Biomedical subjects
Publications and source records attributed to C S Henney.
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The role of protein synthesis in the mechanism of T cell-mediated cytolysis has been re-investigated. Cytolytically active (C57BL/L anti-DBA/2) spleen cells treated with pactamycin (10-7 M to 10-6 M) exhibited suppressed protein synthesis (100 +/- 5%), but unimpeded lytic activity. Drug-treated effector cells, incubated for prolonged (up to 24 hr) periods of time in the presence and absence of antigen, showed no siginificant diminution of lytic activity although the incorporation of 3H-leucine into protein was totally ablated. Studies with emetine, another irreversible inhibitor of protein synthesis, gave identical results. These findings are difficult to reconcile with the hypothesis that effector T cells lysis via a soluble protein mediator.
Spleen cells from C57BL/6 mice immunized with a DBA/2 mastocytoma (P815) were harvested at various stages of the immune response and cultured in vitro in the presence and absence of antigen. Killer T cell activity in immune spleens could not be demonstrated until 6 or 7 days after antigen, but spleen cells harvested as early as 3 or 4 days and cultured for 24 hr at 37 degrees C showed significant cytotoxicity. This increased activity was not augmented further by culturing with antigen. "Memory" T cells, whose in vitro differentiation into killer cells required the presence of antigen, could not be demonstrated until 9 or 10 days after alloantigenic stimulation. Once produced, however, these cells persisted for at least 6 months. Memory cells, like killer T cells bound avidly to homologous allogeneic monolayers. There were indications that the memory T cell pool was heterogeneous. On one hand, when cells harvested 10 days after stimulation were exposed to antigen in vitro, their lytic activity increased within 24 hr but showed no further increases when the culture period was extended. In contrast, 45-day-old immune cells showed increasing lytic activity throughout a 4-day exposure to antigen. Augmentation of lytic activity in both cell populations was independent of DNA synthesis through the first 24 hr of culture. Subsequent increases in the activity of 45-day cells was dependent upon cell proliferation. Both the antigen-independent augmentation of lytic activity which followed culturing of immune cells, and the antigen-induced differentiation of memory cells were reversibly inhibited by a series of drugs which raised lymphocyte cAMP levels.
The lysis of allogeneic cells by cytolytically active T lymphocytes has been shown to involve two stages, Each with distinct cation requirements. Cytolysis required the presence of Ca++ ions, but addition of Mg++ to Ca++ containing medium synergistically enhanced target cell destruction. This synergistic effect resulted form kinetically separable events mediated by these cations. Thus, optimal adhesions between killer and target cells necessitated the presence of Mg++; interactions also occurred, but less efficiently, in the presence of Ca++. Lymphocyte-target cell interactions resulting from the present of Mg++ did not lead to the lysis of the target cell, until Ca++ were added. A "late" stage of the establishment of a target cell lesion is thus uniquely served by Ca++.
Murine cytotoxic T cell activity was evaluated in analogy with the methods used to characterize enzymes. Concordant with this analogy, the relationship between "steady-state" lytic rates and target cell ("substrate") number was described by a hyperbola, the asymptote of which was the maximum rate of lysis ("V") and was directly proportional to the number of killer cells. The number of target cells required to reach a rate of one-half V ("K") was independent of the number of effector cells, but was found to vary with the total cell concentration in the cultures. The kinetic analogy was extended to analyze this variability by studying the inhibition of cytolysis caused by normal syngeneic lymphocytes. A value for K in the absence of non-killer cells ("Kh") was evaluated. This term, which was independent of effector cell frequency, was a measure of lytic efficiency and, as such, was used to compare various effector cell populations. The Kh value for a number of lymphoid cell populations from identically immunized C57BL/6 mice was found to vary considerably, thereby providing direct evidence that effector populations have different lytic efficiencies. Because of this variability, the widely used practice of comparing killer cell populations solely on the basis of their relative lytic activity may often be invalid.
