Human leukocyte antigen (HLA) molecular mismatch is a powerful biomarker of rejection. Few studies have explored its use in assessing rejection risk in heart transplant recipients. We tested the hypothesis that a combination of HLA Epitope Mismatch Algorithm (HLA-EMMA) and Predicted Indirectly Recognizable HLA Epitopes (PIRCHE-II) algorithms can improve risk stratification of pediatric heart transplant recipients. Class I and II HLA genotyping were performed by next-generation sequencing on 274 recipient/donor pairs enrolled in the Clinical Trials in Organ Transplantation in Children (CTOTC). Using high-resolution genotypes, we performed HLA molecular mismatch analysis with HLA-EMMA and PIRCHE-II, and correlated these findings with clinical outcomes. Patients without pre-formed donor specific antibody (DSA) (n=100) were used for correlations with post-transplant DSA and antibody mediated rejection (ABMR). Risk cut-offs were determined for DSA and ABMR using both algorithms. HLA-EMMA cut-offs alone predict the risk of DSA and ABMR; however, if used in combination with PIRCHE-II, the population could be further stratified into low-, intermediate-, and high-risk groups. The combination of HLA-EMMA and PIRCHE-II enables more granular immunological risk stratification. Intermediate-risk cases, like low-risk cases, are at a lower risk of DSA and ABMR. This new way of risk evaluation may facilitate individualized immunosuppression and surveillance.

Purpose Allograft rejection is driven by HLA disparity between donor and recipient despite of the use of various immunosuppression protocols. To better define the risk of allograft failure or complications from overt immunosuppression we applied the epitope-based mismatch approach to quantify the degree of HLA disparity in a in a large multi-center pediatric heart transplant (pHTX) cohort that has been followed for 3-5 years. Methods 274 pHTX recipient/donor pairs were retrospectively HLA genotyped by Next-Generation Sequencing and evaluated for HLA allele (alMM) and eplet mismatch (epMM) loads. HLAMatchmaker version 2.2 was used to determine epMM load at HLA-A/B/C, -DRB1/3/4/5, -DQA1/DQB1 and -DPA1/DPB1. Results Overall, the average total alMM was 13(±2). 76% were ≥5 HLA-A/B/C allele mismatches, 67% were ≥3 HLA-DRB and ≥3 HLA-DQ allele mismatch. The average epMM load was 60 (±17), range 16-112 (Table). 94% of cases had >11 HLA-DR or DQ epMM, a threshold previously shown to be associated with development of HLA donor-specific antibody in renal transplantation. While 159 cases (61.6%) had both DR and DQ epMM >11, only DR epMM or only DQ epMM >11 occurred in 31 (12.0%) and 50 (19.4%) cases, respectively (Figure). Therefore, this cohort can be divided into 3 groups for Class II epMM load: 6% (16) of cases with low, 36% (99) with intermediate and 58% (159) with high load. Conclusion Although this pHTx cohort is highly mismatched, for HLA alleles, there is a large range based on epMM, especially for HLA-DR and -DQ indicating that allele level typing is insufficient for determination of the true degree of mismatching. By correlating these data with clinical parameters, we aim to determine risk stratification based on epMM load in pediatric heart transplantation.

Data on the clinical importance of newly detected donor-specific anti-HLA antibodies (ndDSAs) after pediatric heart transplantation are lacking despite mounting evidence of the detrimental effect of de novo DSAs in solid organ transplantation. We prospectively tested 237 pediatric heart transplant recipients for ndDSAs in the first year posttransplantation to determine their incidence, pattern, and clinical impact. One-third of patients developed ndDSAs; when present, these were mostly detected within the first 6 weeks after transplantation, suggesting that memory responses may predominate over true de novo DSA production in this population. In the absence of preexisting DSAs, patients with ndDSAs had significantly more acute cellular rejection but not antibody-mediated rejection, and there was no impact on graft and patient survival in the first year posttransplantation. Risk factors for ndDSAs included common sensitizing events. Given the early detection of the antibody response, memory responses may be more important in the first year after pediatric heart transplantation and patients with a history of a sensitizing event may be at risk even with a negative pretransplantation antibody screen. The impact on late graft and patient outcomes of first-year ndDSAs is being assessed in an extended cohort of patients.

Post-transplant de novo anti-HLA donor specific antibodies (DSA) indicate a worse outcome in adult transplantation. No large prospective studies have established the prevalence of newly detected DSA after pediatric heart transplant (HT), nor studied their impact on outcomes. We used the CTOTC-04 cohort to determine the incidence, specificity and time course of newly detected DSA in the 1st yr post-HT.

