Monoclonal antibodies against leucoagglutinin-reactive human T-lymphocyte surface components. I. Characterization of cellular binding sites.

The binding specificities of three biologically active anti-lymphocyte monoclonal antibodies (MoAb) (K46M, K3G, and 3-19-2) produced against human T-cell surface components reactive with the mitogenic lectin leucoagglutinin from Phaseolus vulgaris (La) were analysed. K46M is a strong T-cell mitogen, while K3G and 3-19-2 inhibited cell-mediated cytotoxicity. Resting peripheral blood lymphocytes (PBL) contained 4-16% K46M+ cells, 8-35% K3G+ cells, and less than 0.3-4% 3-19-2+ cells. After stimulation with T-cell mitogens the proportion of K46M+ and 3-19-2+ cells increased markedly (mean 59 and 30% positive cells, respectively), while the increase in K3G+ cells was less prominent (38%). K46M-reactive structures were expressed on mature T cells and probably also on B cells. K3G reacted with B and T cells while 3-19-2 showed a broader specificity reacting also with erythrocytes. All three MoAb reacted with lipid extracts of resting and activated PBL as well as with purified neutral glycolipids of lymphoid origin. In addition 3-19-2 reacted with lipid extracts of erythrocytes. K46M immuno-precipitated four surface peptides from lectin-stimulated PBL. Their apparent molecular weights were 53,000, 42,000, and 16,000 (doublet). The 53,000 and 42,000 MW peptides were identified as the alpha and beta chains of the T-cell antigen receptor. The identity of the 16,000 MW peptides is presently unknown. K3G and 3-19-2 did not specifically precipitate any lymphocyte surface peptide.

Monoclonal antibodies against leucoagglutinin-reactive human T-lymphocyte surface components. II. Studies on the mechanism of K46M-induced activation and determination of the frequency of responding cells.

A monoclonal antibody, K46M (IgM kappa), obtained after immunization with leucoagglutinin (La)-reactive T-cell surface components, stimulated human lymphocytes to proliferate. It induced maximal proliferation at greater than 20 micrograms IgM/ml after 3-4 days of culture. Cells stimulated by K46M produced interleukin 2 (IL-2) and gamma interferon (IFN-gamma) and expressed receptors for IL-2 and transferrin. The majority of the activated cells were phenotypically T cells as defined by monoclonal antibodies against CD3 and CD2, and an increase in the K46M-positive cells was also observed during the activation period. K46M-activated cells display major histocompatibility complex (MHC)-unrestricted cytotoxicity against several cultured target cells. The frequencies of the cytotoxic and of the proliferative precursor cells were determined using a limiting dilution assay. K46M seems to activate a larger fraction of cytotoxic precursor cells against Molt 4 than against K562, but the statistical significance of these observations requires further exploration. Both K46M or La activated 40% of PBL to proliferate, whereas 70% of PBL were induced by OKT3. However, the frequency of K46M-activated cells was 40% only when the lymphocytes were plated at low cell densities, i.e. less than 0.5 cells per well. At higher densities an inhibition of proliferation was seen that resulted in a biphasic response curve, indicating that the activation of PBL by K46M was not a single hit event. This was not found with either La or OKT3. Whether K46M, in contrast to OKT3 and La, activates a subpopulation with suppressor activity remains to be established.

Characterization of human CD4+ T-cell clones that secrete helper factor(s) for B-cell proliferation and maturation.

