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Complement 4d (C4d) is a marker of complement activation that has been used to evaluate humoral rejection in renal and heart allografts. Studies suggested a role for C4d detection in liver allografts in diagnosing acute cellular and humoral rejection but none correlated this with the pre-transplant liver disease. Our study analyzed the association of C4d deposition in liver allografts with the pre-transplant liver disease. C4d deposition was evaluated by indirect immunofluorescence and correlated with lymphocytotoxic crossmatch results, post-transplant clinicopathological diagnosis and type of pre-transplant liver disease. Allograft biopsies were classified by the native liver disease. After excluding 20 patients with rare liver diseases; C4d deposition was evaluated in 506 biopsies from 310 patients including 25 with PSC, 198 with viral hepatitis and 87 with other diseases. C4d immunereactivity distribution was not different among biopsies from patients with different lymphocytotoxic crossmatch results. Sinusoidal C4d deposition was noted in 11.9% of biopsies and 15.2% of patients (in one or more biopsies). 26% (9/35) of biopsies from patients with PSC had sinusoidal C4d deposition; more frequently than from patients with viral hepatitis 12% (43/348) (p=0.04) and other liver diseases 7% (8/123) (p=0.005). In conclusion, C4d deposition in liver allografts is independent of the crossmatch results. It occurs with a variety of pathologic abnormalities and underlying liver diseases; but is more frequent in patients with PSC. This suggests that mechanisms other than allo-immunity activate complement. The mechanisms and clinical significance of C4d deposition in liver allografts in patients with PSC remain to be determined.

As of November 2013, 14.5% of the waitlist for a donor kidney comprised patients awaiting a retransplant. We performed a retrospective cohort study of 11,698 adult solitary kidney recipients using national Scientific Registry of Transplant Recipients data transplanted between 2002 and 2011. The aim was to investigate whether outcomes from patients' initial transplants are significant risk factors for patients' repeat transplants or for likelihood of relisting after a failed primary transplant. Retransplant recipients were more likely to be treated for acute rejection [adjusted odds ratio (AOR), 95% confidence interval (CI) = 1.26 (1.07-1.48), p = 0.0053] or hospitalized (AOR = 1.19, 95% CI 1.08-1.31, p = 0.0005) within a year of retransplantation if these outcomes were experienced within a year of primary transplant. Delayed graft function following primary transplants was associated with 35% increased likelihood of recurrence (AOR = 1.35, 95% CI = 1.18-1.54, p < 0.0001). An increase in 1-year GFR after primary transplant was associated with GFR 1 year postretransplant (β = 6.82, p < 0.0001), and retransplant graft failure was inversely associated with 1-year primary transplant GFR (adjusted hazard ratio = 0.74, 95% CI = 0.71-0.76 per 10 mL/min/1.73 m(2) ). A decreased likelihood for relisting was associated with hospitalization and higher GFR following primary transplantation. The increasing numbers of individuals requiring retransplants highlights the importance of incorporating prior transplant outcomes data to better inform relisting decisions and prognosticating retransplant outcomes.

HLA disparity has a negative impact on the outcomes of hematopoietic cell transplantation (HCT). We studied the independent impact of amino acid substitution (AAS) at peptide-binding positions 9, 99, 116, and 156, and killer immunoglobulin-like receptor binding position 77 of HLA-A, B, or C, on the risks for grade 3-4 acute graft-versus-host disease (GVHD), chronic GVHD, treatment-related mortality (TRM), relapse, and overall survival. In multivariate analysis, a mismatch at HLA-C position 116 was associated with increased risk for severe acute GVHD (hazard ratio [HR] = 1.45, 95% confidence interval [CI] = 1.15-1.82, P = .0016). Mismatch at HLA-C position 99 was associated with increased transplant-related mortality (HR = 1.37, 95% CI = 1.1-1.69, P = .0038). Mismatch at HLA-B position 9 was associated with increased chronic GVHD (HR = 2.28, 95% CI = 1.36-3.82, P = .0018). No AAS were significantly associated with outcome at HLA-A. Specific AAS pair combinations with a frequency >30 were tested for association with HCT outcomes. Cysteine to tyrosine substitution at position 99 of HLA-C was associated with increased TRM (HR = 1.78, 95% = CI 1.27-2.51, P = .0009). These results demonstrate that donor-recipient mismatch for certain peptide-binding residues of the HLA class I molecule is associated with increased risk for acute and chronic GVHD and death.

Immune responses to lung-associated self-antigens (SAgs) have been implicated in chronic lung allograft rejection. The goals of this study were to determine the prevalence of pre-existing antibodies (Abs) to the SAgs in pulmonary diseases and the association between pre-existing Abs to SAgs and the development of primary graft dysfunction (PGD), donor-specific antibodies (DSA), and chronic rejection.Pre- and post-transplant sera were analyzed from 317 lung transplant (LTx) recipients between 2000 and 2011 with diagnosis of chronic obstructive disease (n = 161), idiopathic pulmonary fibrosis (IPF; n = 50), cystic fibrosis (CF; n = 55), and others (n = 51). Samples were analyzed for Abs to SAgs by enzyme-linked immunosorbent assay, and DSA and cytokines by Luminex. The clinical diagnosis of PGD and bronchiolitis obliterans syndrome (BOS) was based on International Society for Heart and Lung Transplantation guidelines.The overall prevalence of Abs to SAgs was 22.71%, including 18% in chronic obstructive pulmonary disease (p = 0.033), 34% in IPF (p = 0.0006), 29% in CF (p = 0.0023), and 19.6% in other diagnoses (p = 0.044). The incidence of PGD (88% vs 54%, p0.05), DSA (70% vs 45%, p0.01), and BOS (90% vs 38% (p0.001) after LTx was significantly higher in patients with pre-LTx Abs to SAgs than without. Pro-inflammatory cytokines (interleukin-1β, interleukin-17, and interferon-γ) were elevated in patients who had pre-LTx Abs to SAgs, along with a reduction in anti-inflammatory interleukin-10.Patients with IPF and CF have the highest prevalence of Abs to SAgs. Patients with pre-existing Abs to SAgs are at increased risk for development of PGD, DSA, and BOS. Strategies to remove pre-existing Abs to SAgs should be considered to improve lung allograft outcome.

