Myrthel E. Hargrove

Differential expression of asialo GM1 on alloreactive cytotoxic T lymphocytes and lymphokine-activated killer cells

The present study was undertaken to examine the differential expression of asialo GM1 (AsGM1) on the responding cells and effectors of alloreactive cytotoxic T lymphocytes (CTL) and lymphokine-induced activated killers (LAK). It was found that AsGM1 was expressed on the 3-day-cultured LAK effectors. Its expression gradually disappeared to the extent that AsGM1 became undetectable after 5 to 6 days of culturing. In contrast, AsGM1 was detected on 3-day CTL generated in mixed-lymphocyte cultures (bulk cultures); however, the levels of AsGM1 expression remained the same for at least 7 days. When examining the expression of AsGM1 on the responding cells, the reciprocal results were obtained. AsGM1 was expressed the LAK responders, but we were unable to demonstrate AsGM1 on CTL responders. Depletion of AsGM1+ cells from the responding population reduced subsequent CTL responses; however, CTL responses could be restored by adding conditioned media containing both interleukin 2 (IL-2) and other helper-T-cell factors and could not be restored by purified IL-2 alone adding at comparable doses. Reconstituting the AsGM1-depleted responders with Lyt-2-depleted splenocytes also restored the CTL response. Furthermore, depletion of AsGM1 cells from the responding population did not reduce the precursor frequency of allo-CTL, whereas the precursor frequency of LAK cells was reduced 42-fold. These findings show that the reduction of CTL responses after depletion of AsGM1+ cells was not due to the removal of precursors; instead, the defect appeared to be in the helper population. We further found that the helper defect was not due to impaired IL-2 production, because the endogenous production of IL-2 AsGM1-depleted responders was not reduced. Therefore, AsGM1+ cells may play a role in the helper pathway other than IL-2 production.

Anti-CD3 antibody-induced activated killer cells subsets of killer cells that mediate fast or slow lytic reactions

The present study has characterized two sets of alpha CD3-induced activated killer cells (CD3-AK). A 2 to 4-h 51Cr release assay or triton-treated 125IUdR release assay demonstrated that CD3-AK cultured in the continuous presence of alpha CD3 (CD3-AK+) mediated fast lysis. A 20-h 51Cr or 125I-deoxyuridine release assay demonstrated that CD3-AK cultured in the absence of alpha CD3 (CD3-AK-) mediated slow lysis. Activating the TCR-CD3 complex of CD3-AK- cells with alpha CD3 for 2 h enabled the killer cells to mediate fast lysis. The activation process was inhibited by H-7, a protein kinase C (PKC) inhibitor. Conversely, removal of alpha CD3 from CD3-AK+ cell cultures for 24 h resulted in almost complete loss of the ability of CD3-AK+ cells to mediate fast lysis, but they still retained the ability to mediate slow lysis. It appeared that constant perturbation of CD3 by alpha CD3 maintained the CD3-AK+ cells in an active state, and thus, they were able to mediate fast lysis. On the other hand, activation by a 2-h incubation with PMA could convert the noncytolytic CD3-AK- cells to be cytolytic in slow lysis and to augment the slow lytic reactions mediated by CD3-AK- cell with low cytolytic activity. These results were confirmed by triton-treated 125IUdR release assay which could detect early DNA-release. Thus it appeared that activation of CD3-AK cells with T cell activation signals that bypassed TCR (such as PMA) could induce slow lysis. In the effector phase of lytic reactions, the fast lytic reaction was relatively resistant to inhibition by H-7, whereas the slow lytic reaction was susceptible to H-7 inhibition, indicating that fast lysis was PKC independent and slow lysis was PKC dependent. It was further found that H-7 inhibition was at an early stage of slow lysis, suggesting that a PKC dependent activation process preceded the PKC independent lytic process. These findings indicated that the CD3-AK- cells were in a less activated state which required further activation to turn on their lytic machinery to initiate the lytic reaction.

Asialo GM1 as an accessory molecule determining the function and reactivity of cytotoxic T lymphocytes

The expression and function of asialo-GM1 (AsGM1) in alloreactive cytotoxic T lymphocytes (CTL) was studied. We have shown previously that the cytotoxic reactions mediated by AsGM1+-cloned CTL were blocked by anti-AsGM1 or by purified AsGM1. To further determine the role of AsGM1 in CTL-mediated cytotoxicity, we examined the correlation between this blocking effect and the expression of AsGM1 on effector and target cells. Now we found that the blocking by anti-AsGM1 was largely dependent on the expression of AsGM1 on the effector cells in a dose-dependent fashion. The expression of AsGM1 on target cells had only little effect on the blocking of cytotoxic reactions by anti-AsGM1 or AsGM1. A threefold difference was seen in the blocking of AsGM1+ and AsGM1- targets. The observation was in sharp contrast to the effectors as no blocking was ever seen with AsGM1- CTL. Similar to CTL effectors, we found that the expression of AsGM1 and L3T4 were mutually excluded on mitogen-activated T cells, despite the fact that they could coexpress in resting T cells. The expression of AsGM1 on CTL effectors was associated with the antigen-nonspecific natural killer (NK)-like or lymphokine-activated killer (LAK)-like activity exerted by the alloreactive CTL. All AsGM1+ CTL possessed LAK activity against antigen-unrelated tumor targets, and the AsGM1- CTL only displayed antigen-specific alloreactivity. The LAK activity was associated with the expression of AsGM1 on effectors, and was not related to the AsGM1 expression on target cells. These findings indicate that the AsGM1 expressed on alloreactive CTL may function as an accessory molecule for T-cell receptors in the antigen-specific alloreactive cytotoxicity mediated by AsGM1+ CTL. The expression of AsGM1 may also be related to the activation of an NK-like apparatus in these CTL. Therefore, AsGM1 not only may be involved in cytotoxic reactions mediated by AsGM1+ CTL, it may also modulate the specificity of the CTL cytotoxicity.