Human peripheral blood lymphocytes (PBL) were activated with K46M, a m mitogenic monoclonal antibody against La-reactive T lymphocyte surface structures. The cultures were expanded in the presence of interleukin 2 (IL-2). After 1 month of culture, the activated T cells were cloned by limiting dilution at 0.5 cells/well. Five clones with the CD3+CD4+ phenotype and one clone with the CD3+CD8+ phenotype were obtained. The CD3+CD8+ clone (K99) displayed a strong major histocompatibility complex (MHC)-unrestricted cytolytic activity against MOLT-4 and a weaker reactivity against the bladder tumour cell lines T24 and RT4. The natural killer (NK)-susceptible K562 cells were not lysed. Two of the CD3+CD4+ clones (K91 and K914) showed a helper activity in pokeweed mitogen (PWM)-induced IgG production by B cells. These cells differed in the expression of CD45R and CDw29 antigens, as defined by the monoclonal antibodies 2H4 or D10D11 and 4B4. When stimulated with PWM for 48 or 72 h, clone K91 and an additional CD4-positive clone (K913) secreted a factor into the supernatants which helped B cells to produce IgG. The K913 supernatant also induced some IgM production. The supernatant obtained after similar stimulation of K914 cells was inactive. None of these supernatants induced B cells to proliferate when tested together with phorbol myristate acetate (PMA). However, when K91 and K914 cells were activated with phytohaemagglutinin (PHA) for 48 or 72 h, the supernatant from K91 was strongly helpful in B-cell proliferation, whereas the supernatant from K914 cultures was only moderately active. In conclusion, we have established human T helper clones that release different factors supporting either B-cell proliferation or maturation when stimulated with PWM or PHA.

Enhancement of human spontaneous cell-mediated cytotoxicity by a monoclonal antibody against the large sialoglycoprotein (CD 43) on peripheral blood lymphocytes.

A murine monoclonal antibody, MoAb B1B6 (IgG1 chi), which recognizes the large sialoglycoprotein (LSGP) on human peripheral blood lymphocytes (PBL) effectively enhanced the spontaneous cytotoxicity of these cells against the natural killer (NK)-sensitive target cells K562 and Molt-4. Whereas preincubation of the lymphocytes with MoAb B1B6 resulted in increased cytotoxicity, preincubation of the target cells had no effect, indicating that the MoAb amplified cytotoxicity at the effector cell level. Kinetic analysis of the data revealed no differences between the control and the MoAb-treated lymphocytes with regard to Vmax, usually considered to reflect the overall lytic potential of the cells. The slopes of the saturation curves, however, differed significantly for the two cell populations, indicating a substantial increment in the activity of the MoAb-treated cells. When studied at the single cell level and with K562 as targets, treatment of PBL with the MoAb resulted in the recruitment of new effector lymphocytes from the pool of non-binding cells. In contrast, when Molt-4 cells were employed as targets, no additional effector cells were recruited. These results indicate that the enhanced cytotoxicity induced by MoAb B1B6 is the result of either recruitment of new effector lymphocytes or of an increased recycling capacity of preexisting effector cells. Together with previous observations, these findings support the conclusion that LSGP belongs to the set of surface molecules which regulate human lymphocyte activation.

Studies on the role of lymphocyte function-associated antigen 1 (LFA-1) in T cell activation.

The effect of polyclonal (anti alpha and beta chain) and monoclonal (anti alpha-chain) antibodies against lymphocyte function-associated antigen 1 (LFA-1) on T cell activation was studied. When added at the beginning of activation but not after 24 h or later the antibodies as well as the F(ab')2 or Fab fragments of polyclonal antibodies inhibited concanavalin A (Con A)-induced proliferation, interleukin 2 (IL-2) production, and the expression of receptors for IL-2 and transferrin. The inhibitory effect reached a maximum at the same time as optimal proliferation (72 h). Inhibition of proliferation lasted for 5 days or longer, although IL-2 production was only inhibited during the first 48 h of culture. Receptors for IL-2 and transferrin were re-expressed to the original level after 3 days of activation. Addition of external IL-2 at the beginning of the anti-LFA-1 containing culture prevented the inhibition of IL-2 receptor expression, while inhibition of transferrin receptor expression was unaffected, supporting the conclusion that the expression of these two receptors is regulated by partially independent signals. The polyclonal and monoclonal anti-LFA-1 antibodies also inhibited phorbol ester (PMA)-dependent OKT3 activation of highly purified T cells. The results suggest that the LFA-1 antibodies block an early step in the reactions necessary for IL-2 production, and that the LFA-1 molecule participates not only in T cell-accessory cell interaction but also in T-T interaction during the early phases of the activation process.

T lymphocytes displaying major histocompatibility complex-unrestricted cytotoxicity after activation by K46M, a mitogenic monoclonal antibody against leucoagglutinin-reactive human T lymphocyte surface components.