Summary Recent years have witnessed remarkable expansion in the knowledge of how various immune/inflammatory cells and T helper (Th) cell subsets, including Th1, Th2, Th9, Th17, Th22, follicular T helper (Tfh) and Treg subpopulations, reciprocally regulate each other. This review highlights current understanding of the Th subsets paradigm, who are the old school players, who are the new kids on the block and how does each come to play in different clinical contexts in solid organ transplantation. The article commences with a brief overview of the development and characteristic cytokine profiles of individual members of the paradigm. However, the main focus of this review is on the current understanding of the Th subset paradigm, and how these unique subpopulations impact host responses towards solid organ allografts. More specifically, it will highlight the recent findings that implicate the paradigm in transplantation. The interplay among different subsets is discussed collectively in the clinical context of pretransplant immunological risk factors such as alloimmunization as well as post-transplant immunological consequences such as rejection. Accumulating evidence suggests that Th17 cells play a role in the development of chronic allograft injury in transplantation of various organs. In vitro, tacrolimus suppressed Th1 and Th2 cells but not Th17 cells. Animal studies suggest that regulatory T cells (Treg)-based therapies could be effective as mechanisms of long-term drug-free transplant tolerance in humans. Indeed, a dual role for TGF-β and Foxp3 in induced tolerance has been proposed, in which TGF-β stimulates Foxp3 expression and is associated with the induction of Treg-facilitating acquisition of tolerance. Exploiting Th subsets' regulatory functions could potentially offer opportunities for immunological interventions in solid organ transplantation.

Summary This report describes the project to identify the global distribution of extended HLA haplotypes, a component of 16th International HLA and Immunogenetics Workshop (IHIW), and summarizes the initial analyses of data collected. The project aims to investigate extended HLA haplotypes, compare their distribution among different populations, assess their frequency in hematopoietic stem cell unrelated donor registries and initiate an international family studies database and DNA repository to be made publicly available. HLA haplotypes compiled in immunogenetics laboratories during the evaluation of transplant candidates and related potential donors were analysed. Haplotypes were determined using the pedigree analysis tool publicly available from the National Marrow Donor Program (NMDP) website. Nineteen laboratories from 10 countries (11 laboratories from North America, five from Asia, two from Latin America and one from Australia) contributed data on a total of 1719 families comprised of 7474 individuals. We identified 10 393 HLA haplotypes, of which 1682 haplotypes included high-resolution typing at HLA-A, B, C, DRB1 and DQB1 loci. We also present haplotypes containing MICA and other HLA loci and haplotypes containing rare alleles seen in these families. The project will be extended through the 17th IHIW, and investigators interested in joining the project may communicate with the first author.

Data from HLA typing studies have made significant contributions to genetic theories about the Austronesian diaspora and the health of descendant populations. To help further unravel pattern and process elements, we have typed HLA and MICA loci at high resolution in DNA samples from well defined groups of Maori and Polynesian individuals. Our results show a restricted set of HLA class I alleles compared with other well characterised populations. In contrast, the class II HLA-DRB1 locus seems to be diverse in Maori and Polynesians and both groups show high frequencies of HLA-DRB1 ∗ 04:03 , -DRB1 ∗ 08:03 , - DRB1 ∗ 09:01 and - DRB1 ∗ 12:01 . Our survey also provides the first ever MICA datasets for Polynesians and reveal unusual distributions and associations with the HLA-B locus. Overall, our data provide further support for a hybrid origin for Maori and Polynesians. One novel feature of our study is the finding that the gene sequence of the HLA-B ∗ 40:10 allele in Polynesians is a recombinant of HLA-B ∗ 55:02 and - B ∗ 40:01 . HLA-B ∗ 40:10 is in close association with HLA-C ∗ 04:03 , an allele identified as a hybrid of HLA-C ∗ 04 and -C ∗ 02 . In this respect, our data resemble those reports on Amerindian tribes where inter-allele recombination has been a common means of generating diversity. However, we emphasize that Amerindian gene content per se is only a very minor element of the overall Polynesian genepool. The wider significance of HLA and MICA allele frequencies across the Pacific for modern day health is also discussed in terms of the frequency relative to reference populations of disease known to be associated with specific HLA and MICA markers. Thus, Polynesians and Maori are largely unaffected by “European autoimmune diseases” such as ankylosing spondylitis, uveitis and coeliacs disease, yet there are several Maori- and Polynesian-specific autoimmune diseases where the HLA and MICA associations are still to be determined.

