ALLOGENEIC FIBROBLASTS USED TO GROW CULTURED EPIDERMAL AUTOGRAFTS PERSIST IN-VIVO AND SENSITIZE THE GRAFT RECIPIENT FOR ACCELERATED 2ND-SETREJECTION

Introduction: Cultured epidermal autografts (CEAs) have been used for wound coverage in patients with massive burns and other skin defects, However, CEAs often display late breakdown, which may be immunological ly mediated and initiated by persistent foreign fibroblasts used as a feeder layer to optimize keratinocyte growth, This study investigates whether these fibroblasts, previously shown to persist in vitro, survi ve after grafting and induce host sensitization to alloantigen. Method s: CEAs from CBA donors (H-2(k)) were grown ore allogeneic NIH 3T3 (H- 2(q)) or syngeneic LTK (H-2(k)) fibroblasts, which were removed by try psinization 7 days later, CBA mice (n = 85) were flank-grafted with NI H allografts (positive control), CEA/3T3s, CEA/LTKs, or CBA autografts (negative control). Hosts were challenged with second set NIH tail al lografts 3 weeks later, Median graft survival was compared between gro ups by Wilcoxon rank and chi(2) analysis, Additional CBA mice (n = 15) received CEAs that were biopsied 0, 4, and 8 days after grafting. The presence of allogeneic fibroblasts was determined by Western immunobl otting, using KL295, a monoclonal antibody that recognizes H-2(q) (but not H-2(k)) class II histocompatibility antigens. Results: Allogeneic fibroblasts persisted after grafting but decreased over time, as dete rmined by alloantigen expression on Western immunoblots. Accelerated t ail graft rejection occurred in hosts primed by NIH allografts (9 days , p < 0.05), as well as by CEAs grown with an allogeneic (10 days, p < 0.05) but not a syngeneic feeder layer (12 days, NS), Mice receiving flank autografts rejected second set tail allografts at 12 days. Concl usions: Immunogenic fibroblasts used to grow CEAs survive in vivo and sensitize the graft recipient for accelerated second-set rejection. Th ese persistent cells may initiate an inflammatory response that may re sult in late graft breakdown and limit the utility of CEAs grown with a foreign fibroblast feeder layer.