Generation of FASL-expressing antigen-presenting cells for suppression of alloreactive T cells in the rat

Abstract

CONTRARY TO EARLY expectations, Fas Ligand (FasL) expression in organ allografts compromised the transplanted tissue and did not prolong graft survival. It is known that antigen-presenting cells (APCs) strongly stimulate allogenic T cells. Recent data in a mouse model revealed that the T cell response to adenovirus could be suppressed by treating the recipient with APCs that previously were infected with recombinant FasL-adenovirus and consequently expressed FasL. It was thought that adenoviral antigens are presented by APCs to T cells and that concomitant expression of FasL induces apoptosis of specific T cells. Based on this finding, we speculated that FasL-expressing APCs would induce apoptosis of allogenic T cells and thus suppress the subsequent response to alloantigen. In vivo one could possibly inject donor APCs expressing FasL into an allogenic recipient and thereby suppress the T cell response to a subsequent graft. In the present experiment we tested this hypothesis by infecting allogenic rat macrophages with FasL-adenovirus and using the infected cells for stimulation of allogenic T cells in cell culture experiments. MATERIALS AND METHODS Construction of Ad-mFasL Recombinant adenoviruses carrying the murine FasL (mFasL) gene were generated as previously described. Briefly, the mFasL gene was cloned into a shuttle vector and recombined with an adeno-5 vector in bacteria. Adeno-mFasL viruses were propagated in 911 cells. The correct mFasL insert of the recombinant construct was proven by sequence analysis. Transcription of the mFasL gene in infected cells was verified by RT-PCR.