The expression of H-2Kk antigens in a (C3H × DBA/2)F1 lymphoma cell line growing in vitro was investigated with monoclonal antibodies specific for a public antigen of the H-2Kk region (H-2.m3) in fluorescence analysis and microcytotoxicity assays and in cell-mediated cytotoxicity with allogeneically stimulated effector cells. Estimates of relative levels of H-2Kk-antigen expression obtained by the different methods were highly correlated. The uncloned, unselected population gradually lost H-2Kk surface antigen expression under culture conditions. This was due to the appearance of H-2Kk negative variants. Fifteen cloned sublines of a population enriched for cells expressing antigen H-2.m3 in the fluorescence activated cell sorter contained either two distinct populations, one consisting of H-2.m3 negative and one of H-2.m3 positive cells, or consisted of H-2.m3 negative cells only. The expression of the H-2.m3 determinant of H-2Kk paralleled that of other serological H-2Kk determinants and of H-2Kk target determinants for cell-mediated cytotoxicity. In nearly all clones where two populations could be detected, the proportion of H-2.m3 negative cells increased with time in culture. The amounts of H-2Kk antigen expressed by the clones appeared not to be correlated to the amounts of H-2Dk antigens on the cell surface as judged by cell-mediated cytotoxicity. In at least one clone and in the uncloned population, H-2Kk-antigen expression detectable by fluorescence analysis could be stimulated by growing the cells in the peritoneal cavities of (C3H × DBA/2)F1 mice or by adding mouse interferon preparations to the cell cultures. The increase in susceptibility to cell-mediated lympholysis of cells grown in vivo paralleled the increase in H-2 expression detected by fluorescence. In contrast, cells growing in the presence of interferon in vitro showed reduced sensitivity to lysis by alloreactive lymphocytes, although H-2 antigens were strongly expressed as measured by fluorescence.
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