Two monoclonal anti-CD3 antibodies can induce different events in human T lymphocyte activation.

Two monoclonal antibodies, WT32 and CLB-T3/4.2a, directed against the CD3 complex were used to study the mechanism of activation of human peripheral T lymphocytes. WT32, a mouse monoclonal IgG2a antibody with a low avidity (much less than OKT3) for the CD3 complex, effectively induces mitogenesis of purified T lymphocytes when used in the 1 ng-10 micrograms range in the presence of monocytes or recombinant interleukin 2 (IL2). In contrast, CLB-T3/4.2a, a mouse monoclonal antibody of the same isotype with a high avidity (much greater than OKT3) for the CD3 complex, induces IL2 receptor expression and IL2 responsiveness only at very low concentrations (less than 5 ng/ml), yet in the presence of monocytes this antibody induces proliferation within a similar dose range as WT32. Apparently, in the absence of accessory cells which can cross-link the antibody CD3 complexes, the binding properties (avidity) of an antibody and thereby the number of receptors that are occupied are important parameters for induction of IL2 responsiveness. Furthermore, we show that Ca2+ mobilization only occurs when the cells are stimulated by saturating amounts of antibody, so that, when the conditions are optimal for the induction of IL2 responsiveness, no Ca2+ mobilization will be detected.

The role of interleukin-2 in proliferative responses in vitro of T cells from patients after bone marrow transplantation. Evidence that minor defects can lead to in vitro unresponsiveness.

We have studied lectin-induced interleukin-2 (IL-2) production and proliferation of peripheral blood mononuclear cells from patients who had undergone a successful allogeneic bone marrow transplantation. Shortly after transplantation, the T cells show a decreased proliferative response and a decreased IL-2 production. However, addition to the culture of exogenous IL-2 does not result in restoration of the proliferative response, which indicates that the low proliferative response is not due to decreased IL-2 production alone. Longitudinal studies show a substantial variation between patients in the time in which the capacity to produce IL-2 is restored; however, in all patients there is a period in which IL-2 production is still diminished, but the proliferative capacity, as measured upon addition of exogenous IL-2 to the culture, is almost within the normal range. Also during this period, the proliferative response of the T cells can be restored by the addition of irradiated "feeder cells" obtained from the bone-marrow donors, as these cells secrete IL-2 without consuming it. Because peripheral blood samples from patients after bone marrow transplantation show great imbalances in the distribution of T4/T8 subpopulations, we have studied the influence of an artificially produced "reverse T4/T8" ratio on the proliferative response to mitogen and (allos-)antigen stimulation of healthy donor T lymphocytes. Even at very low proportions of T4 cells, normal responses were obtained in the proliferation assays with polyclonal mitogens. Only the response to soluble antigens, such as tetanus toxoid, was impaired. However, a low proportion of T4 cells resulted in a low IL-2 production so that, when IL-2 is a limiting factor due to intrinsic defects of patient cells, an inverse T4/T8 ratio can cause a nonresponsiveness in in-vitro assays.

The role of IL-2 and T4+ cells in the generation of human influenza virus-specific CTL activity.

Stimulation of human peripheral blood lymphocytes (PBL) with influenza A virus leads to the generation of virus-specific cytotoxic T lymphocyte (CTL) activity as well as natural killer (NK)-like activity. In this study, we show that exogenous IL-2 augments the in vitro generation of virus-specific CTL activity, only when added some days after the initiation of the culture. Apparently, the endogenously produced IL-2 can be a limiting factor in the in vitro generation of CTL activity. The increase of influenza virus-specific CTL activity after addition of exogenous IL-2 does not affect the restriction pattern of the CTL response. So, the preferential use of certain HLA antigens as restriction elements is not due to a limiting amount of endogenously produced IL-2. Depletion of T4+ cells completely abrogates the generation of virus-specific CTL activity. Addition of exogenous IL-2 to T4+-cell-depleted cultures fully restores the generation of HLA-restricted virus-specific CTL activity. We conclude that in the in vitro generation of virus-specific CTL activity in bulk cultures of human PBL the sole function of T4+ cells in human virus-specific CTL generation is the production of IL-2, no cognitive cell interaction of T8+ CTL precursors with T4+ cells is required, and in bulk cultures T8+ cells themselves are not able to produce sufficient amounts of IL-2 to ascertain the maturation of virus-specific CTL precursors into cytolytic T cells. Finally, we show that exogenous IL-2 also has a stimulatory effect on the NK-like or lymphokine-activated killer activity, which is always concomitantly induced in virus-specific CTL generation cultures, but has no influence on the levels of IFN produced in such cultures.

Monocyte-dependent induction of proliferation of human peripheral T cells by recombinant interleukin 2.

