Characterization of Monoclonal Antibodies Against Cell Surface Molecules Associated with Cytotoxic Activity of Natural and Activated Killer Cells and Cloned CTL Lines

Abstract

Supernatants of hybridomas from fused spleen cells of mice immunized with either a T4+ or a T8+ CTL clone, were screened for their ability to inhibit the cytotoxic activity of the T4+ and the T8+ CTL clone. Eight monoclonal antibodies (MAbs) with blocking activity were obtained and a preliminary characterization of these antibodies was carried out. The MAbs SPV-L1 and SPV-L5 were found to react with thymocytes and all peripheral blood leukocytes. SPV-L1 and SPV-L5 precipitated a molecular complex consisting of two noncovalently bound chains of molecular weights of 95 and 160 kD indicating that they recognize the human equivalents of the recently described leukocyte function associated (LFA-1) antigens. The SPV-L1 and SPV-L5 antibodies inhibited the reactivity of six CTL clones tested in this study. In addition, SPV-L1 and L5 blocked the lectin dependent cellular cytotoxicity mediated by these CTL clones. Natural killer cell activity mediated by fresh peripheral blood lymphocytes and activated killer cell activity generated in MLC both measured against K562 was also inhibited by SPV-L1 and SPV-L5. Two other antibodies, SPV-L3 and SPV-L4 blocked strongly the cytotoxic activity of two T4+ CTL clones, whereas the reactivity of the three T8+ CTL clones and one T4+ CTL clone was not or only moderately inhibited. These antibodies recognized an Ia antigen as judged from tissue distribution and immunoprecipitation studies. However, these latter studies also suggest that SPV-L3 and SPV-L4 recognize HLA-DC rather than HLA-DR antigens. Finally, three antibodies (SPV-T3a, SPV-T3b, SPV-T3c) were obtained directed against the T3 molecular complex and one MAb (SPV-T8) was found to react with T8. The anti-T3 reagents were shown to be mitogenic for peripheral blood lymphocytes, with the exception of SPV-T3a. The results presented here indicate that the antigens recognized by SPV-L1 and SPV-L5 are involved in various cytotoxic reactions. In contrast, SPV-L3, SPV-T3 and SPV-T8 only seem to play a role in antigen specific cytotoxic reactions.

Role of p150,95 in adhesion, migration, chemotaxis and phagocytosis of human monocytes

“…LFA-1 has been described to act as a cellular adhesion molecule with an important role in lymphoid cell-cell interac tions [13][14][15]. The anti-LFA-1 mAb used here did not block the adhesion of monocytoid cells to artificial substrates such as plastic surfaces (Table 3).…”

“…In addition, Dana et al described two functional domains in CR3, one involved in C3bi binding and the other in stimula tion of granulocyte adhesion-dependent functions [11]* LFA-1 is present on virtually all leukocytes [12,13], and acts as an adhesion molecule involved in cell-cell interactions [14,15]. Anti-LFA-1 mAb have been demonstrated to inter fere with the lytic activity of cytotoxic T lymphocytes (CTL) [13,15,16] and natural killer (NK) cells [13,[17][18][19] and are able to block T helper cell-monocyte-associated functions [2] by preventing cell-cell contact.…”

Biochemical and functional characteristics of the human leukocyte membrane antigen family LFA‐1, Mo‐1 and p!50,95

“…Twenty |xl containing 0.4 [xCi = 14.8 kBq tritiated thymidine ([3H]dThd) was added during the last 4 h of incubation. The cultures were harvested with an automatic cell harvester, MASH-II, and counted as described previously [3].…”

“…LFA-1 is expressed on lymphocytes, monocytes and granulocytes [1], whereas Mo-1 and pl50,95 are present on monocytes, granulocytes and "null" cells [2], Recently, it has been shown that these molecules contribute to the adhesion reaction of these cells. Blocking experiments with anti-LFA monoclonal antibodies (mAb) demonstrated that LFA-1 inhibits the adhesion step between effector and target cells in cytotoxic T lymphocyte, natural killer and lectin-dependent cellular cytotoxicity [1,3,4], LFA-1 also participates in monocyte-dependent lectin-induced T cell proliferation suggesting that it also acts as an adhesion molecule between monocytes and T cells. Mo-1, which is the receptor for the inactivated form of C3b, mediates the adhesion of soluble complexes or particles selec tively opsonized with C3bi [5,6].…”

“…Studies on the biosynthesis of complement proteins (reviewed in [1]) have shown that the liver and cells of the monocyte/ macrophage series are the major sites of synthesis, although other cell types also synthesize certain complement proteins [2,3]. Complement protein D of the alternative pathway has been the subject of limited biosynthetic study [4][5][6], however, recent work has demonstrated that U937 cells, a human mono cyte cell line, synthesize D [7].…”