Background and Aims Early post-transplant hepatic fibrosis is associated with poor outcomes and may be influenced by donor/recipient genetic factors. The rs368234815 IFNL4 polymorphism is related to the previously described IL28B polymorphism, which predicts etiology-independent hepatic fibrosis. The aim of this study was to identify the impact of donor and/or recipient IFNL4 genotype on early fibrosis among patients transplanted for hepatitis C (HCV). Methods Clinical data were collected for 302 consecutive patients transplanted for HCV. 116 patients who had available liver biopsies and donor/recipient DNA were included. 28% of these patients with stage 2 fibrosis or greater were compared to patients without significant post-transplant fibrosis with respect to clinical features as well as donor/recipient IFNL4 genotype. Results The IFNL4 TT/TT genotype was found in 26.0% of recipients and 38.6% of donors. Patients who developed early post-transplant fibrosis had a 3.45 adjusted odds of having donor IFNL4 TT/TT genotype (p = 0.012). Donor IFNL4 TT/TT genotype also predicted decreased overall survival compared to non-TT/TT genotypes (p = 0.016). Conclusions Donor IFNL4 TT/TT genotype, a favorable predictor of spontaneous HCV clearance pre-transplant, is associated with increased early post-transplant fibrosis and decreased survival.

We describe a novel HLA-DPB1 allele identified in an ASHI proficiency testing (PT) sample. We tested the sample by rSSO (One Lambda), SSP (One Lambda), Real Time-PCR (Linkage Biosciences), SBT (Conexio) and NGS (Immucor). Initial results by rSSO and SSP resulted in no assignment because probes and primers detected shared recognition sites between DPB1 ∗ 01:01 & 40:01 in exon 2 and a group of alleles including DPB1 ∗ 01:01 but not 40:01 in exon 3. Typing by Real Time-PCR & SBT resulted in DPB1 ∗ 01:01:01, 40:01 based on exon 2 sequences (Fig. 1-A). Mia Fora software auto-assigned DPB1 ∗ 01:01:01, 40:01 based on exon 2 as IMGT currently does not have reference sequences for DPB1 ∗ 40:01 beyond exon 2. However Mia Fora provides sequence data for exon 3 and most of exon 4 for DPB1 ∗ 40:01 (Fig. 1-B). The non-DPB1 ∗ 01:01:01 allele has 0 mismatches in exon 2 but 7 mismatches in exon 3 with DPB1 ∗ 40:01. We identified DPB1 ∗ 39:01 to have 0 mismatches in exon 3 and 1 mismatch in exon 2. This variant has a novel non-synonymous G > T substitution at the 2nd nucleotide of codon 84 (Gly > Val) in DPB1 ∗ 39:01 or a recombination between exon 2 of DPB1 ∗ 40:01 and exon 3 of DPB1 ∗ 39:01. DPB1 ∗ 39:01v also has a synonymous C > T substitution at the 3rd position of codon 3 in exon 1 (Data not shown). Exclusion of exon 3 probes in rSSO assigns DPB1 ∗ 01:01:01, 40:01 (Fig. 1-C). We designed hemizygous primers to sequence DPB1 ∗ 39:01v by Sanger’s SBT and results are concordant with NGS for both substitutions (Fig. 1-D). The sequence was submitted to Gene Bank (KX121039) and IMGT (HWS10026456). The haplotype carrying DPB1 ∗ 39:01v is predicted as A ∗ 30:01, C ∗ 04:01, B ∗ 53:01, DRB1 ∗ 03:02, DRB3 ∗ 02:02, DQA1 ∗ 04:01, DQB1 ∗ 04:02, DPA1 ∗ 02:01. With exception of our lab 56/57 labs participating in this PT who reported results for this allele made the assignment of DPB1 ∗ 01:01, 40:01 (98% consensus). Table 1 shows unsupervised assignments by different methods in our, number of adjustments needed to make different assignments & number of labs using various methods who participated in this HLA Class II PT. Download high-res image (470KB) Download full-size image

The global organ shortage is the strongest factor for the increase in transplant wait time and deaths on waitlists. Here we describe a model for involving high school students in education research around organ donation and transplantation and capitalize on the strength of a pre-existing educational program offered by the local organ procurement organization (OPO). While training in education research at Cleveland Clinic, a high school student embarked on a collaborative project with the local OPO. The project involved evaluating three educational programs, selecting the most appropriate program for administration at her school, coordinating with the student's school administration and teachers, administering an assessment tool for the effectiveness of the program, and analyzing the results. The local OPO program that was selected for implementation consisted of a video presentation entitled "Share your life, share your decision" prepared by the United States Health Resources and Services Administration (HRSA), lectures by invited speakers and an educational assessment (pre- and post-education). The assessment survey included 3 multiple choice and 7 true/false questions. Compared to the over 2500 programs administered in the last 5 years by the local OPO, this program had a higher volume of participation (n = 353 compared to an average of 150 students/day). Students correctly classified transplantation status of more organ and tissues post-education (P < .0001 for both). For 5 out of the 7 true/false questions, students correctly answered questions more frequently post-education (P ≤ .002 for all). This experience included for the first time a formal assessment of the program which will be utilized to address targeted areas for specific improvements. This student collaborative model of involving students in organ donation and transplantation related education research has the potential to promote and maximize the effectiveness of educational programs targeting their peers.

We describe two novel alleles, MICA*058 and MICA* 002:03.

