GENERATION OF NKT-DEPENDENT AND INDEPENDENT REGURATORY T CELLS IN CYCLOPHOSPHAMIDE (CP)-INDUCED TOLERANCE

Abstract

P947 Aims: Recent reports provided the evidence that NK T cells play important roles in the induction of transplantation tolerance. We have previously reported a method of CP-induced tolerance. Our protocol consists on injection of 1x108 allogeneic spleen cells (SC) on day 0, and 200mg/kg CP on day 2. As a consequence, skin allograft tolerance can be induced across MHC-matched minor antigen-mismatched combinations. Furthermore, we have demonstrated the three major mechanisms, i.e., clonal destruction for tolerance induction, intrathymic clonal deletion and the appearance of regulatory T cells for the maintenance of tolerance (JEM 1990, JI 1990). In this study, we examined whether NKT cells were involved in the generation of regulatory T cells. Methods: Balb/c (WT; H-2d, Ly1.2, Mls-1b) or NKT (Jα281) knockout (NKT KO; Balb/c background) mice were thymectomized on day –14 (ATx). Recipient mice were primed with 1×108 DBA/2 (H-2d, Ly1.1, Mls-1a) SC on day 0 and treated with 200mg/kg CP on day 2. Recipient mice were grafted with donor skins on day 28. Mixed chimerism and clonal destruction was analyzed by using anti-Ly1.1 mAb, and antiVb6 mAb. The regulatory T cells generated in the tolerant mice were assayed in the following ways. Untreated WT mice were irradiated with 3 Gy, and injected i.v. with 1x108 SC from thymectomized WT or NKT KO recipients that had accepted DBA skin grafts for over 100 days. Skin grafting was performed 1 day following the transfer of the SC. Results: Skin allograft tolerance was induced in WT mice but not in NKT KO mice. Recent study demonstrated that skin allograft tolerance can be induced in thymectomized NKT KO mice treated with DBA SC and CP. The adoptive transfer experiment revealed the following results. In the irradiated control WT mice, DBA skin grafts were rejected within 30 days after grafting (n=6, MST±SD=23.3±6.8 days, median=24 days). The survival of the DBA skin grafts was further prolonged in the irradiated BALB WT mice by transferring the SC from thymectomized WT mice that had accepted DBA skin grafts (n=6; MST±SD= 62.3±7.6 days, median=63 days). Similarly, in the irradiated BALB WT mice which received the SC transferred from thymectomized NKT KO mice that had accepted DBA skin grafts, the survival of DBA skin grafts was moderately prolonged (n=6; MST±SD=48.5±17.9 days, median=54 days). However, there was a statistically significant difference between the graft survivals in irradiated BALB WT mice receiving SC transfers from thymectomized WT and NKT KO mice that had accepted DBA skin grafts (P< 0.05). These skin allograft prolongations were tolerogen-specific since third party skin B10.D2 (H-2d) allografts were rejected within 24 days after grafting. Conclusions: The results demonstrated that regulatory cells are generated without the contribution of NKT cells. Also, regulatory cells are generated via NKT cell-dependent mechanism since the survival of DBA skin grafts was significantly longer in irradiated recipients receiving 1x108 SC from tolerant WT mice than those receiving 1x108 SC from tolerant NKT KO mice.