associated with an increased risk of grades II-IV acute GVHD (HR, 1.34; 95% CI, 1.13-1.59; P = 0.001). In contrast, HLA-B62 was significantly associated with a decreased risk of grades II-IV (HR, 0.73; 95% CI, 0.62-0.87; P < 0.001) and III-IV acute GVHD (HR, 0.63; 95% CI, 0.46-0.87; P = 0.005). The risk of leukemia relapse was significantly higher in HLA-B62-positive patients than in HLA-B62-negative patients (HR, 1.23; 95% CI, 1.05-1.43; P = 0.01). Both HLA-B60 and -B62 did not affect overall survival. The findings of this study may by implication suggest the possibility that the effects of specific HLAs on transplant outcomes may reflect inherent biological features, and thus consideration of specific HLAs may be helpful to predict transplant outcomes.

Distinct molecular and immunological properties of circulating exosomes isolated from pediatric lung transplant recipients with bronchiolitis obliterans syndrome - a retrospective study.

Long-term success following human lung transplantation is poor due to chronic rejection. We demonstrated circulating exosomes of lung origin during acute and chronic lung allograft rejection. We analyzed plasma from pediatric lung transplant recipients (LTxRs) enrolled in the CTOT-C-03 to determine whether circulating exosomes are released into circulation during bronchiolitis obliterans syndrome (BOS). Plasma exosomes were isolated, and human leukocyte antigens (HLA) were detected. Exosomes were analyzed for lung self-antigens (SAgs), co-stimulatory molecules transcription factors, major histocompatibility complex class II (MHC-II), adhesion molecules, and 20S proteasome. Mice were immunized with exosomes from BOS or stable to determine their immunogenicity. Circulating exosomes from BOS LTxRs contained increased levels of SAgs, donor HLA class I, MHC-II, transcription factors, co-stimulatory molecules, and 20S proteasome compared with stable. Serial analysis of exosomes containing SAgs demonstrated that exosomes are detectable in the circulation before BOS. Mice immunized with exosomes from BOS, or stable, demonstrated that exosomes from BOS are distinct in inducing both humoral and cellular immune responses to SAgs. Circulating exosomes from BOS LTxRs elicit distinct humoral and cellular response. In addition, detection of SAgs on circulatory exosomes 12months before diagnosis of BOS suggest that exosomes could serve as biomarker.

Stem cell donor HLA typing improves CPRA in kidney allocation.

The Organ Procurement and Transplantation Network (OPTN) Kidney Allocation System provides a priority to sensitized candidates based on the calculated panel reactive antibody (CPRA) value. The human leukocyte antigen (HLA) haplotype reference panel used for calculation of the CPRA by the United Network for Organ Sharing (UNOS), the OPTN contractor, has limitations. We derived a novel panel from the National Marrow Donor Program HLA haplotype data set and compared the accuracy of CPRA values generated with this panel (NMDP-CPRA) to those generated from the UNOS panel (UNOS-CPRA), using predicted and actual deceased donor kidney offers for a cohort of 24282 candidates. The overall accuracy for kidney offers was similar using NMDP-CPRA and UNOS-CPRA. Accuracy was slightly higher for NMDP-CPRA than UNOS-CPRA for candidates in several highly sensitized CPRA categories, with deviations in linkage disequilibrium for Caucasians and the smaller size of the UNOS panel as contributing factors. HLA data derived from stem cell donors yields CPRA values that are comparable to those derived from deceased kidney donors while improving upon several problems with the current reference panel. Consideration should be given to using stem cell donors as the reference panel for calculation of CPRA to improve equity in kidney transplant allocation.

CAR-Tregs as a Strategy for Inducing Graft Tolerance.

The adoptive transfer of alloantigen-specific regulatory T cells (Tregs) following organ transplantation is an emerging treatment paradigm that may induce tolerance and reduce the risk for graft rejection. In particular, redirecting Treg specificity via expression of synthetic chimeric antigen receptors (CARs) has demonstrated therapeutic promise in several preclinical studies. In this review, we highlight recent progress and remaining barriers to the clinical translation of CAR-Treg therapies. CAR Tregs targeting human leukocyte antigen (HLA)-A2 showed antigen-specific in vitro activation and superior in vivo protective function relative to polyclonal Tregs. Adoptively transferred anti-HLA-A2 CAR Tregs prolonged the survival of HLA-A2-positive grafts in humanized mouse models. Donor HLA molecules are attractive candidate antigens to target with CAR Tregs in transplantation due to mismatched HLA only expressed on the transplanted organ. The feasibility of this approach has been demonstrated by several independent groups in recent years. However, substantial challenges in CAR design and preclinical modeling must be more extensively addressed prior to clinical application.

