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 in vitro time‐lapse microscopy in a mouse transplant model, we investigated whether transplant outcome would result in changes in the proportion of CD 4 + T cells forming prolonged interactions with donor dendritic cells. By blocking CD 4– MHC class II and CD 28–B7 interactions, we defined immunologically relevant interactions as those ≥500 s. 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 ≥500 s 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 CD 4 + T cells using high‐throughput imaging flow cytometry.