SCA1(+) MAC1(+) NITRIC OXIDE-PRODUCING CELLS IN THE SPLEENS OF RECIPIENTS EARLY FOLLOWING BONE-MARROW TRANSPLANT SUPPRESS T-CELL RESPONSES IN-VITRO/

Spleen cells collected from allogeneic chimeras early after bone marro w transplantation (BMT) consistently showed suppressed proliferative r esponses to interleukin-2 in vitro and failed to proliferate in mixed lymphocyte reaction (MLR) assays. However, isolation of Thy 1.2(+) T c ells from the heterogeneous spleen cell suspension prior to culture re sulted in heightened proliferation, suggesting the presence of cells c apable of suppressing T cell responses in vitro. When separated into s ubpopulations by negative and positive selection with specific monoclo nal antibodies, a non-T, non-B population with immunosuppressive prope rties was identified. The suppressive cells were found in the spleens of both allogeneic and syngeneic chimeras, but not normal donor mice. Suppressor activity was transient and typically declined by 3 weeks po st-BMT. The cells suppressed the response of alloactivated T cells iso lated from BMT chimeras as well as naive donor T cells in MLR assays i n a dose-dependent manner. To explore the mechanism(s) involved in the suppression, the effects of interferon-gamma (IFN-gamma)-specific mAb and the nitric oxide (NO) synthase inhibitor N-G-methyI-L-arginine we re examined. The results support a role for both IFN-gamma and NO in t he suppressive activity. Separation of cells based on Mac-1 expression indicated that there were both Mac-1-enriched and Mac-1-depleted cell s capable of producing NO, but that the Mac-1-depleted cells were the most potent suppressors in MLR assays. The Mac-1-depleted cells still contained a residual population of Mac-1(dim) cells which showed incre ased levels of Mac-1 expression after overnight culture. Intracellular staining with an inducible nitric oxide synthase (iNOS)-specific mAb indicated that the NO-producing cells expressed the cell surface marke rs Mac-1 and Sea-1. When iNOS knockout transgenic mice were used as 't ransplant donors, in vitro suppression of T cell responses was reduced but not eliminated, suggesting that other mechanism(s) could contribu te to the suppression. Collectively, these results demonstrate that Sc a-1(+)/Mac-1(+) cells capable of producing NO are present in the splee ns of recipients early after BMT and suggest that these cells may have immunoregulatory roles in vivo. (C) 1998 Academic Press.