A novel variation of the mixed lymphocyte reaction for measuring T cell responses to skin-specific antigens of pigs

Skin and vascularized composite allografts (VCA) containing skin are transplanted to restore form and function of tissues after major injuries. Skin has long been recognized as being particularly immunogenic, causing high risk of rejection and immune sensitization. Due to skin-specific antigens, donor skin is often rejected even in animals that are tolerant of the remaining donor tissue. To study the reaction of lymphocyte subsets against these minor and/or tissue-specific skin antigens in swine made tolerant to allogeneic donors through hematopoietic stem cell transplants (HSCT), we developed a skin-adapted variation of the mixed lymphocyte reaction (MLR). We processed porcine skin into single cell suspensions to be used as stimulators. Peripheral blood mononuclear cells were used as responders. We first optimized the concentrations of skin stimulators to achieve T cell proliferation with minimal self-background reactivity. The assay was then tested in two pigs that had received combined VCA/ HSCT. The first pig had not rejected any part of the VCA, and the second pig was actively rejecting the epidermis of the VCA at the time of the assay. Despite lack of anti-donor MLR responses against donor lymphocytes in the peripheral blood in either animal, the second pig demonstrated a specific response against donor skin cells. Our results suggest that the assay will be useful to study recipient sensitization against skin antigens, even in otherwise tolerant animals. This assay may have both diagnostic and therapeutic implications for immune responses specific to the skin.