Global Proteomics Analysis of Circulating Extracellular Vesicles Isolated from Lung Transplant Recipients.

Lung transplant recipients (LTxRs) with acute rejection (AR) and chronic rejection (bronchiolitis obliterans syndrome [BOS]) induce circulating exosomes known to contain donor human leukocyte antigens and lung-associated self-antigens. Here, we sought to identify proteomic signatures in circulating extracellular vesicles (EVs) that differentiate LTxRs in 4 groups: stable, AR, BOS, or respiratory viral infection (RVI). EVs were isolated from plasma from patients in each group via ultracentrifugation. EV protein cargoes were prepared for shotgun proteomics using liquid chromatography–tandem mass spectrometry. We identified 2 unique proteins for AR, 4 for RVI, 24 for BOS, and 8 for stable LTxRs. Differential analysis of AR, BOS, RVI, and stable proteins identified significantly deregulated proteins (p < 0.05, log2(fold change) > ±1) in each condition (31, 2, and 2, respectively). EVs from LTxRs with AR contained proteins involved in immunoglobulin, complement regulation, coagulation, and innate and adaptive immune response pathways. EVs from LTxRs with BOS revealed enriched immunoglobulin receptors and a carboxypeptidase N catalytic chain. EVs from LTxRs with RVI had an enriched macrophage-stimulating factor. We found unique signatures in LTxRs with AR, BOS, and RVI, highlighting complex immune mechanisms underlying lung allograft rejection. Proteomic signatures in LTxRs’ circulating EVs provided insights into immunological mechanisms of graft rejection and RVI.

Impact and production of Non-HLA-specific antibodies in solid organ transplantation.

Organ transplantation is an effective way to treat end-stage organ disease. Extending the graft survival is one of the major goals in the modern era of organ transplantation. However, long-term graft survival has not significantly improved in recent years despite the improvement of patient management and advancement of immunosuppression regimen. Antibody-mediated rejection is a major obstacle for long-term graft survival. Donor human leucocyte antigen (HLA)-specific antibodies were initially identified as a major cause for antibody-mediated rejection. Recently, with the development of solid-phase-based assay reagents, the contribution of non-HLA antibodies in organ transplantation starts to be appreciated. Here, we review the role of most studied non-HLA antibodies, including angiotensin II type 1 receptor (AT1 R), K-α-tubulin and vimentin antibodies, in the solid organ transplant, and discuss the possible mechanism by which these antibodies are stimulated.

Profiling non-HLA antibody responses in antibody-mediated rejection following heart transplantation.

Antibody-mediated rejection (AMR) driven by the development of donor-specific antibodies (DSA) directed against mismatched donor human leukocyte antigen (HLA) is a major risk factor for graft loss in cardiac transplantation. Recently, the relevance of non-HLA antibodies has become more prominent as AMR can be diagnosed in the absence of circulating DSA. Here, we assessed a single-center cohort of 64 orthotopic heart transplant recipients transplanted between 1994 and 2014. Serum collected from patients with≥pAMR1 (n=43) and non-AMR (n=21) were tested for reactivity against a panel of 44 non-HLA autoantigens. The AMR group had a significantly greater percentage of patients with elevated reactivity to autoantigens compared to non-AMR (P=.002) and healthy controls (n=94, P<.0001). DSA-positive AMR patients exhibited greater reactivity to autoantigens compared to DSA-negative (P<.0001) and AMR patients with DSA and PRA>10% were identified as the subgroup with significantly elevated responses. Reactivity to 4 antigens, vimentin, beta-tubulin, lamin A/C, and apolipoprotein L2, was significantly different between AMR and non-AMR patients. Moreover, increased reactivity to these antigens was associated with graft failure. These results suggest that antibodies to non-HLA are associated with DSA-positive AMR although their specific role in mediating allograft injury is not yet understood.

Proteomics Analysis of Circulating Extracellular Vesicles Isolated from Plasma of Human Lung Transplant Recipients

