Tu82 - Detecting Alloreactive T Cells in Transplant Recipients with a Trogocytosis Assay

Organ transplantation success is dependent on prevention of alloimmune graft rejection with immunosuppressant drug therapy, which unfortunately also increases the risk of infection and cancer. Drug regimens are adjusted and minimized based on graft health, which is currently evaluated by infrequent and invasive biopsies. A less-invasive, blood-based assay to monitor allograft rejection could reduce the need for regular biopsies and allow for more frequent alloimmune monitoring. Mixed lymphocyte reactions co-culturing donor tissue and recipient blood show that alloreactive T cell abundance inversely correlates with graft health; however, these assays are challenging to standardize and require difficult-to-acquire donor material. We hypothesized that a new way to measure allo-HLA-reactive T cells could be developed using the principle of trogocytosis, whereby T cells acquire cognate HLA proteins from target cells and re-incorporate them as a part of their own membranes. We generated K562 cell lines that express a donor HLA protein fused to a fluorescent reporter and designed assays to quantify allo-HLA-specific trogocytosis, defining alloreactive T cells as cells that become positive for the fluorescent allo-HLA protein. Assay conditions were optimized with human CD8+ T cells engineered to express an HLA-A2-specific TCR, and background trogocytosis was minimized by removing K562 co-stimulation and supplementary IL-2. Blood collected from an HLA-A2-negative kidney transplant recipient during biopsy-confirmed graft rejection of an HLA-A2-positive graft had a higher frequency of T cells that trogocytosed HLA-A2 than blood collected prior to transplantation. Experiments are ongoing to test the sensitivity and specificity of the assay in longitudinal patient cohorts.