Cytomegalovirus reactivation is common after reduced-intensity conditioning allogeneic hematopoietic stem cell transplant. Natural killer and T cells mediate immunity against viruses including cytomegalovirus. The alloreactivity of natural killer cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors with target cell ligands. This study sought to assess whether donor inhibitory or activating killer immunoglobulin-like receptor genotypes may influence post-transplant cytomegalovirus reactivation in transplant recipients.We analyzed 64 patients who underwent T-cell replete, matched sibling donor reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation at our institution. Transplant recipients were categorized according to their HLA inhibitory killer immunoglobulin-like receptor ligand groups. Donor killer immunoglobulin-like receptor genotypes were determined and then were assessed for correlations with cytomegalovirus reactivation in transplant recipients.No differences in cytomegalovirus reactivation were observed when comparing those with or without missing inhibitory killer immunoglobulin-like receptor ligands. When considering the number of donor activating killer immunoglobulin-like receptor genes, those with 5 or 6 had less cytomegalovirus reactivation than those with 1 to 4 (19% vs 48%; P = .029). The difference could not be attributed to baseline patient or transplant characteristics. No specific activating killer immunoglobulin-like receptor genotype was found to be associated with cytomegalovirus reactivation.These observations indicate that assessment of donor killer immunoglobulin-like receptor genotype may have important implications for predicting cytomegalovirus reactivation after T-cell replete, matched sibling donor reduced intensity conditioning allogeneic hematopoietic stem cell transplant.

Aim Predicting virtual crossmatch (XM) results for patients with DQ antibodies has been challenging and a number of explanations have been set forth including structural variations of the DQA/DQB heterodimers, non-native epitopes among solid phase beads, and level of expression of HLA-DQ molecules on donor cells. We compared reactivity of anti-DQ antibodies using Luminex bead assays to reactivity with B cells using flow cytometry. Methods The reactivity of 4 anti-DQ specific sera (Table 1) were evaluated by Luminex Single Antigen (LSA) and phenotype-panel beads, and flow cytometry. Results Serum TE reacted strongly with beads carrying DQ4, 5, and 6 specificities. In contrast its reactivity with B cells carrying these specificities varied significantly based on the DQA/DQB allele combinations (Table 2). Strikingly, cell# 8, 9, 10, and 11 carrying the DQB1∗05:01, DQA1∗01:01 alleles were not reactive with serum TE (Table 2) despite a high level of surface expression of these molecules on these cells as assessed by serum AT (Table 3). Serum AA also failed to react with a DQB1∗05:01, DQA1∗01:01 cell (Table 3). Conclusion The expression of HLA-DQ epitopes on SA beads is not always concordant with that of B cells carrying the same DQA1∗/DQB1∗ alleles. Thus, reactivity of anti-DQ antibodies with SA beads does not always accurately predict their reactivity with B cells expressing the same alleles.

s S189 None of the time of transplant BALS had growth of Aspergillus, however there were 2 BALs with weak positive LFD results (see table 2). There were no obvious confounding factors such as growth of non-pathogenic fungi. 16 of these patients had repeat BALs. All repeat BALS had negative LFD results, including the 2 that were previously positive. Conclusion: The LFD appears to be a quick easy to use test for the diagnosis of invasive pulmonary aspergillosis with high sensitivity. The rapidity of the test compared to fungal culture could reduce patient mortality by reducing delays in starting treatment. Table 2. LFD and Culture Results for Time of Transplant Patients Patient sex and age (years) LFD result Aspergillus growth from BAL culture LFD result from repeat BAL Aspergillus growth from culture of repeat BAL M 56 Negative No Negative No F 39 Positive No Negative No F 44 No Negative No M 16 Negative No Negative No M 59 Negative No Negative No F 28 Negative No Negative No F 27 Negative No Negative No F 60 Negative No Negative No F 56 Negative No Negative No F 48 Positive No Negative No F 23 No Negative No M 58 Negative No Negative No F 54 Negative No Negative No F 50 Negative No M 45 Negative No Negative No M 58 Negative No F 32 Negative No M 44 Negative No Negative No M 64 Negative No Negative No F 58 Negative No

Introduction. In recent years, the use of higher risk donor organs and steroid avoidance regimens with potential increased acute rejection risk have increased. We hypothesized that these patterns adversely affect changes in posttransplant renal function. Methods. By using Scientific Registry of Transplant Recipients data and multivariable generalized linear models, we examined factors associated with slopes of estimated glomerular filtration rate (GFR; modification of diet in renal disease) from 6 to 12 months and 6 to 24 months posttransplant in solitary adult renal transplant recipients transplanted between 2003 and 2008 (n=91,241). We estimated GFR at each interval, analyzed changes within patients between follow-up intervals, and evaluated changes in 1-year graft and patient survival during the study period. Results. GFR intercept at 6 months averaged 54.3 mL/min/1.73 m 2 (standard deviation [SD] 18.2 mL/min/1.73 m 2 ). Decline in GFR between 6 and 12 months posttransplant averaged 0.69 mL/min/1.73 m 2 (SD 10.9 mL/min/1.73 m 2 ) and between 6 and 24 months averaged 2.45 mL/min/1.73 m 2 (SD 15.7 mL/min/1.73 m 2 ). However, the GFR decline was significantly attenuated during the study period among both deceased and living donor transplant recipients. Factors significantly associated with steeper GFR decline were increased pretransplant dialysis time, older donor age, diabetes as a primary diagnosis, low body mass index, African American race, retransplants, nonprivate insurance, and increased panel reactive antibody percent. Baseline or slope in renal function did not differ substantially by immunosuppressive regimen. One-year overall graft survival increased during the study period from 92.3% to 93.9%. Conclusions. GFR slopes and 1-year survival rates have improved in the United States independent of donor quality and immunosuppressive regimen. Findings may reflect increased skill in medical management and need further evaluation to determine whether short-term improvements translate to improved long-term survival.