We demonstrated induction of circulating exosomes in lung transplant recipients (LTxRs) with acute/chronic rejection (bronchiolitis obliterans syndrome [BOS]) containing donor human leukocyte antigen and lung self-antigens (SAgs) (Kα-1 Tubulin, Collagen V), costimulatory molecules (CD80, CD86), transcription factors (NFkB, 20S proteasome) (Am J Transplant 17(2):474-484, 2017). In the current study, we identified unique proteomic signatures in circulating extracellular vesicles (EVs) that can differentiate human LTxRs with acute rejection (AR), BOS, respiratory viral infection (RVI), and stable. EVs were isolated using ultracentrifugation from LTxRs plasma (stable, AR, BOS or RVI) following transplant. EVs were characterized for the presence of lung SAgs (Kα-1 Tubulin, Collagen V) and size (50-200nm) was determined using NanoSight. EV protein cargoes were prepared for shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified unique proteins (2 AR, 24 BOS, 4 RVI, 8 stable cohort) using LC-MS/MS. Differential analysis of the proteins from AR, BOS and RVI to stable yielded significant unique proteins (p-value <.05, fold-change >2) in each clinical cohort (27 AR, 2 BOS, 2 RVI). EVs from AR contained proteins involved in immunoglobulin, complement regulation, innate and adaptive immune response pathways. EVs from BOS revealed enrichment of immunoglobulin receptors and carboxypeptidase N catalytic chain. EVs from RVI were enriched for macrophage stimulating factor. Our results demonstrate unique signatures in acute/chronic rejection and RVI highlighting complex immune mechanisms underlying lung allograft rejection. Presence of complement proteins and immunoglobulins in circulating EVs from AR can be helpful in differentiating cellular rejection with a mixed phenotype (cellular and humoral). We identified immunoglobulins and complement proteins in BOS in support of significance of Abs in the pathogenesis of BOS. Macrophage stimulating factor contained in EVs specific to RVI can be useful for the differential diagnosis of RVI from AR. To conclude, proteomic signatures of circulating EVs from LTxRs provided insights into immunological mechanisms at play in graft rejection and RVI leading to increased risk for chronic lung allograft dysfunction.

A Comparison of Intra-Operative Human Leukocyte Antigen Antibody Removal Techniques in Pediatric Heart Transplantation

Many potential pediatric heart transplant (HT) recipients develop sensitization to potential donor Human Leukocyte Antigens (HLA) from prior tissue and blood product exposure. HT in the face of allosensitization carries elevated risk of morbidity and mortality. We sought to determine and compare the degree and timing of HLA-antibody removal between centrifuge-based plasmapheresis (cPP) and membrane-based plasmapheresis (mPP) during cardiopulmonary bypass (CBP). Two mPP and two cPP systems were each incorporated into CPB circuits. The prime consisted of highly sensitized pooled donor whole blood. Blood was considered sensitized if any HLA-antibody exceeded 3000 mean fluorescence intensity (MFI) units by Luminex assay. Fresh frozen plasma was utilized to replenish the plasmapheresis circuits. Samples to determine the panel reactive antibody (PRA) were taken at baseline, then at 30 minute intervals until the end-point at 2 hours. The pooled donor blood was sensitized to HLA Class II but not Class I per institutional threshold. Reduction of anti-HLA class II antibody to below threshold levels (MFI <3000) was achieved in both the cPP and mPP systems. In the cPP circuits, there was a gradual decrease in antibody levels at each 30-minute interval, reaching a nadir at 90 minutes with a 2 to 3.5-fold reduction at completion. In the mPP circuit, there was an initial rapid decrease in antibody levels at 30-minute mark followed by a plateau corresponding to a 1.7 to 2.5-fold decrease in MFI at 2 hours. The incorporation of cPP or mPP systems into the CPB circuit can effectively reduce circulating HLA-antibodies. This can be accomplished within the time period of CPB for implantation before graft reperfusion and may be done more efficiently using cPP. This technology may have important utility for sensitized transplant recipients at increased risk for early antibody-mediated rejection.

International Retrospective Study of Allogeneic Hematopoietic Cell Transplantation (HCT) for Activated Phosphoinositide 3-Kinase Delta (PI3K) Syndrome

