Tolerance induction across M1s and minor histocompatibility complex by inhibiting activation of T helper type 1 in early period

We have previously succeeded in inducing persistent donor-specific toleranc e across Mis plus multiple minor histocompatibility barriers by portal veno us (p.v.) injection of donor spleen or bone marrow cells plus cyclophospham ide (CY) treatment. Microchimerism was established in the lymph-hemopoietic organs of the tolerant recipients. However, the mechanisms, particularly t he roles of CY in the tolerance induction, have not been clarified. We exam ined the tolerance induction using other anti-mitotic agents and evaluated the in vitro proliferative responses and cytokine expression of T cells fro m the recipients after stimulation with donor alloantigens. The administrat ion of not only CY but also mitomycin C (MMC) and cytosin arabinoside (Ara C) elicited a prolongation of skin graft survival. CY induced tolerance whe n it was administered 2 days after the p.v. injection, but not immediately or 4 days after the p.v. injection. T cells collected from the tolerant rec ipients showed no proliferative responses as a result of stimulation with d onor alloantigens whereas the responses of T cells from non-tolerant recipi ents were significantly enhanced. Interferon-gamma (IFN gamma) was extensiv ely expressed in the non-tolerant T cells from 24 to 48 h after the stimula tion with donor alloantigens. In contrast, the expression of IFN gamma was observed in the tolerant T cells from 72 h after the stimulation, Also, the tolerant T cells showed the expression of interleukin-10 (IL-10) and trans forming growth factor-beta 1 (TGF-beta1) from 72 h after the stimulation wh ereas the non-tolerant T cells did not. These data suggest that CY, when ad ministered 2 days after the p.v. injection, induces persistent tolerance by inhibiting T helper type 1 (Th1) activity in the early period but not the Th1 activity in the later periods. (C) 2001 Elsevier Science B.V. All right s reserved.