*Purpose: Solid organ transplant is lifesaving for children with end-stage heart, liver, or kidney disease. Protection of the transplanted organ from rejection requires long-standing immunosuppression. However, despite our arsenal of immunosuppressive agents, 25% of pediatric transplant recipients have a rejection episode in the first-year post-transplant. The goal of our study was to identify novel and robust surrogate endpoints of allograft status.

*Methods: Blood was obtained from pediatric recipients of liver, heart, kidney, or intestinal grafts enrolled at seven sites in the NIAID-sponsored Clinical Trials of Organ Transplantation in Children (CTOTC)-06, a prospective multi-institutional study. PBMC from 52 children were analyzed, from patients who had stable graft function (n=24) in the 12-month period surrounding sample collection and patients who had an episode of biopsy-proven rejection (n=28) within 30 days after sample collection. These cohorts were well-balanced with respect to clinical characteristics. PBMCs were thawed, barcoded, pooled, and stained with 37 metal-conjugated antibodies against cell surface and intracellular proteins expressed in immune cell lineages and analyzed by single-cell mass cytometry (cytometry by time-of-flight; CyTOF). To identify differences in population frequency between stable and rejection cohorts, we computed cell-type proportions for each population by calculating the number of cells of each population divided by the total number of cells in the respective lineage. Linear discriminant analysis (LDA) was performed to assess which cell-type proportions maximally separate stable and rejecting patients.

*Results: Several CD4+ populations were potentially important in discriminating stable from rejection cohorts by LDA. Regulatory T cells (Treg; CD45⁺ CD3⁺ CD19^– CD4⁺ CD8^– CD25⁺ FoxP3⁺) were decreased in the rejection cohort however the difference was not significant. Dimensionality reduction by UMAP revealed a distinct subpopulation of cells adjacent to Treg cells that shared many phenotypic markers. Interestingly this T cell population (CD45⁺ CD3⁺ CD19^– CD4⁺ CD8^– CD25^lo CD5⁺ CD38^– FoxP3^lo CD45RA^–) was significantly (p < 0.05) increased in the stable vs the rejection pediatric transplant recipients.

*Conclusions: Single-cell CyTOF analysis identified a T cell population that is significantly decreased prior to rejection. Additional studies will determine if this T cell population can be harnessed to predict or prevent allograft rejection.

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