This study interrogates the antigen-specificity of inflammatory infiltrates in renal biopsies with BK polyomavirus (BKPyV) viremia (BKPyVM) with or without allograft nephropathy (BKPyVN). PBMC from 5 healthy HLA-A0101 subjects were stimulated by peptides derived from the BKPYV proteome or polymorphic regions of HLA. Next generation sequencing (NGS) of the T-cell receptor (TCR) cDNA was performed on peptide stimulated PBMC and 23 biopsies with T-cell mediated rejection (TCMR) or BKPyVN. Biopsies from patients with BKPyVM or BKVPyVN contained 7.7732 times more alloreactive than virus reactive clones. Biopsies with TCMR also contained BKPyV-specific clones, presumably a manifestation of heterologous immunity. The mean cumulative T-cell clonal frequency was 0.1378 for alloreactive clones and 0.0375 for BKPyV reactive clones. Samples with BKPyVN and TCMR clustered separately in dendrograms of V-family and J-gene utilization patterns. Dendrograms also revealed that V-gene, J-gene, and D-gene usage patterns were a function of HLA type. In conclusion, biopsies with BKPyVN contain abundant allospecific clones that exceed the number of virus reactive clones. The T-cell component of tissue injury in viral nephropathy appears to be mediated primarily by an ‘innocent bystander’ mechanism in which the principal element is secondary T-cell influx triggered by both anti-viral and anti-HLA immunity.

Purpose of review Transplant pathology contributes substantially to personalized treatment of organ allograft recipients. Rapidly advancing next-generation human leukocyte antigen (HLA) sequencing and pathology are enhancing the abilities to improve donor/recipient matching and allograft monitoring. Recent findings The present review summarizes the workflow of a prototypical patient through a pathology practice, highlighting histocompatibility assessment and pathologic review of tissues as areas that are evolving to incorporate next-generation technologies while emphasizing critical needs of the field. Summary Successful organ transplantation starts with the most precise pratical donor–recipient histocompatibility matching. Next-generation sequencing provides the highest resolution donor–recipient matching and enables eplet mismatch scores and more precise monitoring of donor-specific antibodies (DSAs) that may arise after transplant. Multiplex labeling combined with hand-crafted machine learning is transforming traditional histopathology. The combination of traditional blood/body fluid laboratory tests, eplet and DSA analysis, traditional and next-generation histopathology, and -omics-based platforms enables risk stratification and identification of early subclinical molecular-based changes that precede a decline in allograft function. Needs include software integration of data derived from diverse platforms that can render the most accurate assessment of allograft health and needs for immunosuppression adjustments.

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.

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