Partial tolerance of subcutaneously transplanted xenogeneic tumour cell graft by Fas-mediated immunosuppression

TAKAHIRO SAWADA,*{ TAKEHIKO KOJI,{ YOSHITAKA HISHIKAWA,{ KOJI KISHIMOTO,* TAKESHINAGAYASU,* TAKAO TAKAHASHI,* TADAYUKI OKA* & HIROYOSHI AYABE* *The First Department of Surgery,Nagasaki University School of Medicine and {The Department of Histology and Cell Biology, Nagasaki University School ofMedicine, JapanSUMMARYCertain anti-Fas antibodies, such as RMF2, induce apoptosis of Fas-expressing cells. We appliedthe Fas/anti-Fas system to induce killing of Fas-expressing immunocytes with resultant immuno-suppression. W7TM-1 tumour cells, a rat T-cell line, were inoculated subcutaneously in BALB/cmice and tumour growth was monitored in untreated mice and in mice treated with RMF2. Prior totreatment with RMF2, we examined the expression of Fas in isolated splenocytes and in tumour-infiltrating lymphocytes by flow cytometry and immunohistochemistry, respectively. There wasa remarkable increase in Fas-positive lymphocytes, including natural killer (NK) cells, amongsplenocytes at day 5 after tumour cell inoculation. The number of Fas-positive infiltratinglymphocytes also increased markedly, from day 5 to day 10. We then examined whether RMF2could induce apoptosis of Fas-positive activated lymphocytes isolated from the spleen at day 5in vitro. Terminal deoxy (d) -UTP nick end labelling (TUNEL) and Annexin V staining methodsshowed apoptosis of isolated cells when incubated with RMF2, and typical apoptotic features wereconfirmed by 4k,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining. Furthermore,suppression of cellular and humoral immunity was noted in RMF2-treated mice by mixedlymphocyte reaction and assay of serum levels of immunoglobulin G, respectively. Finally,treatment of animals with RMF2 daily from day 5 to day 9 could maintain the tumour size, whilethe tumour mass began to diminish in untreated mice immediately after reaching a maximum size.We confirmed the enhancing effects of long-term treatment with RMF2, through the induction ofimmunosuppression, on the growth of unvascularized xenogeneic tumour cell grafts.INTRODUCTIONXenotransplantation is the most promising therapy for recon-structing the function of damaged organs. However, at present,such an approach ultimately results in graft rejection. Plattet al.