Allogeneic Fibroblasts Used to Grow Cultured Epidermal Autografts Persist in Vivo and Sensitize the Graft Recipient for Accelerated Second-Set Rejection

Abstract
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 immunologically 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, survive after grafting and induce host sensitization to alloantigen. CEAs from CBA donors (H-2k) were grown on allogeneic NIH 3T3 (H-2q) or syngeneic LTK (H-2k) fibroblasts, which were removed by trypsinization 7 days later. CBA mice (n = 85) were flank-grafted with NIH allografts (positive control), CEA/3T3s, CEA/LTKs, or CBA autografts (negative control). Hosts were challenged with second set NIH tail allografts 3 weeks later. Median graft survival was compared between groups 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 immunoblotting, using KL295, a monoclonal antibody that recognizes H-2q (but not H-2k) class II histocompatibility antigens. Allogeneic fibroblasts persisted after grafting but decreased over time, as determined by alloantigen expression on Western immunoblots. Accelerated tail graft rejection occurred in hosts primed by NIH allografts (9 days, p < 0.05), as well as by CEAs growth 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. Immunogenic fibroblasts used to grow CEAs survive in vivo and sensitize the graft recipient for accelerated second-set rejection. These persistent cells may initiate an inflammatory response that may result in late graft breakdown and limit the utility of CEAs grown with a foreign fibroblast feeder layer.