Background. We report our initial experience in using the proteasome inhibitor, bortezomib, to treat established antibody-mediated rejection (AMR) in 20 patients. Methods. There were 16 kidney-only and 4 kidney-combined organ recipients with de novo donor-specific antibody (DSA) and histologic evidence of AMR with peritubular capillaries C4d deposition. AMR was diagnosed 19.8 months (range 1-71 months) posttransplant. Patients received intravenous corticosteroids followed by a 2-week cycle on days 1-4-8-11 of plasmapheresis and 1.3 mg/m 2 bortezomib; then 0.5 mg/kg intravenous immunoglobulin four times. Results. De novo class I DSA was detected in 11 (55%) and class II DSA in 18 (90%) recipients. The absolute mean difference between peak-nadir dominant DSA was 68,171 molecules of equivalent soluble fluorochrome (P

HLA polymorphism remains a major hurdle for hematopoietic stem cell transplantation (HSCT). In 2004, Elsner et al. proposed the HistoCheck Web-based tool to estimate the allogeneic potential between HLA-mismatched stem cell donor/recipient pairs expressed as a sequence similarity matching (SSM). SSM is based on the structure of HLA molecules and the functional similarity of amino acids. According to this algorithm, a high SSM score represents high dissimilarity between MHC molecules, resulting in a potentially more deleterious impact on stem cell transplant outcomes. We investigated the potential of SSM to predict high-risk HLA allele mismatch combinations responsible for severe acute graft-versus-host disease (aGVHD grades III and IV) published by Kawase et al., by comparing SSM in low- and high-risk combinations. SSM was calculated for allele mismatch combinations using the HistoCheck tool available on the Web (www.histocheck.org). We compared ranges and means of SSM among high-risk (15 combinations observed in 722 donor/recipient pairs) versus low-risk allele combinations (94 combinations in 3490 pairs). Simulation scenarios were created where the recipient's HLA allele was involved in multiple allele mismatch combinations with at least 1 high-risk and 1 low-risk mismatch combination. SSM values were then compared. The mean SSM for high- versus low-risk combinations were 2.39 and 2.90 at A, 1.06 and 2.53 at B, 16.60 and 14.99 at C, 4.02 and 3.81 at DRB1, and 7.47 and 6.94 at DPB1 loci, respectively. In simulation scenarios, no predictable SSM association with high- or low-risk combinations could be distinguished. No DQB1 combinations met the statistical criteria for our study. In conclusion, our analysis demonstrates that mean SSM scores were not significantly different, and SSM distributions were overlapping among high- and low-risk allele combinations within loci HLA-A, B, C, DRB1, and DPB1. This analysis does not support selecting donors for HSCT recipients based on low HistoCheck SSM scores.

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer and natural killer T cells are involved in activation of these cells and can influence antiviral immunity in the liver. This study investigated the association between KIR genetic diversity and sustained virologic response (SVR) to Peginterferon and Ribavirin (Peg/RBV) therapy in liver transplant (LT) recipients with hepatitis C virus (HCV) recurrence. We tested KIR genotypes in 44 HCV-infected LT recipients treated with Peg/RBV for 48 weeks. Patients were categorized as having KIR genotypes A/A or B/x and analyzed for association with SVR. Fifteen of 44 (34%) patients had SVR. Only 2 of 18 (11%) who lacked KIR2DS2/KIR2DL2 achieved SVR compared to 13 of 26 (50%) who carried these two genes (odds ratio: 8.0, 95% confidence interval: 1.5-42.0, P = 0.008). The association between lack of KIR2DS2/KIR2DL2 and SVR remained significant after exclusion of 10 patients with non-genotype 1 HCV. No correlation was found with other activating or inhibitory KIR genes. Absence of KIR2DS2 and/or KIR2DL2 is associated with failure of Peg/RBV therapy in patients with recurrent HCV after LT. These findings support the role of natural killer and natural killer T cells in HCV clearance after LT and might be generalizable to treatment of HCV infection outside the setting of LT.

Achievement of complete donor chimerism (CDC) after allogeneic nonmyeloablative hematopoietic stem cell transplantation (NMHSCT) is important for preventing graft rejection and for generating a graft-vs-malignancy effect. The alloreactivity of NK cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors (KIRs) with target cell HLA/KIR ligands. The influence of KIR matching on the achievement of T-cell CDC after NMHSCT has not been previously described. We analyzed 31 patients undergoing T-cell replete related donor NMHSCT following fludarabine and 200 cGy TBI. Recipient inhibitory KIR genotype and donor HLA/KIR ligand matches were used to generate an inhibitory KIR score from 1 to 4 based upon the potential number of recipient inhibitory KIRs that could be engaged with donor HLA/KIR ligands. Patients with a score of 1 were less likely to achieve T-cell CDC (P=0.016) and more likely to develop graft rejection (P=0.011) than those with scores greater than 1. Thus, patients with lower inhibitory KIR scores may have more active anti-donor immune effector cells that may reduce donor chimerism. Conversely, patients with greater inhibitory KIR scores may have less active NK cell and T-cell populations, which may make them more likely to achieve CDC.