Activation of human peripheral blood lymphocytes (PBL) with the mitogenic monoclonal antibody (MoAb) K46M, which recognizes 1-5% of PBL, resulted in the expansion of cells with cytolytic activity. Thus, after culture of the activated lymphocytes in medium containing interleukin 2 (IL-2), they lysed a variety of cultured cell lines. The majority of the activated lymphocytes reacted with MoAb to CD8, CD3, and to the T cell antigen receptor heterodimer (Ti) but not with antibodies to antigens expressed on natural killer (NK) cells. The cytotoxicity was not inhibited by MoAb to CD3 or Ti. However, the killing of K562, but not of other cell lines, was enhanced by three to four times in the presence of anti-Ti antibodies. Anti-CD3 or other control antibodies had no effect. Cold target inhibition experiments indicated that the cytolytic lymphocytes recognized closely related structures on the target cells. Phenotypically and functionally similar effector cells emerged after activation of PBL with the anti-CD3 MoAb OKT3. Taken together, the results indicate that activation of PBL with MoAb K46M induces cytotoxic cells that differ from classical NK cells but that resemble mature cytotoxic T lymphocytes (CTL). However, unlike CTL, cytotoxicity was not MHC-restricted and the conventional T-cell receptor complex (CD3/Ti) appeared not to be involved in target cell recognition and cytolysis.

Inhibition of proliferative and cytotoxic activities of human T lymphocytes with rabbit antibodies directed against leucoagglutinin-reactive T cell surface components.

Three rabbit antisera (870, 872 and 873) were raised against leucoagglutinin-reactive components from the surface of human T cells. The antibodies reacted with two major glycoproteins of 175 kDa and 105 kDa. None of the antibodies triggered peripheral blood lymphocytes or T cells to proliferation when tested under different culture conditions. All antibodies inhibited the proliferative response to concanavalin A or to allogeneic lymphocytes in mixed lymphocyte culture when whole IgG fractions were used. Complete inhibition of cytotoxic activity was obtained in cell-mediated lympholysis and in natural killer cell cytotoxicity (NK) when fresh peripheral blood lymphocytes were used as effector cells. Weak inhibition was also obtained in NK when mixed lymphocyte culture-activated effector cells were used. The inhibition was stronger, when NK activity was determined against MOLT4 target cells as compared to K562. Whereas F(ab')2 fragments of 873 IgG inhibited cytotoxic T lymphocyte activity completely, Fab fragments of 873 IgG neither inhibited proliferation nor cytotoxic T lymphocyte activity, but gave some inhibition of NK against MOLT4 targets. The results indicate that antibodies against these leucoagglutinin-reactive structures reacted with polypeptides similar to or identical with the human "leukocyte function-associated antigen-1" (LFA-1) considered to be an important mediator of cell-cell interactions and nonspecific adherence.

The use of supernatants from neuraminidase and galactose oxidase (NAGO)-treated lymphocytes as a source of T cell growth factor.

Human peripheral blood or tonsil lymphocytes produce T cell growth factor (TCGF), when activated with neuraminidase (NA) and galactose oxidase (GO). Partial purification of NAGO-TCGF on Sepharose G-100 columns gave a TCGF-active fraction within the same molecular weight range as the conventional lectin-induced TCGF (approximately 15,000 Da). Human T cells, activated in mixed lymphocyte culture (MLC) with irradiated allogeneic EB-virus transformed B-cells (LCL) could be maintained in continuous culture for several months with retained functional activities. The cells showed similar growth patterns when cultured in the presence of either NAGO-TCGF or PHA-TCGF. The growing cells were characterized by means of monoclonal antibodies. After 4 weeks of culture 98% of these were OKT3+ and 87% were also OKT8+. The cytolytic activities of the cultures were tested in cell-mediated lympholysis (CML) against allogeneic LCL as target cells, in natural cytotoxicity (NK) against K562 cells and in antibody dependent cytotoxicity (ADCC) against bovine erythrocytes. Cultures displaying one or several of these functions were obtained. The results indicate, that TCGF obtained from supernatants of NAGO-activated lymphocytes is as potent as the T cell growth promoting factor obtained by lectin stimulation. One major advantage of using NAGO-generated TCGF is that contamination with lectin is avoided.