Polyfunctional CD8+ T Cells are Associated with Biopsy Proven Acute Cellular Rejection in Pediatric Heart Transplant Patients

PURPOSE Polyfunctional T cells (PTC) have the ability to secrete ≥2 cytokines per cell when stimulated and increased presence of PTC is associated with favorable outcomes in patients undergoing chimeric antigen receptor (CAR) T cell therapy for cancer. In pediatric heart transplant recipients (PHT), the development of PTC may represent increased exposure to donor specific antigens with the potential to induce greater allograft damage through elevated cytokine production; however, the ability of circulating PTC to predict acute cellular rejection (ACR) in PHT is unknown. The goal of this pilot study was to determine the frequency of circulating PTC and secreted cytokine profiles of T lymphocytes in PHT with varying degrees of ACR, with the hypothesis that an elevation in circulating PTC is associated with ACR. METHODS Peripheral blood mononuclear cells were collected from PHT concurrently with clinically indicated cardiac biopsy. Single-cell expression profiles of 32 cytokines (Isoplexis Isocode Chip) were quantified in CD4+ and CD8+ T cells from each patient. Samples were stratified by ISHLT biopsy grade for ACR. The polyfunctional strength index (PSI) was calculated as the %PTC multiplied by the intensities of each secreted cytokine. RESULTS PSI of CD8+ T cells in PHT with ACR 1R (n=4) and 2R (n=2) was increased compared to ACR 0 (n=3; Figure 1). PSI in CD4+ T cells in PHT with ACR was also increased, but less than in CD8+ T cells. Cytokines that drove an increased PSI were granzyme B, macrophage inflammatory protein (MIP)-1a, MIP-1b and sCD137 in CD8+ T cells and MIP-1a, MIP-1b and tumor necrosis factor (TNF)-α in CD4+ T cells. Correspondingly, stimulated cytokine production (CD8+ > CD4+) was increased in PHT with ACR vs no ACR. CONCLUSION PHT with ACR had distinct circulating T lymphocyte profiles, specifically with increased polyfunctionality of circulating CD8+ T cells. Quantification of CD8+ PTC may represent a novel biomarker of ACR in PHT and warrants further investigation.