INHIBITION OF DNA-SYNTHESIS AND IL-2 BIOACTIVITY IN MLR BY SPLENIC PREGNANCY-ASSOCIATED NATURAL SUPPRESSOR CELLS INVOLVES THE PRODUCTION OF A TGF-BETA-1-LIKE MOLECULE AND A 2ND DISTINCT INHIBITORY FACTOR

Natural suppressor cells exhibiting a double-negative, immature T cell phenotype have been identified in maternal spleen during syngeneic murine pregnancy. In the present study, splenic pregnancy-associated natural suppressor (SPANS) cells are shown to express alpha/beta T cell receptors. SPANS cell-mediated inhibition of DNA synthesis by spleen cells responding in mixed lymphocyte reactions (MLR) is associated with a reduction in interleukin (IL)-2 bioactivity beginning after 96 h of culture. Although culture supernatants from suppressed MLR exhibit diminished ability to support the growth of IL-2-dependent CTLL-2 cells, SPANS cells themselves are unable to inhibit IL-2-driven CTLL-2 proliferation, suggesting that SPANS cells down-regulate IL-2 synthesis in MLR. IL-2 utilization in MLR is also inhibited by SPANS cells, since the addition of exogenous IL-2 fails to relieve the inhibitory effect of SPANS cells on lymphoproliferative responses in MLR. Flow cytofluorometric analysis reveals that MLR performed in the presence of SPANS cells contain normal percentages of CD4 and IL-2 receptor-bearing spleen cells. Thus, SPANS cells do not inhibit cellular proliferation in MLR by selectively interfering with clonal expansion of IL-2-producing helper T cells or by down-regulating IL-2 receptor expression. We have determined that SPANS cells inhibit DNA synthesis in MLR via the production of a transforming growth factor (TGF)-beta1-like suppressor factor, since cellular proliferation in MLR is restored to normal levels in the presence of anti-TGF-beta1 neutralizing antibody. However, IL-2 bioactivity in these cultures remains low in comparison to control MLR, suggesting the presence of a second distinct suppressor factor. Although the identity of this second inhibitory molecule has yet to be determined, neutralizing antibody studies have ruled out IL-10.