Aim Recent years have witnessed remarkable advances in the ability to detect and characterize HLA antibodies and ability to predict results of flow crossmatch virtually. In this study we compare the crossmatch results and HLA antibody profile among one heart and one kidney transplant recipients. Methods Pre-Tx and serial post-Tx HLA antibody testing was performed using Luminex based assays (One Lambda Thermo fisher) for detection of total HLA IgG, IgG subclasses, IgM, C1q. Sera were tested for total IgG at neat and 1:8 dilution. Antibody testing results were used to predict virtual crossmatch results. Crossmatches were performed by flow cytometry and extended incubation CDC. Results The following table shows pre-Tx and most recent post-Tx DSA characteristics, crossmatch results, biopsy results and graft outcomes in the 2 cases. Conclusion These data provide a cautionary note that pre-Tx DSA characteristics and virtual crossmatch predictions even when consistent with physical crossmatch results (both Pos and Neg), may not correlate with clinical outcomes particularly in highly allosensitized patients. Download : Download full-size image

Background HLA-DQ Donor Specific Antibody (DSA) is implicated in the negative impact of allograft function after lung transplantation. Non-HLA antibody to Protein Kinase C- ζ [(PKC- Z ) Ab] was reported to associate with graft injury in the absence of C4d deposition. Here we demonstrate DQ DSAs on chronic and acute graft injury elevated by (PKC- Z ) Ab following lung transplants. Case Report A 28-yo old male patient with cystic fibrosis underwent 1st Bilateral Lung Transplant (BLT) in 8/2011 with negative HLA DSA and cross-matches. Low titer (PKC- Z ) Ab was detectable. Graft function performed well in room air after BLT. In 9/2011 bronchoscopy biopsy showed A1B1 along with the appearance of DQ6 de novo DSA, but both biopsy and DSA turned negative following prednisone taper treatment. In 10/2014, he was admitted for cellular and antibody mediated rejection (CMR and AMR) with bronchoscopy biopsy A3B2R and significant increase of DQ6 without C4d deposition. At that time, (PKC- Z ) Ab was unchanged. One month later, (PKC- Z ) Ab abruptly increased to around 2.5-fold and DQ6 peaked (Fig. 1A1, A2). Patient lost allograft and received 2nd BLT on 11/19/2014 with negative cross-matches, DQ9 preformed DSA, and high titer (PKC- Z ) Ab. He was readmitted in 3/2015 for aggressive therapy for acute AMR with ×5 plasmapheresis, rituximab and bortezomib, and IVIG ×5 due to the significant drop of allograft function. AMR was aborted with remarkable decline of DQ9 DSA and (PKC- Z ) Ab on 4/21/2015 (Fig. 1B1, B2) resulting in improved graft function. Summary De novo DQ6 DSA with concurrent CMR resulted in chronic allograft rejection while preformed DQ9 DSA lead to acute allograft rejection after HLT. This negative impact of DQ DSAs to lung allograft injury and/or dysfunction was exacerbated by high titer (PKC- Z ) Ab. Download : Download full-size image

This report describes the design of the eplet version of HLAMatchmaker to determine class II compatibility at the structural level. This matching algorithm is based on the hypothesis, developed from molecular modeling of crystallized antigen-antibody complexes, that functional epitopes are represented by patches of surface-exposed nonself-amino acid residues surrounded by residues within a 3-A radius. Patch determinations with a molecular viewer of crystalline structural models downloaded from the Entrez Molecular Modeling Database Web site led to the identification of 44 DRB, 33DQB, 29 DQA, 20 DPB, and 9 DPA unique combinations of polymorphic positions. The residue compositions of these patches were then determined from amino acid sequences. This analysis resulted in a repertoire of 146 DRB, 74 DQB, 58 DQA, 45 DPB, and 19 DPA eplets. In many eplets, the residues are in short linear sequences, but many other eplets have discontinuous sequences of residues that cluster on or near the molecular surface. This analysis has also shown that all serologically defined DR and DQ antigens detectable by monospecific antibodies have unique eplets. Other eplets are present in groups of class II antigens, many of which appear as cross-reacting. The eplet version of HLAMatchmaker should be considered as a hypothetical model for the structural assessment of donor-recipient compatibility and the determination of mismatch acceptability for sensitized patients. This computer algorithm can be downloaded from the HLA Matchmaker Webside at http://tpis.upmc.edu.

Aim We monitored donor lymphoid chimerism (DLC) long term patterns by STR to investigate their clinical relevance to aGvHD. Methods 81 out of 879 liver transplant patients (2005-2013) who clinically suspected as aGvHD were tested for DLC. Short tandem repeat (STR) assay for DLC was run on the peripheral blood, T, NK, and CD8 + T cells. Cases with positive DLC in any preparation (>5%) were monitored until resolution of DLC or patient expiration. Results Three characteristic patterns of subset DLC were observed. The first pattern (5 patients) is characterized by low level D% that is detected only in CD8 + T cell on initial positive test (Fig. 1). In the second pattern, four patients on the initial positive DLC, had relatively higher CD8 + T cell D% combined with positive T cell D% (Fig. 1). These two patterns were associated with effective aGvHD control with complete resolution of DLC within a mean of 21 days. In the third pattern, four patients with positive DLC in all CD8 + T cells, NK cells and T cell were associated with longer duration for DLC resolution (114 days) (Fig. 2) and failure of treatment (Fig. 2). Conclusions Detection of DLC in both NK and CD8 + T cell preparations is associated with longer duration to DLC resolution and higher mortality rate. These results suggest that multi-subset DLC testing is a useful tool to support the diagnosis of aGvHD and predict the response to treatment.

