LAUSR

Background and Aims: In otherwise near‐normal appearing biopsies by routine light microscopy, next‐generation pathology (NGP) detected close pairings (immune pairs; iPAIRs) between lymphocytes and antigen‐presenting cells (APCs) that predicted immunosuppression weaning failure in pediatric liver transplant (LTx) recipients (Immunosuppression Withdrawal for Stable Pediatric Liver Transplant Recipients [iWITH], NCT01638559). We hypothesized that NGP‐detected iPAIRs enrich for true immune synapses, as determined by nuclear shape metrics, intercellular distances, and supramolecular activation complex (SMAC) formation. Approach and Results: Intralobular iPAIRs (CD45high lymphocyte–major histocompatibility complex II+ APC pairs; n = 1167, training set) were identified at low resolution from multiplex immunohistochemistry–stained liver biopsy slides from several multicenter LTx immunosuppression titration clinical trials (iWITH; NCT02474199 (Donor Alloantigen Reactive Tregs (darTregs) for Calcineurin Inhibitor (CNI) Reduction (ARTEMIS); Prospective Longitudinal Study of iWITH Screen Failures Secondary to Histopathology). After excluding complex multicellular aggregates, high‐resolution imaging was used to examine immune synapse formation (n = 998). By enriching for close intranuclear lymphocyte–APC distance (mean: 0.713 μm) and lymphocyte nuclear flattening (mean ferret diameter: 2.1), SMAC formation was detected in 29% of iPAIR‐engaged versus 9.5% of unpaired lymphocytes. Integration of these morphometrics enhanced NGP detection of immune synapses (ai‐iSYN). Using iWITH preweaning biopsies from eligible patients (n = 53; 18 tolerant, 35 nontolerant; testing set), ai‐iSYN accurately predicted (87.3% accuracy vs. 81.4% for iPAIRs; 100% sensitivity, 75% specificity) immunosuppression weaning failure. This confirmed the presence and importance of intralobular immune synapse formation in liver allografts. Stratification of biopsy mRNA expression data by immune synapse quantity yielded the top 20 genes involved in T cell activation and immune synapse formation and stability. Conclusions: NGP‐detected immune synapses (subpathological rejection) in LTx patients prior to immunosuppression reduction suggests that NGP‐detected (allo)immune activity usefulness for titration of immunosuppressive therapy in various settings.