The in vitro proliferation of human peripheral lymphocytes induced by interleukin 2 (IL2), a product of cDNA, cloned in E. coli has been studied. The maximal mitogenic signal is given by concentrations greater than or equal to 2.5 U/ml. Due to growth factor consumption, at least 10 U/ml are needed to maintain a logarithmic response until day 6. The anti-Tac antibody, directed against the IL2 receptor, effectively blocks this response, but we could not obtain a decrease of IL2-reactive cells by depletion of putative in vivo activated Tac+ cells, using this antibody and a fluorescence-activated cell sorter. Depletion of Leu7+ and Leu11b+ cells does not cause a decrease of the response, which indicates that the responding cells are not confined to the natural killer lineage. By simultaneous staining of cell-surface markers and DNA, the nature of the proliferating cell was determined. More than 90% of the dividing cells expressed HLA class II and the Tac antigen, whereas the lymphocyte populations, defined by the surface markers Leu2, Leu3, Leu4, Leu7 and Leu11b, were all represented in the dividing cells. The magnitude of the response was proportional to the number of monocytes present in the culture. Depletion of monocytes completely abrogated the response, whereas an increase in the number of monocytes to a 1:1 ratio with lymphocytes caused a 2-fold increase in proliferation. However, purified T cells do proliferate to IL2 when cultured in the presence of a supernatant that was harvested from a 2-day culture of adherent monocytes. The proliferation-inducing activity in the supernatant eluted with apparent molecular weights of 15 000 and 75 000 on an Ultrogel AcA-54 column. Therefore, we conclude that in vitro, in the presence of an IL1-like activity produced by monocytes, IL2 is mitogenic for a population of T cells.

T cell triggering by lectins. I. Requirements for interleukin 2 production; lectin concentration determines the accessory cell dependency.

The requirements for lectin-induced interleukin 2 (IL2) production by human T cells have been investigated. With two different types of T cells, the Jurkat T cell lymphoma and highly purified HLA class II- peripheral T cells, the amount of IL2 produced was strongly dependent on the lectin concentration used. Addition of accessory cells caused a shift in the dose-response curve, resulting in strongly enhanced IL2 production at low concentrations. Thus, the (absolute) accessory cell dependency for T cells to produce IL2 is defined by experimental conditions. Only at lectin concentrations that were found to be optimal in the presence of accessory cells, removal of these cells abrogates IL2 production. Furthermore, after depletion of monocytes IL 2 production by peripheral T cells became almost completely dependent on the presence of thiols in the culture medium. In contrast, the IL2 production by the Jurkat line was not influenced by addition of thiols. The Jurkat model was used to study the nature of accessory cell because this cell line does not show any reactivity to allogeneic cells. Various myeloid and B lymphoid cell lines were tested as accessory cells. The capacity to function as accessory cell was not related to the monocytic origin of the cell. B cell lines were far more effective than monocytes, as two HLA class II- monocytic cell lines were not active. Even after HLA class II determinants were induced on these cells by incubation with an interferon-gamma-containing conditioned medium, they failed to act as as accessory cells. These experiments question the importance of HLA class II molecules and monokines, such as IL1, for lectin-induced IL2 production.

T cell triggering by lectins. II. Stimuli for induction of interleukin 2 responsiveness and interleukin 2 production differ only in quantitative aspects.

We have investigated the requirements for lectin-induced proliferation of highly purified human T cells. To study activation, independent of growth factor production, we cultured the cells in the presence of an excess of interleukin 2 (IL2), which was a product of cDNA cloned in E. coli. In the presence of IL2, the same cooperative effect of lectin and accessory cells was found that we have previously described for IL2 production. Thus, analogous to induction of IL2 production, the acquisition of responsiveness to IL2 can be completely monocyte dependent, but a 10-fold increase in lectin concentration completely abolishes the requirement for accessory cells. Furthermore, two stimuli (IL 1 and phorbol myristate acetate), which are able to replace monocytes at the level of IL2 production, also induce responsiveness to IL2 under accessory cell-dependent conditions. Thus, very similar conditions are required for proliferation and for the induction of IL2 production. There is only a quantitative difference: proliferation of cells in the presence of exogenous IL2 occurs already at low lectin concentrations, whereas IL2 production and consequently proliferation in the absence of exogenous IL2 requires higher lectin concentrations. At high lectin concentrations, when IL2 production has become the only limiting factor, purified T cells cannot be induced to proliferate in the absence of exogenous IL2 because the lectin concentration that induces IL2 production independent of accessory cells inhibits mitogenesis. However, after addition of thiols to the medium, which enhances the IL2 production, a very narrow range of lectin concentration can be found which is just below toxic values and still high enough to induce IL2 production in the absence of accessory cells. Under these conditions, accessory cells are no longer a prerequisite for lectin-induced T cell proliferation.

Isolation of functionally different human monocytes by counterflow centrifugation elutriation.

Human peripheral blood monocytes were isolated by counterflow centrifugation elutriation (CCE). This technique was modified in such a way that various monocyte fractions (viability greater than 99%) could be elutriated by increasing the density of the CCE-medium in steps of 0.0027 g/ml. All monocytes showed the same size distributions as determined by electronic sizing, which indicated that they differed in their density only. Both cytoplasmic esterase and peroxidase activity increased with the density of the cells. Furthermore, the monocytes with the highest density were 2.3-4 times more active in an antibody-dependent cellular cytotoxicity (ADCC) assay than those with the lowest density. In contrast, the monocyte with the highest density were less capable to induce the proliferation of lymphocytes in mixed leukocyte cultures (MLC) than those with the lowest density. This observation could not be attributed to differences in the expression of HLA-DR determinants, since a monoclonal antibody directed against HLA-DR antigens reacted equally well with the monocytes in different fractions. These results provide evidence for the existence of functionally different subsets of monocytes or different states of differentiation or maturation.