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C57BL/6 mice immunized i.p. with alloantigen (P815 mastocytoma cells) develop cytolytically active thymus-derived (T) splenic lymphocytes. The definition of specific histamine receptor sites on effector T cells has been studied by measuring the in vitro effects of the hormone on cytolytic activity. Histamine was found to inhibit cytolysis reversibly and to increase lymphoid cell cyclic AMP levels. Both of these histamine activities were reversed by burimamide and metiamide; neither activity was affected by diphenhydramine or pyrilamine. These findings indicate that modulation of effector T cell activity by histamine is mediated only by one of the subtypes of tissue histamine receptors, designated a histamine-type 2 receptor. This receptor appears to be present on cytolytically active cells; there is no evidence for activation by histamine of auxiliary or "suppressor" cells. The estimated dissociation constant (KB) for the burimamide-receptor complex (9 times 10-minus 6 tm) and for the metiamide-receptor complex (8 times 10-minus 7 M) indicated that the histamine receptor on T cells is quite similar to histamine-type 2 receptors in other tissues. Cells bearing such receptors could not be isolated by passage through a column of histamine-coated tsepharose beads. The cytolytic activity of spleen cells taken from mice early (days 7-9) after immunization is virtually unaffected by histamine in vitro. In contrast, the activity of spleen cells taken from mice later in the immune response is progressively more susceptible to inhibition by histamine. After reaching a maximum at day 11, the spleen cell cytolytic activity falls in a pattern that parallels the increase in susceptibility to histamine. The susceptibility of effector T cells to histamine appears also to reflect their site of origin, for peritoneal exudate effector cells were found to be significantly less sensitive than spleen cells to inhibition by histamine. The progressive increase in inhibition by histamine apparently reflects the appearance of greater numbers of specific histamine-type 2 receptors, and is probably a general phenomenon, for spleen cells from A/J or C3H mice immunized with either P815 mastocytoma (H-2d) or EL-4 (H-2b) cells showed the same effect. However, the appearance of histamine receptors could be altered by prior immunization with an unrelated alloantigen: thus, when A/J mice are preimmunized with EL-4, a subsequent immunization with mastocytoma cells results in peak spleen anti-H-2d activity at day 9 instead of days 11-13, and the appearance of significant (greater than 40 percent) inhibition by histamine as early as day 8 instead of day 16. The physiological role of the histamine receptors is as yet undefined, though their unexpected rate of appearance on effector T cells, coincident with a decline in the number of lytically active cells in vivo, may be a significant hint that hormone receptors play a role in the control of T-cell proliferation.
Guinea pig lymphotoxin, which was produced by stimulating immune lymph node cells with the specific antigen, ovalbumin, was partially purified by the sequential use of Pevikon block electrophoresis, ion exchange chromatography on DEAE-cellulose, and polyacrylamide disc gel electrophoresis. The lymphotoxin recovered from the final step was purified approximately 200- to 300-fold as compared to the starting material, but nevertheless contained at least one major contaminant protein. Rabbits were immunized with the partially purified lymphotoxin, and an antiserum capable of neutralizing the cytotoxic effect of lymphotoxin was obtained. The anti-lymphotoxin activity was shown to be due predominantly to IgM immunoglobulin. The antiserum neutralized concanavalin A-induced lymphotoxin with equal efficiency as antigen-induced lymphotoxin. On the other hand, the anti-lymphotoxin serum did not neutralize preformed mitogenic factor or migration inhibitory factor (MIF) which had been produced by antigen-stimulated immune lymph node cells. Thus these activities appear to be due to substances which are antigenically distinct from lymphotoxin. Likewise, anti-lymphotoxin serum, when present in cultures of antigen-stimulated immune lymphoid cells, caused no diminution in the amount of mitogenic factor or MIF which was produced, indicating that it had no direct deleterious effect on the function of the lymphoid cells responsible for producing these lymphokines.
A leukocyte population consisting of approximately 85% lymphocytes, prepared from human peripheral blood by centrifugation through a Ficoll-Hypaque gradient, was studied for its capacity to destroy antibody-coated human liver (Chang) cells in vitro. Cytolysis was a rapid event: increased ionic flux (86Rb) from the target cell occurred within 10 min of the addition of effector cells. Kinetic analysis of target cell destruction (51 Cr release) was compatible with a "one hit" hypothesis, thereby indicating that cytolysis resulted from a single collision was an effector cell. The initial rate of cytolysis was linear and related to the number of leukocytes added, but lysis at all of the leukocyte to target cell ratios tested ceased after 5 hr. The number of target cells killed at that time was directly proportional to the number of leukocytes added. While the lytic capacity of the effector population was totally depleted after incubation with antibody-coated target cells, cytotoxicity was not affected by co-culturing leukocytes with Chang cells treated with pre-immune serum. The cytotoxic effector cells functioning in this antibody-dependent lytic system are thus to be contrasted with killer T cells, whose lytic activity is not compromised by interaction with homologous target cells. It was estimated that approximately 4% of the leukocyte population employed could kill antibody-coated Chang cells, a figure consistent with the estimated frequency of "null" cells within human peripheral lymphocytes.
