ROLE OF NKT CELLS IN THE SUPPRESSION OF INTRATHYMIC EFFECTOR CELLS FOR THE ESTABLISHMENT OF TOLERANCE IN CYCLOPHOSPHAMIDE (CP)-INDUCED TOLERANCE

Abstract

P946 Aims: Recent reports provided the evidence that NKT 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 administration of 200mg/kg CP on day 2. As a consequence, skin allograft tolerance can be easily 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 reguratory T cells for the maintenance of tolerance (JEM 1990, JI 1990). The unsolved mechanism of CP-induced tolerance system is the regulatory role against the effector T cells which are generated in the thymus from clonal destruction in the periphery through the establishment of intrathymic clonal deletion. The aim of the present study was to investigate these regulatory role by using NKT KO mice. Methods: Balb/c (WT; H-2d, Ly1.2, Mls-1b) or NKT (Jα281) knockout (NKT KO; Balb/c background) mice were primed i.v. with 1×108 DBA/2 (H-2d, Ly1.1, Mls-1a) SC on day 0 and treated i.p. with 200mg/kg CP on day 2. In some group to deplete intrathymic T cells, recipients were thymectomized on day –14 (ATx) or received thymic irradiation (TI; 700 rad). Skin grafting was performed at 4weeks. A mixed chimeric state (T cell chimerism) was evaluated using FITC-conjugated anti-Ly1.1 and PE-conjugated anti-Ly1 (1.1+1.2) mAb at various times. Clonal destruction was examined by analyzing the expression of Mls-1a-reactive CD4+CD8-Vβ6+ T cells in the spleen or thymus. Results: In WT mice treated with SC and CP, skin allograft tolerance and mixed chimerism were induced. In the NKT KO mice treated with SC and CP, however, survival of DBA skin grafts were moderately prolonged (median 35). Wken NK T KO mice were thymectomized on day –14 (or reconstituted with NKT cells from WT mice on day –14), DBA skin grafts were permanently accepted in the tolerogen-specific manner. Donor derived T (Ly1.1+) cells were clearly detectable from 2-14 weeks after treatments in tolerance NKT KO mice treated with ATx or TI, SC and CP (without NKT reconstitution). On the other hand, chimerism were detectable at 2 weeks and became undetectable by 14 weeks in NKT KO mice treated with SC and CP. The level of chimerism in the NKT KO mice treated with ATx or TI, SC and CP were significantly higher than that in the NKT KO mice treated with SC and CP. As to the mechanisms, clonal destruction was induced in SC of all recipients. On the other hand, however, clonal destruction in the thymus of WT or NKT KO mice treated with SC and CP was not observed. Conclusions: The present study strongly indicated the regulatory role of NKT cells. Effector T cells in the thymus were not destructed by the treatment with SC and CP. New effector T cells migrate from the thymus into the periphery until clonal deletion (associated with mixed chimerism) occurs. Without the existence of NKT cells, these effector T cells gradually break the tolerant state.