Chou-Chik Ting

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.

IMMUNOGENICITY OF TUMOR ANTIGENS

A central theme in experimental tumor immunology is the immunization with tumor antigens. The objectives of the studies on this subject have usually been the induction of either transplantation protection against tumor challenge or of in vitro immune reactivity. Few detailed studies have been performed to determine mechanisms for in vivo immunogenicity, by relating those data to results obtained in vitro. In this paper, we will describe a series of experiments performed primarily with virus-induced tumors of mice and rats, in which some of the factors that may influence in vivo immunogenicity are analyzed. It has become clear from these studies that some “nonimmunogenic” tumors or tumor cell preparations are antigenic and under some circumstances can induce an immune response in the host. We have placed particular emphasis on lymphoproliferative responses and on cell-mediated cytotoxic reactions, after primary or secondary exposure to tumor antigens in vivo or in vitro, and these experiments have been quite helpful for the analysis of immunogenicity. A number of factors have been identified that may interfere with immunogenicity of antigenic tumors: Suppressor cells in the host, immunosuppressive viruses in the tumors, and specific and nonspecific serum factors. Careful dissection of factors influencing immunogenicity of tumor antigens should help to provide practical solutions to problems related to immunoprevention and immunotherapy of cancer, which have not been solved by empirical studies.

Studies of the mechanisms for the induction of in vivo tumor immunity: I. Induction of primary and secondary cell-mediated cytotoxic responses by adoptive transfer of lymphocytes

Abstract Cell-mediated immunity to FBL-3, a syngeneic Friend virus-induced leukemia in C57BL/6 mice, could be adoptively transferred. Characteristic primary and secondary cytotoxic responses could be induced by adoptive transfer of normal and presensitized lymphocytes, respectively. In vivo tumor immunity could also be produced by adoptive transfer of presensitized lymphocytes. Both the primary and secondary cell-mediated cytotoxic reactions were T-cell dependent. The specificity of these reactions was primarily directed against F (Friend) type-specific antigen and FMR (Friend, Moloney, Rauscher) common antigen. The cytotoxic responses produced by adoptive transfer experiments gave better correlation to in vivo tumor immunity than those generated by in vitro mixed lymphocyte tumor cell culture reactions.

Differential requirement of protein tyrosine kinase and protein kinase C in the generation of IL-2-induced LAK cell and αCD3-induced CD3-AK cell responses

This study examined the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the signal transduction pathways for lymphocyte activation through IL-2R to generate LAK cells and through TCR-CD3 to generate CD3-AK cells. Two PTK inhibitors [herbimycin A and genistein (PTK-I)] and two PKC inhibitors [calphositin C and staurosporine (PKC-I)] were used in the experiments. It was found that the primary activation pathway through IL-2R was PTK-dependent; that is, generation of both the IL-2-induced proliferative and the cytotoxic responses was completely abrogated by PTK-I and not by PKC-I. Quite different results were obtained with the alpha CD3-induced CD3-AK cell response. First, the alpha CD3-induced proliferation was only partially inhibited by PTK-I or PKC-I alone. Second, generation of CD3-AK cytotoxic response was primarily PKC-dependent; that is, only PKC-I induced significant inhibition. Genistein was found to reduce protein tyrosine phosphorylation in both LAK cells and CD3-AK cells, indicating that CD3-AK cells were also susceptible to PTK-I treatment. Further studies showed that PTK-I and not PKC-I suppressed perforin mRNA expression and N-2-benzyoxycarbonyl-L-lysine thiobeneylester esterase production in LAK cells, and the opposite was true for CD3-AK cells. These results indicate that different pathways were employed in lymphocyte activation through IL-2R and TCR-CD3. The former pathway is primarily PTK-dependent. Activation through TCR-CD3 is a more complex event.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Effect of interleukin 2 on cytotoxic effectors: II. Long-term culture of NK cells

Abstract The present study reports the establishment of an NK (natural killer) cell line, designated as IL2-CE1. This cell line was maintained in active growth in culture with the supplementation of interleukin 2 (IL2). The cells gave cytotoxicity against a variety of murine tumors. There was no detectable cytotoxicity against three human tumors tested. The reactivity obtained with the murine tumors was generally correlated with the sensitivity of these tumor cells to NK cytotoxicity. With few exceptions, the reactivity was much higher for NK-sensitive targets like YAC and RL♂ 1 cells. There was very little cytotoxicity against NK-resistant targets like P815 and HFL/d. The reactivity of the effectors against YAC was susceptible to trypsin treatment and this effect was reversible. In determining the surface markers of IL2-CE1 cells, it was shown that over 90% of the cells had Thy-1.2 antigen despite their resistance to the lytic effect of anti-Thy-1.2 antibody. There were no detectable surface Ig or Fc receptors. Therefore, the IL2-CE1 cells were consistent with being NK cells. Although these cells gave high levels of cytotoxicity against murine tumors, in the tumor neutralization tests the IL2-CE1 cells failed to give any protection against an immunosensitive leukemic line, FBL-3. After the IL2-CE1 cells were cloned, it was found that different NK clones showed variations in their fine specificity. Our finding supports the presence of different populations or subsets of NK cells. Establishment of these NK clones in long-term culture should be helpful for the detailed characterization of the NK cells and aid in the determination of their significance in the immune surveillance against neoplasia.

Studies of the mechanisms for the induction of in vivo tumor immunity. VI. Induction of specific and nonspecific cell-mediated immunity in tumor-bearing hosts and its correlation with transplantation tumor immunity.

Abstract Studies were performed to determine the development of cell-mediated cytotoxic response at tumor site in C57BL/6 mice bearing progressively growing FBL-3 ascites leukemia. The effectors isolated from tumor ascites are found to be highly cytotoxic for leukemic target cells. The levels of cytotoxicity obtained with effectors isolated from tumor site are generally higher than those obtained with immune mice. This cytotoxicity is both specific and nonspecific. The specific cytotoxicity against tumor-associated antigen is mainly mediated by T cells and the nonspecific cytotoxicity against unrelated tumor cells is mediated largely by macrophages. The T-cell-enriched preparation did not give significant natural killer activity. When testing the ability of these effectors to produce in vivo immunity against the challenge of FBL-3, it was found that only T cells could confer the transplantation-type immunity, but the immunity was transient. The macrophage-enriched preparation isolated from tumor ascites failed to give in vivo protection. These findings indicate that in FBL-3 system, mice with progressively growing tumors are able to develop immune response against tumor cells. However, this immunity is probably interfered with by a suppressor factor(s) or suppressor cells which restrict their activity to eliminate the tumor cells effectively.