Mastocytoma cells (P815 of the DBA/2 strain) treated with increasing concentrations of glutaraldehyde were concurrently evaluated for their ability to incorporate exogeneous uridine, thymidine, and amino acids. The antigenic expression and membrane integrity of these treated cells were assayed by measuring their susceptibility to lysis by antibody and complement and by T-effector cells. The concentrations of glutarladehyde required to effect target cell antigen display were greater than those required to inhibit totally the cell's protein and nucleic acid synthetic processes. Thus, P815 cells treated with 0.15% glutaraldehyde for 10 sec were unable to incorporate either amino acids or nucleotides into macromolecules, but were readily lysed by effector T cell populations, and by alloantibody in the presence of complement. Target cells treated with glutaraldehyde concentrations in excess of 0.25% for 10 sec were resistant to both forms of immune lysis. In keeping with these observations, monolayers of P815 cells treated for 10 sec with 0.15% glutaraldehyde, were capable of specifically depleting T-effector cells from a cytolytically-active spleen cell population. After treatment with higher concentrations of glutaraldehyde (0.3%), however, the monolayers lost their capacity to adsorb effector cells. Although P815 cells treated with glutaraldehyde continued to exhibit H-2d alloantigen, neither these cells nor glutaraldehyde-treated DBA/2 spleen cells induced significant blastogenesis or stimulated the production of cytolytically active effector cells in mixed leukocyte cultures.
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The lysis of DBA/2 mastocytoma cells (P815) by alloimmune C57BL/6 thymus-derived lymphocytes was inhibited by cytochalasin A (CA) and cytochalasin B (CB). Although CB prevented incorporation of glucose into lymphocytes, the effector cell susceptibility to the drug was independent of the hexose content of the culture medium. Indeed, cytolysis occurred in the absence of exogenous hexose. Futhermore CA which, like CB, inhibited the adsorption of effector spleen cell populations onto homologous allogeneic monolyers and prevented the migratory movement of lymphoid cells did so without affecting incorporation of glucose into lymphocytes. These findings suggest that the inhibition of cytolysis by CA and CB is mediated by common inhibitory effects on membrane mobility rather than by affecting glucose uptake.
Adult C57BL/6 mice immunized i.p. with an allogeneic mastocytoma cell line (P815 of the DBA/2 strain) develop cytolytically active T cells. Activity peaks at 10 to 11 days and falls rapidly thereafter. We have observed an anomalous in vitro behavior of effector cell populations harvested during this decline. The lytic activity of spleen cells harvested 12 to 18 days after alloantigen was markedly augmented after culture for 24 hr at 37 degrees C. The augmented lytic activity was caused by T cells and specificity exhibited was identical to the pre-culture population. No augmentation of lytic activity occurred when cells were cultured at 4 degrees C or when they were treated with a protein synthetic inhibitor (pactamycin) at concentrations which did not compromise cytotoxic expression by the starting population. Augmentation of cytolysis was independent of DNA synthesis and did not require the presence of added antigen. When immune lymphoid cell populations were depleted of effector cells by adsorption onto allogeneic monolayers and then cultured, new killer cells differentiated within a 24-hr period. This process was not augmented by the addition of antigen to the nonadherent cell population. We interpret these demonstrations to show: i) that there is a pre-killer T cell whose differentiation into an effector cell requires de novo protein synthesis but not cell proliferation, and ii) that the in vivo fall in lytic activity during the primary immune response is at least partially caused by suppression of the differentiation of this pre-killer cell. Furthermore, we conclude that the pre-killer cell is distinct from a memory T cell because: i) its conversion to an effector cell is antigen-independent and ii) because, unlike the memory cell, pre-killers do not bind avidly to allogeneic cell monolayers.
Experiments have been designed to test the hypothesis that soluble mediator production and T-cell-mediated cytotoxicity are necessarily related phenomena, and that soluble mediators may be involved in the mechanism of cytolysis. To this end, agents known to inhibit T-cell-mediated lysis in vitro have been studied for their effects on the production of two lymphocyte-derived mediators, lymphotoxin (LT) and migration inhibitory factor (MIF). A clear dissociation between mediator production and cell-mediated cytolysis was found using inhibitors of protein synthesis. Pactamycin and emetine, in doses of 10(-7) M to 10(-6) M, suppressed production of MIF and LT with only slight effect on killing of mastocytoma cells by immune T cells. On the other hand colchicine and vinblastine inhibited T-cell-mediated cytolysis in a dose-related manner but had no significant effect on either MIF or LT production, A striking dichotomy was also observed after augmentation of intracellular cyclic 3'5' adenosine monophosphate (cAMP) levels with cholera enterotoxin. Increased cAMP levels were associated with abrogation of direct lytic activity, but were without significant effect on MIF or LT production in guinea pigs or mice. These findings indicate that mediator production and direct lymphocyte-mediated cytolysis can be experimentally dissociated and represent independent cell-mediated immune functions.
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