PROLONGED SURVIVAL OF CULTURED KERATINOCYTE ALLOGRAFTS IN THE NONIMMUNOSUPPRESSED MOUSE

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Abstract
The effect of in vitro culture on the survival of allografts of epidermal keratinocytes has been examined using a mouse model. Female BALB/c tail epidermal cells were cultured from single cell suspensions to form confluent sheets that were grafted onto male CBA recipients using a transplantation technique that ensured separation of donor graft from host skin. Animals were killed at defined intervals, and the status of the grafts determined histologically. Full thickness skin allografts rejected at 13–15 days. Allografts of epidermis (obtained by enzymatic cleavage at the dermoepidermal junction) rejected at 19–20 days. Cultured keratinocyte allografts were not rejected at least within 70 days and had a histological appearance identical to syngeneic controls. The expression of MHC class I and class II determinants and the leukocyte common (Ly5) surface marker on the donor cells before and after culture were examined using indirect immunofluorescence and monoclonal antibodies. These and other cytochemical studies showed that freshly dissociated tail epidermal cells contained 0.3% of cells that expressed membrane-bound ATP-ase activity, la antigens, and the Ly5 surface antigen. These are the Langerhans' cells of the epidermis. In culture, these cells decrease so that by day 8 of culture, no such cells can be detected. At confluence, there are no la positive cells, but all cells express MHC class I antigens and stain with an antikeratin antibody. The loss of la expression on culture correlates with a decreased stimulation of the class II H-2d-restricted T cell clone D7.1 by cultured keratinocytes compared with freshly dispersed epidermal cells. Furthermore, cultured keratinocytes fail to prime allogeneic mice as determined by the survival of whole thickness skin grafts, whereas freshly dispersed cells induce an accelerated rejection. The results suggest that the survival of cultured keratinocyte allografts is due to the elimination of cells expressing la antigens and supports the passenger leukocyte theory of graft rejection.

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