Reversal of multiple-site tumor cell-induced immunosuppression by specific cytokines and pharmacological agents

The present study explores a model for tumor cell-induced immunosuppression and reversal of suppression by cytokines and other pharmacological agents. To simulate tumor-cell-induced suppression, a panel of suppressor agents which included CsA (cyclosporin A), SSP (staurosporine), BSO (L-buthionine-[S,R]-sulfoximine) and PMA, and a panel of anti-suppressor agents which included IL-2, IL-4, GSH (glutathione) and amiloride, were tested. These suppressor/anti-suppressor agents acted differently on four specific sites of the immune arm that affected the alpha CD3-induced T cell proliferative and cytotoxic responses. They included (1) IL-2 production, (2) PKC-regulated cytolytic granule production, (3) GSH-regulated maturation of functional granules, and (4) granule exocytosis. When a single suppressor agent was used, all the suppressor agents tested in this study inhibited the generation of alpha CD3-induced activated killer cells (CD3-AK), whereas alpha CD3-induced proliferation was inhibited by CsA, BSO, and EL-4 tumor cells. Except for EL-4, suppression induced by a single suppressor agent could be corrected by an appropriate single anti-suppressor agent. Multiple suppressor agents induced profound suppression of CD3-AK response. In most cases, multiple anti-suppressor agents were required to correct the immune defects induced by multiple suppressor agents. Finally, EL-4 tumor-cell-induced immunosuppression could not be corrected by any single anti-suppressor agent tested, but a combination of IL-4, GSH and amiloride fully restored the CD3-AK response. These results suggest that tumor cells may induce multiple immune defects that require multiple anti-suppressor agents for correcting the defects to restore the host immunocompetence.

IL-4 regulation of a protein kinase C independent pathway for the generation of αCD3-induced activated killer cells

alpha CD3 induced the generation of activated killer cells from resting T cells. Pretreatment of the splenic responders with PMA, a phorbol ester, depleted protein kinase C and induced unresponsiveness to the generation of alpha CD3-induced activated killer (CD3-AK) cells. Addition of exogenous IL-4 (1 U/ml) restored the cytotoxic response, with the maximal effect achieved with 30 to 100 U/ml. The phenotypes of CD3-AK cells maintained in IL-2 or in IL-4, with or without PMA, were the same: Thy1+ and CD8+. These results were reproduced with purified T cells and purified CD8+ cells, indicating that both the effectors and precursors were CD8+ cells and IL-4 had a selective effect to upregulate the CD8+ cells. Similar results were obtained by using SSP (staurosporine), another PKC inhibitor. At 2 days prior to testing, switching the lymphokine added to 2-week PMA- and IL-2-maintained CD3-AK cells reversed their cytolytic activity: switching from IL-2 to IL-4 restored cytolytic activity, and switching from IL-4 to IL-2 reduced cytolytic activity. The cytolytic activity of these CD3-AK cells correlated with their ability to produce BLT-esterase. In the absence of PMA, CD3-AK cells cultured in either IL-2 or IL-4 were cytolytic and contained high levels of BLT-esterase. In contrast, in the presence of PMA, only the IL-4-maintained CD3-AK cells were cytolytic and produced significant amounts of BLT-esterase. The effect of IL-4 was abrogated by the alpha IL-4 antibody 11B11, which reduced the cytolytic activity of CD3-AK and the ability to produce BLT-esterase. The requirement of IL-2 was less stringent and its major role appeared to be maintaining the cell growth. These findings indicate that IL-4 may participate in the regulation of a PKC-independent pathway for the generation of CD3-AK cells by regulating the production of cytolytic granules.

Anti-CD3 antibody-induced activated killer cells: Cytokines as the additional signals for activation of killer cells in effector phase to mediate slow lysis

This study examines the role of cytokines in activating the effector cells to mediate slow lysis. After activation of splenocytes by alpha CD3, further culturing the cells in the absence of alpha CD3 resulted in the generation of activated killer cells (CD3-AK-) to mediate slow lysis. In contrast to fast lysis which was not affected by a PKC inhibitor H-7, slow lysis was inhibited. These findings suggested that a PKC-dependent activation phase preceded the lytic phase in slow lysis. To explore the mechanism for activating the lytic machinery in slow lysis, we examined the roles of cytokines in these reactions. First, it was found that alpha IL-2 or an alpha IL-2/alpha IL-4 combination inhibited slow lysis but had no effect on fast lysis. Secondly, IL-2, IL-4, or TNF alpha converted a noncytolytic CD3-AK- cells to mediate slow lysis, but they did not augment fast lysis. IL-2 and IL-4 had additive effect, and TNF alpha synergized with IL-2 to further augment the CD3-AK- cytolytic activity. Exogenous IL-6 and INF did not have any appreciable effect on the cytolytic activity of the killer cells. Besides TNF alpha, these cytokines were not directly cytotoxic to the target cells, indicating that they were not cytotoxic factors per se. Treatment with cycloheximide for 24 hr abrogated the cytolytic activities of CD3-AK cells, suggesting that a cytotoxic factor(s) was continuously synthesized to be stored in activated killer cells and was catabolized within 24 hr. Our results indicated that in the effector phase of slow lysis, after activating the CD3-AK- cells by the first signal (appropriate target cells), IL-2 and/or IL-4 appeared to be the second signal to initiate a cascade of events which triggered the release of other cytokines (e.g., TNF). This process resembles the secondary (memory) type of immune response. These events lead to full activation of the killer cells and converted the preformed cytotoxic factors into active form to initiate the lytic reaction and completed the lytic process.

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.