C. Ashokkumar1, A. Zeevi2, C. Bentlejewski3, J. Dobberstein4, G. Mazariegos4, K.M. Abu-Elmagd5, R. Sindhi6 1Transplant Surgery, CHILDREN’S HOSPITAL OF PITTSBURGH AND UNIVERSITY OF PITTSBURGH, PITTSBURGH/PA/UNITED STATES OF AMERICA, 2Surgery / Transplant, Thomas E. Starzl Transplantation Institute, Pittsburgh/PA/UNITED STATES OF AMERICA, 3Pathology, University of Pittsurgh, Pittsburgh/UNITED STATES OF AMERICA, 4Transplant Surgery, Children’s hospital of Pittsburgh of UPMC and University of Pittsburgh, Pittsburgh/PA/ UNITED STATES OF AMERICA, 5Surgery, University of Pittsburgh, Pittsburgh/PA/UNITED STATES OF AMERICA, 6Transplant Surgery, Children’s Hospital of Pittsburgh of UPMC and University of Pittsburgh, Pittsburgh/PA/UNITED STATES OF AMERICA

UNLABELLED:To determine whether early acute cellular rejection (ACR) is associated with sub-optimal immunosuppression in children with liver transplants (LTx).METHODS:Twenty-five children with primary LTx after pre-transplant rabbit anti-thymocyte globulin (rATG), and steroid-free tacrolimus (TAC) were evaluated. Mitogen-stimulated T- and B-cell responses and mixed lymphocyte response to donor and third-party antigens were performed at several time points between two consecutive TAC doses. TAC concentrations (C) associated with half-maximal effect (EC(50)) on lymphocytes was determined by pharmacodynamic equations.RESULTS:Mean age was 7.2 +/- 6.2 years, mean time to lymphocyte function studies was 25 +/- 19 days. Acute rejection occurred at a mean interval of 31 +/- 19 days after LTx. Rejectors (n = 16) demonstrated significantly higher EC(50) of TAC for the intra-cellular IFN-gamma in T cells (p = 0.005) and its CD8+ sub-population (p = 0.027) as well as the co-stimulatory/activation receptor CD54 on B cells (p = 0.0001). The response of recipient lymphocytes to donor antigen was significantly higher in rejectors, compared with non-rejectors (p = 0.015). The patient groups demonstrated no differences in third-party MLR, or in C of TAC.CONCLUSIONS:Independent of the amount of immunosuppressant, ACR of liver allografts in children is associated with enhanced donor-specific alloreactivity. This is accompanied by a cytotoxic T-cell sub-population with increased requirement for TAC.

O147* Aims: Recipients of whole organ allografts were treated in accordance with two therapeutic principles of (1) recipient pretreatment with Thymoglobulin (THY, 3-6 mg/kg) or Campath (CAM, 30 mg) before graft reperfusion and (2) minimum posttransplant immunosuppression consistent with graft survival and function using tacrolimus monotherapy (target trough level of 10 ng/ml). Other agents (e.g. steroid, rapamycin) were added only to treat biopsy-proven rejection. This report described changes in immunological and hematological parameters with a minimum followup of >12 months. Methods: Blood samples were obtained at 1, 3, 6, and 12 months after transplantation for four-color flow cytometry of leukocyte lineages, in vitro cell proliferation assays, and ELISPOT using cadaveric donor splenocytes or live donor PBMC as alloantigens. Results: Pretreatment with THY or CAM resulted in significant posttransplant lymphocyte depletion, which slowly recovered over the next 6-12M. CAM had stronger and prolonged impacts on lymphocyte depletion. Recovery of CD4 population was slower than CD8, resulting in remarkably low CD4/CD8 ratios (<0.8) at 6-12M with a higher incidence in the CAM group. Likewise, DC1 and DC2 populations considerably decreased after transplantation. DC1 gradually recovered and became nearly normal by 12M (49% vs. 57% in normal volunteers); however, recovery of DC2 was remarkably slow, particularly in the CAM group, and was 5.5 ± 5.0% at 12M compared to 15.0 ± 9.0% in the normal. CAM, but not THY, also effectively depleted B cells, and significantly low B cell counts persisted for nearly 12M. At 1 year, 48 of 155 (31%) functioning kidney recipients were on daily immunosuppression with single or multiple drugs. The remaining 107 (69%) were on tacrolimus, cyclosporine, or rapamycin monotherapy, with doses spaced from one every other day to once weekly. Of 95 liver recipients, 26 (27%) are on daily therapy with single or multiple agents, while 69 (73%) are on spaced monotherapy. Detailed in vitro studies (MLR, CML, limiting dilution, ELISPOT) were performed in 20 kidney and 16 liver recipients with >1 year follow-up. Kidney recipients on continuous multiple drugs showed global suppression to mitogens and alloantigens. In contrast, two-thirds of recipients on spaced weaning showed donor-specific hyporeactivity with vigorous responses to mitogens and 3rd party alloantigens. The remaining recipients on spaced monotherapy had intact anti-donor reactivity by MLR; but, CML responses were low with CTLp frequencies of less than 1:90,000-300,000. Patients (n=2) with modest killing (10-16%) with CTLp of 1:60,000-150,000 showed a low frequency of γ-IFN positive cells in ELISPOT. On the contrary, although the majority of liver recipients were in more advanced stages of weaning, correlation between immunosuppression and in vitro results was less clear probably due to the complicated immunological status in this population (e.g. hepatitis). Approximately 50% showed donor-specific hyporeactivity or total suppression in MLR and CML. The remaining half showed reactivity to mitogens and alloantigens (donor and 3rd party) in MLR with a significantly higher background. Donor killing in CML was 8-43% with low CTLp frequencies of 1:<200,000. Conclusions: Donor-specific immune modulation was observed in a subset of allograft recipients pretreated with THY or CAM and given minimal posttransplant immunosuppression. In contrast, patients on continuous multiple drugs showed overall suppression.