Mutations resulting in increased PI3K signaling confer increased risk of B cell lymphoma, recurrent infections, poor viral control, and autoimmunity. Allogeneic HCT is curative, but the optimal approach to HCT for these patients is still evolving as experience grows. Herein, we present the clinical outcomes of 27 patients transplanted for activating PI3K mutations. Required approvals were obtained by contributing centers. Nineteen <i>PIK3CD</i> patients and 8 <i>PIK3R1</i> patients received 22 and 10 HCTs respectively, at median age 12 years (range 2-66) at time of first HCT. Significant pre-HCT comorbidities were common, Fig 1. Conditioning platforms varied in intensity; most included serotherapy (84%). With median survivor follow up of 26.3 months from first HCT (range 2.4-71.8), overall survival was 85% (3 infectious deaths and 1 due to organ toxicity); graft failure (GF)-free survival (GFFS) was estimated at 80% at 1 year but declined to 68% by 2 years, Fig 2. Neutrophil engraftment occurred at median 15.5 days (range 11-33) with autologous recovery in 1 patient at day +14 and another deceased prior to engraftment. Four engrafted patients are alive and well despite mixed donor chimerism (<95%). Nine (33%) patients required 24 total subsequent unplanned donor cell infusions (DCI) for mixed chimerism (n=15), poor graft function (n=2), infection (n=1), GF (n=3), malignancy relapse (n=1), or to improve immune reconstitution (n=2). Grade 2-4 acute graft versus host disease (GVHD) occurred in 27% patients, with Grade 3-4 acute GVHD and limited self-resolved chronic GVHD in 1 (4%). Organ toxicities and infectious complications are summarized in Fig 3; CMV infection requiring treatment occurred in 11 (41%) patients with disease in 3 (11%). Serotherapy use, conditioning intensity, and degree of donor human leukocyte antigen (HLA) match were not significantly associated with cumulative incidence of GF or subsequent unplanned DCI (with death as a competing risk), while mTOR inhibitor use post-HCT was associated with subsequent unplanned DCI for graft augmentation or GF (p=0.006). An mTOR inhibitor was used within the year following 12 HCTs, for primary GVHD prophylaxis (n=7) or to treat disease manifestations or GVHD; loss of chimerism and/or need for subsequent DCI occurred in 9 of these. Patients with activating PI3K mutations are at overall significant risk of GF or need for subsequent DCI. While we found no association with GF or DCI for discrete elements such as serotherapy use, donor source, or conditioning intensity, small numbers preclude conclusions regarding the optimal HCT approach, and more HCTs with longer follow up are needed to refine these preliminary findings. Post-HCT mTOR inhibitor exposure was associated with need for subsequent unplanned DCI, perhaps by providing a survival advantage to host lymphocytes, and may thus be detrimental in these patients during the early post-HCT timeframe.

Using Nanopore Whole-Transcriptome Sequencing for Human Leukocyte Antigen Genotyping and Correlating Donor Human Leukocyte Antigen Expression with Flow Cytometric Crossmatch Results

Transplant centers are increasingly using virtual crossmatching to evaluate recipient and donor compatibility. However, the current state of virtual crossmatching fails to incorporate donor human leukocyte antigen (HLA) expression in the assessment, despite numerous studies that have demonstrated the impact of donor HLA expression on physical crossmatch outcomes. Whole-transcriptome sequencing (RNA-Seq) for HLA enables simultaneous determination of HLA genotyping and relative HLA expression. Ultimately the RNA-Seq needs to be faster to be incorporated into the virtual crossmatching process. However, to demonstrate feasibility, the utility of the MinION sequencer (Oxford Nanopore Technologies, Oxford, UK) was evaluated in combination with RNA-Seq to generate HLA genotypes and to determine HLA class I expression. Although HLA class I expression varied among individuals, the pattern of HLA expression remained relatively consistent (HLA-B>HLA-A=HLA-C). HLA-A and -C had similar expression profiles. The impact of donor HLA expression was evaluated using serum samples containing a single donor-specific antibody (DSA). By making DSA consistent, donor HLA expression variability could be assessed. With consistent DSA mean fluorescence intensity, there was a direct relationship between the donor HLA expression to which the DSA is against and flow cytometric crossmatch median channel shifts.

Epitope matching in kidney transplantation: recent advances and current limitations.

Evolution of human leukocyte antigen (HLA) molecular typing techniques has progressively enabled more accurate determination of the three-dimensional building blocks that form the antibody accessibility and binding sites of each HLA allele. These immunogenic HLA regions known as epitopes are composed of polymorphic sequences of amino acid residues termed eplets. This review provides a critical appraisal of the current understanding of epitope compatibility in kidney transplantation. There is a tendency to suggest that epitope matching is likely to be superior to broad antigen HLA matching such that the allocation of donor kidneys to patients with a more favorable epitope compatibility profile may lead to better allograft outcomes. A growing body of work has highlighted the association between a greater number of eplet mismatches and adverse allograft outcomes, and approaches using eplet matching have been successfully implemented in organ allocation programs. However, our understanding of epitope compatibility remains in its infancy, requiring further and more in-depth evaluation. Critically, it remains unclear how best to translate findings derived at the population level to the care of individual patients. Questions that need to be answered include a lack of consensus in the definition and interpretation of epitope compatibility, are class I and II compatibility of similar clinical importance, how best to define predetermined mismatch thresholds for utilization in organ allocation, and whether other properties such as differences in electrostatic potential between donor and recipient HLA alleles are also important in determining immunological compatibility. Epitope matching likely represents a valid progression in understanding donor-recipient HLA compatibility. However, more clinical data and a better understanding about differences in methods to determine epitope compatibility are required before the approach can be widely applied in clinical practice.