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Background. Enhanced B-cell presentation of donor alloantigen relative to presentation of HLA-mismatched reference alloantigen is associated with acute cellular rejection (ACR), when expressed as a ratio called the antigen presenting index (API) in an exploratory cohort of liver and intestine transplant (LT and IT) recipients. Methods. To test clinical performance, we measured the API using the previously described 6-h assay in 84 LT and 54 IT recipients with median age 3.3 y (0.05–23.96). Recipients experiencing ACR within 60 d after testing were termed rejectors. Results. We first confirmed that B-cell uptake and presentation of alloantigen induced and thus reflected the alloresponse of T-helper cells, which were incubated without and with cytochalasin and primaquine to inhibit antigen uptake and presentation, respectively. Transplant recipients included 76 males and 62 females. Rejectors were tested at median 3.6 d before diagnosis. The API was higher among rejectors compared with nonrejectors (2.2 ± 0.2 versus 0.6 ± 0.04, P value = 1.7E-09). In logistic regression and receiver-operating-characteristic analysis, API ≥1.1 achieved sensitivity, specificity, and positive and negative predictive values for predicting ACR in 99 training set samples. Corresponding metrics ranged from 80% to 88% in 32 independent posttransplant samples, and 73% to 100% in 20 independent pretransplant samples. In time-to-event analysis, API ≥1.1 predicted higher incidence of late donor-specific anti-HLA antibodies after API measurements in LT recipients (P = 0.011) and graft loss in IT recipients (P = 0.008), compared with recipients with API

Background. Transcriptional regulation of liver transplant (LT) rejection may reveal novel predictive and therapeutic targets. The purpose of this article is to test the role of differential DNA methylation in children with biopsy-proven acute cellular rejection after LT. Methods. Paired peripheral blood DNA samples were obtained before and after LT from 17 children, including 4 rejectors (Rs) and 13 nonrejectors (NRs), and assayed with MethylC capture sequencing approach covering 5 million CpGs in immune-cell–specific regulatory elements. Differentially methylated CpGs (DMCs) were identified using generalized linear regression models adjusting for sex and age and merged into differentially methylated regions (DMRs) comprising 3 or more DMCs. Results. Contrasting Rs versus NRs, we identified 2238 DMCs in post-LT and 2620 DMCs in pre-LT samples, which clustered in 216 and 282 DMRs, respectively. DMCs associated with R were enriched in enhancers and depleted in promoters. Among DMRs, the proportion of hypomethylated DMRs increased from 61/282 (22%) in pre-LT to 103/216 (48%, P < 0.0001) in post-LT samples. The highest-ranked biological processes enriched in post-LT DMCs were antigen processing and presentation via major histocompatibility complex (MHC) class I, MHC class I complex, and peptide binding (P < 7.92 × 10−17), respectively. Top-ranked DMRs mapped to genes that mediate B-cell receptor signaling (ADAP1) or regulate several immune cells (ARRB2) (P < 3.75 × 10−08). DMRs in MHC class I genes were enriched for single nucleotide polymorphisms (SNPs), which bind transcription factors, affect gene expression and splicing, or alter peptide-binding amino acid sequences. Conclusions. Dynamic methylation in distal regulatory regions reveals known transplant-relevant MHC-dependent rejection pathways and identifies novel loci for future mechanistic evaluations in pediatric transplant subcohorts.