Quantification of CD4+ T Cell Alloreactivity and Its Control by Regulatory T Cells Using Time-Lapse Microscopy and Immune Synapse Detection

Assays designed to select transplant recipients for immunosuppression withdrawal have met with limited success, perhaps because they measure events downstream of T cell-alloantigen interactions. Using invitro time-lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD4(+) T cells forming prolonged interactions with donor dendritic cells. By blocking CD4-MHC class II and CD28-B7 interactions, we defined immunologically relevant interactions as those 500s. Using this threshold, T cell-dendritic cell (T-DC) interactions were examined in rejection, tolerance and T cell control mediated by regulatory T cells. The frequency of T-DC contacts 500s increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. Regulatory T cells reduced prolonged T-DC contacts. Importantly, this effect was replicated with human polyclonally expanded naturally occurring regulatory T cells, which we have previously shown can control rejection of human tissues in humanized mouse models. Finally, in a proof-of-concept translational context, we were able to visualize differential allogeneic immune synapse formation in polyclonal CD4(+) T cells using high-throughput imaging flow cytometry. The authors measure mouse and human CD4+ T cell alloreactivity using time-lapse microscopy and imaging flow cytometry techniques, and demonstrate that these measurements predict graft outcome in tolerized and rejecting animals. See the supporting video at amjtransplant.com/videos.