CD8(+) ACTIVATED T-LYMPHOCYTES PRODUCE AN IN-VITRO SKIN GRAFT-VERSUS-HOST REACTION IN AN ORGANOTYPIC SKIN CULTURE MODEL

We adapted organotypic skin cultures to the dog as a model for skin graft-versus-host reaction (GVHR) to explore the relative roles of T cells and cytokines. To produce GVHR, activated lymphocytes from bulk mixed leukocyte cultures (bMLC) from 2 dog leukocyte antigen-unrelated dogs were injected into organotypic skin cultures. Additionally, effects of separated CD4(+) and CD8(+) activated lymphocytes as well as cytokine-containing (TNF alpha and IFN gamma) supernatants from bMLC were studied. Noninjected cultures as well as cultures injected with autologous (cultured and uncultured) lymphocytes and allogeneic uncultured lymphocytes served as controls. The unseparated bMLC-activated cell populations induced histopathological changes similar to in vivo skin GVHR along with very prominent class II antigen expression on keratinocytes. Separated CD8(+) cells were directly involved in tissue damage by producing necrosis of epidermis at the site of injection, with less class II antigen expression on keratinocytes, and predominantly distributed intraepidermally. CD4(+) cells, located mostly in the dermal regions, induced prominent class II antigen expression on keratinocytes, but no histological changes of GVHR. High levels of TNF alpha and IFN gamma were found in the supernatant of allogeneic bMLC cultures, although when the supernatant was injected into the organotypic skin cultures, keratinocytes failed to express surface class II antigen and histologically did not show changes of skin GVHR. This study demonstrated that organotypic skin cultures can serve as a model for studying the etiology of GVHR, and indicated direct involvement of CD8(+) cells in tissue damage.