Reactivity of monoclonal antibody 1E5.B5 with a novel phenotypic marker expressed on a murine natural suppressor cell subset

Natural suppressor (NS) cells are antigen-nonspecific, MHC-independent immunoregulatory cells that are typically found in murine bone marrow (BM), newborn (NB) mouse spleen, and in splenic tissue of adult mice during pregnancy and following cyclophosphamide (CY) treatment. There has been a pressing need for the development of NS cell-specific monoclonal antibodies (mAb) since NS cells are generally described as null cells which lack the usual phenotypic markers of mature T cells, B cells, and macrophages. Here we present evidence that mAb 1E5.B5, which was raised in rats against murine splenic pregnancy-associated NS (SPANS) cells, recognizes a unique antigenic marker expressed by some, but not all, murine NS cells. In the presence of complement, mAb 1E5.B5 effectively eliminates SPANS activity, and diminishes NS activity of CY-treated spleen cells in mixed lymphocyte reactions (MLR). However, cytotoxic pretreatment with mAb 1E5.B5 had minimal effects on NS activity of BM and NB spleen cells. We also show that pregnancy spleen cells and CY-spleen cells with moderate NS activity in MLR can be positively selected for by 'panning' with mAb 1E5.B5. In contrast, only weakly inhibitory cells are isolated from BM and NB spleen by this procedure. Cellular ELISA and flow cytometry confirm that mAb 1E5.B5 has specificity for pregnancy spleen cells and CY-spleen cells, as well as for NB spleen and BM cell preparations. Western blot analysis reveals that mAb 1E5.B5 reacts with a novel 50 kDa NS cell-associated antigen which we have termed NS-1. The NS- 1 antigen is not present on other null cells such as natural killer (NK) cells and natural cytotoxic (NC) cells since cytotoxic pretreatment of pregnancy spleen cells with mAb 1E5.B5 does not affect antibody-dependent cell-mediated cytotoxicity, NK or NC activity.