The current paradigm explaining skin graft rejection is that alloreactive T-cells are first activated in graft-draining lymph nodes (dLNs) by dendritic cells (DCs) that have migrated from the graft. Activated T-cells then migrate to the graft where they initiate/contribute to graft rejection. In adult animals up to half of the T-cells recognising allogeneic MHC may already be of effector/memory phenotype, capable of directly entering sites such as the graft bed without prior activation in dLNs. There is increasing evidence that heterologous immunity mediated by these memory alloreactive T-cells is a significant barrier to graft acceptance in the clinic.
To investigate the contribution of heterologous immunity to graft rejection, we utilised intravital multiphoton microscopy to visualise cell migration and dynamic cell-to-cell interactions within a skin-graft model. The model uses TCR-transgenic 5C.C7 T-cells that recognise Moth-Cytochrome-C (MCC) in association with I-Ek and cross-react with alloantigen I-As.
5C.C7 T-cells were harvested from H-2k donors primed with MCC at least 1 month earlier. T-cells were fluorescently-labeled and adoptively transferred into H-2k recipients that received a skin graft from an H-2s donor 1-3 days earlier. T-cells entered directly into the graft following adoptive transfer, and could be identified migrating within the graft bed for up to 5 days, but never tracked into the donor tissue itself. We also observed that DCs from donor tissue (CD11c-YFP donor mouse) were unable to exit the graft, but instead localized to the graft/host interface where they persisted for at least 7 days after grafting. MHC-II+ non-DC cells (MHC-II-GFP donor mouse) were observed migrating from the donor tissue into the graft bed, where they appeared to be interacting with blood vessels. We are currently using several additional fluorescent mouse strains to identify these cells and to investigate their contribution to graft rejection.