Fractionation of lymphocyte populations with monoclonal antibodies specific for LYT-2.2 and LYT-3.1.

Abstract We have characterized two new cytotoxic, monoclonal IgM antibodies specific for the murine T lymphocyte differentiation antigens Lyt-2.2 and Lyt-3.1. Initially, we determined the fractions of thymocytes and lymph node T cells that react with the antibodies; then we studied their effect on a functional subpopulation of T cells, namely cytotoxic T lymphocytes (CTL) specific for major and minor histocompatibility antigens. The antibodies lyse 45% of lymph node T cells and 90% of thymocytes of C57BL/6 series mice, in agreement with the original findings of Cantor and Boyse. However, in BALB/c series mice, only about 30% of lymph node T cells are sensitive to the antibodies. This difference probably reflects a genetic difference between C57BL/6 and BALB/ c mice that affects the relative proportions of T cell subsets. Thymocytes from an Lyt-3.1 congenic strain of BALB/ c were fractionated into peanut lectin agglutinable and nonagglutinable populations and tested with anti-Lyt-3.1 monoclonal antibodies. Twenty percent of peanut lectin nonagglutinable thymocytes, 70% of peanut lectin agglutinable thymocytes, and 70% of unseparated thymocytes were sensitive to monoclonal anti-Lyt-3.1 antibodies. Therefore, the immature peanut lectin agglutinable fraction of thymocytes contains Lyt-3 negative cells. Both hybridoma antibodies eliminate >95% of anti-H-2 and anti-minor H specific CTL activity generated in 5-day mixed lymphocyte cultures (MLC). Furthermore, pretreatment of spleen cells with the monoclonal antibodies and complement almost completely eliminated the generation of CTL, but left most of the T cells that proliferate in MLC. However, a small fraction (<5%) of CTL activity from primary MLC was resistant to elimination by monoclonal anti-Lyt-3.1 antibodies. By pre-treating spleen cells with monoclonal anti-Lyt-3.1 and complement and stimulating the surviving cells in MLC a number of times, we isolated a stable subpopulation of CTL that were completely resistant to any concentration of anti-Lyt-3.1 monoclonal antibodies, but were sensitive to anti-Thy-1.2 monoclonal antibodies. The specificity of this minor subpopulation of CTL, in terms of H-2 restriction and target antigen specificity, did not seem to be different from that of the bulk of CTL. Furthermore, the CTL resistant to lysis by monoclonal anti-Lyt-3.1 antibodies were sensitive to lysis by high concentrations of conventional anti-Lyt-3.1 serum. On this basis, we postulated that lysis by the monoclonal anti-Lyt-3.1 antibodies is very sensitive to determinant density, and that the resistant fraction of CTL have a sub-threshold density of the determinant. To investigate the effect of lowering the determinant density, we compared the lytic behavior of monoclonal and conventional antibodies on target cells homozygous or heterozygous at the Lyt-3 locus. Unlike the case with conventional serum, the titer of the monoclonal anti-Lyt-3.1 ascites dropped drastically on heterozygous target cells. Thus, it seems likely that the CTL resistant to monoclonal anti-Lyt-3.1 antibodies are a stable subpopulation with low Lyt-3.1 density.