Purpose The development of donor-specific HLA antibodies (DSA) after lung transplantation (LT) is a significant risk factor for bronchiolitis obliterans syndrome (BOS). The HALT study aimed to determine the incidence of DSA development & characterize DSA profiles early after LT. Methods and Materials We conducted a prospective observational study at 6 US LT centers that used similar induction & maintenance immunosuppression. Recipients were screened for DSA using the LABScreen® Single Antigen assay at 10, 30, 60, 90, & 120 days after LT, & if clinically indicated. DSA was defined as bead reactivity with an absolute MFI>1000, & an increase in MFI>30% & an increase in absolute MFI≥500 when compared to pre-LT reactivity. Results 115 patients were enrolled at the 6 sites between 12/2011 and 6/2012.[table1]Study follow-up will be complete in 11/2012. An interim analysis through 8/2012 showed that 40 of the 115 (35%) recipients developed DSA after LT; 6 (5%) had class I DSA only, 22 (19%) had class II DSA only, & 12 (10%) had class I & class II DSA. DSA were identified a mean 29.6±24.4 days after LT.[ figure 1 ]The median DSA MFI was 4534 (range: 1068-18150). Conclusions DSA development is common early after LT & most DSA are directed at class II HLA. Future studies should evaluate long-term outcomes after early DSA development & the potential benefit of preemptive DSA depletion. The HALT Study is funded by the NHLBI (R34 HL105412). Recipient Demographics (n = 115) Variable Age, mean (SD) 56.8 (12.4) Female, n (%) 47 (41%) Diagnosis, n (%) COPD 37 (32%) ILD 51 (44%) CF 17 (15%) PAH 2 (2%) Other 8 (7%) Bilateral, n (%) 86 (75%) Allosensitized pre-LT, n (%) 16 (14%) Number of HLA mismatches for A, B, C, DRB1, DQ, median (IQR) 8 (3)

Although outcomes after allogeneic hematopoietic cell transplantation (alloHCT) for AML have improved, this has mainly been attributed to a reduction in transplant-related mortality rather than reduced leukemia relapse. NK cell alloreactivity is regulated by inhibitory and activating signals mediated through cell-surface receptors including the killer immunoglobulin-like receptors (KIRs). Group A and B KIR haplotypes have distinct centromeric (Cen) and telomeric (Tel) gene-content motifs and donor Cen group B KIR haplotypes have been reported to be associated with decreased relapse and improved survival in AML patients undergoing unrelated donor alloHCT. We hypothesized that donor KIR genotype may also be predictive of outcomes after matched related donor (MRD) alloHCT. We evaluated 93 AML patients in CR1/CR2 who underwent T-cell replete alloHCT using HLA- matched related donors at our institution from 1/2000-3/2013. Sixty-six had myeloablative conditioning (MAC) that that was busulfan/cyclophosphamide-based and 27 had reduced-intensity conditioning (RIC) with fludarabine/total body irradiation or busulfan/fludarabine. Donors were KIR genotyped to assign haplotypes A/A vs. B/X and the distinctive Cen and Tel gene-content motifs of group A and B KIR haplotypes according to the presence or absence of one or more B haplotype-defining KIR genes. KIR B–content score for each KIR genotype was defined as the number of Cen and Tel gene-content motifs containing B haplotype–defining genes (range, 0-4). As compared to those with haplotypes B/X (n=40; B content scores of 1-4) those with haplotype A/A (n=25; B content score of 0) undergoing MAC had significantly lower 100-day, 6-, 12- and 24-month non-relapse mortality (NRM) (8% vs. 0%, 13% vs. 0%, 15% vs. 0%, 25% vs. 0%, respectively, Figure 1) which was confirmed on multivariable analysis (HR 9.19, p=0.03). There were no differences between these groups regarding patient and transplant-related characteristics, or for acute or chronic GVHD, relapse, or survival. The causes of death in the group with haplotypes B/X were most commonly attributed to infection and then GVHD. However, within the group with B/X haplotypes, the B motif content score (1-4) was not associated with significant differences in NRM (HR 0.79, p=0.56). No difference in outcomes was observed for those undergoing RIC. The number of donor activating KIR genes (2SD1, 2DS2, 2DS3, 2DS4, 2DS5, and 3DS1) was then assessed. As compared to those with 3-6 activating KIR genes (n=20) those with 0-2 (n=41) undergoing MAC had significantly lower 100-day, 6-, 12- and 24-month non-relapse mortality (NRM) (15% vs. 0%, 15% vs. 5%, 15% vs. 5%, 29% vs. 8%, respectively, Figure 2) which was confirmed on multivariable analysis (HR 4.07, p=0.01). There were no differences in other post-transplant outcomes when comparing these groups or when considering those undergoing RIC. An increase of 1 donor activating KIR also was highly associated with NRM (HR 1.37, p=0.008). Overall, these results suggest that in the MRD MAC alloHCT setting donor KIR genotype may be predictive of increased NRM risk, particularly for those with B/X haplotypes and greater numbers donor activating KIRs. No comparable effects were observed in the RIC setting. Future strategies to further enhance immune reconstitution post-transplant may be appropriate to pursue for these higher risk patients. These results may have potential implications to improve donor selection for those AML patients with multiple HLA-matched related donors and need to be validated in larger cohorts. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

The effect of killer cell immunoglobulin-like receptor (KIR) ligand incompatibility on outcomes after unrelated cord blood transplantation (UCBT) has been controversial. Eurocord found that KIR ligand mismatching was associated with decreased relapse incidence (RI) and improved overall (OS) and leukemia-free survival (LFS) for patients with acute lymphoblastic leukemia (ALL) and for those with acute myeloid leukemia (AML). Recently, the Japanese registry found no association between KIR ligand matching and LFS or OS in 643 UCBT recipients with acute leukemia. However, both studies have analysed the KIR ligand matching effect using low resolution typing of HLA-A,-B,-C and HLA-DRB1 high resolution. With the aim to clarify the KIR effect on outcomes (mainly RI and OS) in a larger series of single-UCBT recipients in the era of HLA-allele typing, we have analysed 1098 patients with AML and ALL reported to Eurocord and CIBMTR. All patients received single UCBT and myeloablative conditioning regimen. HLA matching was defined using high resolution typing or imputation for HLA-A,-B,-C and DRB1. Patients and donors were categorized by their KIR-ligand expression for HLA C group 1 or 2 and Bw4 as KIR ligand matched or mismatched. Patients included in the earlier Eurocord analysis were excluded. Univariate and multivariate models were built to analyse the effect of KIR ligand matching on outcomes. Results: None of the 8/8 HLA-matched transplants were KIR ligand mismatched, therefore they were excluded. Since HLA-match and KIR ligand mismatch were confounded, we conducted 2 separate analyses: a) 6-7/8 HLA-matched (n=501) and b) 3-5/8 HLA-matched transplants (n=586). In the group of 6-7/8 HLA matched, 291 recipients (58%) were KIR ligand-matched and 210 (42%) were mismatched. There were no statistically significant differences, between these two groups for gender, age, CMV serostatus, type and remission disease status, cell dose, conditioning regimen, in vivo T-cell depletion, transplant period and follow-up (around 40 months). In the group of 3-5/8 HLA matched, 176 recipients (30%) were KIR ligand-matched and 410 (70%) were mismatched. In this HLA group (i.e 3-5/8), KIR ligand-matched patients were younger ( Disclosures No relevant conflicts of interest to declare.

Aim Strong pre-formed HLA DSA (p-DSA) is detrimental to short and long-term visceral allograft survival particularly of the liver-free allograft. This limits the opportunity to obtain an immunologically compatible graft for highly sensitized patients with clinical urgency for intestinal transplant. Here we report our preliminary and short term outcome following successful intestinal transplant under alemtuzumab (ALT), bortezomib (BTZ) and IVIG pretreatment. Methods Four patients with cPRA = 100% underwent intestinal transplantation. All patients had strong (MFI ⩾ 5000) p-DSA (either C1q + or −) and +T/B cell flow cytometer cross-match (FCXM) only or both FCXM and cytotoxicity cross-match (CDCXM). Luminex single antigen and C1q assays were done for DSA detection. All patients received ALT (30 mg, iv single dose), BTZ (1.3 mg/m[2], iv /week for 4 weeks) and IVIG (2 gm/kg, iv, single dose) pretreatment combined with tacrolimus based immunosuppression. Results Two out of 4 patients were transplanted in the presence of single strong p-DSA (-C1q), both with a T/B cell +FCXM and -CDCXM. The third patient was transplanted in the presence of multiple strong class I and II p-DSAs. In this patient, class I antibodies were +C1q and were both T/B cell +FCXM/+CDCXM. The MFI of DSA turned negative ( Fig. 1 A). To date, graft survivals are 8, 21 and 39 weeks respectively. The fourth patient was transplanted with multiple strong p-DSAs, one of which was +C1q. This patient had +FCXM and -CDCXM at the time of transplant. There was no reduction in C1q or DSA during the early post-operative day (POD). A negative C1q antibody turned positive in 1.5 weeks with graft lost ( Fig. 1 B). Biopsy confirmed +C4d. Conclusions Rapid reduction of p-DSAs under ALT, BTZ, and IVIG pretreatment can reduce the risk of antibody mediated rejection after isolated intestine transplant. Close monitoring of reduction in DSA trend during the early POD may help to adjust treatment strategies and improve clinical outcomes.

Background NGS technologies have significant potential to generate high resolution HLA typing in a high throughput fashion. However, implementation in clinical immunogenetics laboratories involves extensive validation of a multitude of interdependent procedures, instruments, reagents, and software. To address these challenges we participated in March of 2014 in a multicenter Alpha study lead by the Immunogenetics Laboratory of a large academic institute that has developed an NGS-based protocol for HLA typing on the Illumina MiSeq. Methods We participated in the Alpha study with 2 main objectives: (1) validate the in-house developed NGS genotyping protocol for the HLA-A, B, C, DRB1 and DQB1 loci; (2) identify key critical steps in the protocol that we had problems with in our own lab prior NGS implementation. This abstract focuses only on key technical lessons learned from the study. The Alpha study results are independently submitted in another abstract. Results Participation in the Alpha study identified some critical steps in NGS workflow: (1) the quantitation of genomic DNA needs to be assessed by using methods that directly measure double stranded DNA concentration during primary sample preparation prior to target enrichment, (2) the library preparation reagents need to ensure adequate and uniform coverage throughout the length of the HLA targeted genomic region, (3) use appropriate methods for fragment size selection including magnetic beads and/or specialized electrophoretic instrumentation after library preparation, (4) quantitation and normalization of DNA of each amplicon to a standard DNA amount prior to amplicon pooling, (5) use two independent genotyping algorithms for data analysis to avoid systematic errors of genotype assignments caused by individual analysis software. Specific details about these lessons learned and more technical tips will be provided in this presentation. Conclusion In spite of the technical complexity of NGS workflow, identifying key critical steps in the process and identifying troubleshooting strategies are critical in implementing NGS in the clinical laboratories. Inter-laboratory validation studies and seeking the guidance from laboratories who developed NGS expertise is invaluable for expediting the learning curve for the implementation of this technology. M. Askar: Employee; Company/Organization; Cleveland Clinic.