In vitro inhibition of HIV-1 by Met-SDF-1beta alone or in combination with antiretroviral drugs.

Compounds that can block the CXCR4 chemokine receptor are a promising new class of antiretroviral agents. In these experiments we studied the effect of a modified form of the native stromal cell-derived factor-1 (SDF-1), Met-SDF-1beta. The in vitro susceptibility of two different CXCR4-tropic HIV-1 strains was determined. Antiviral effect was assessed by the reduction of p24 antigen production in PHA-stimulated peripheral blood mononuclear cells with exposure to the modified SDF-1 molecule. The 50% inhibitory concentrations (IC50) were derived from six separate experiments. The IC50 against the two HIV-1 isolates was in 1.0-2.8 microg/ml range for Met-SDF-1beta. Met-SDF-1beta showed synergy to additivity with either zidovudine or nelfinavir at IC75 IC90 and IC95. Additivity was seen when Met-SDF-1beta was combined with efavirenz. No cellular toxicity was observed at the highest concentrations when these agents were used either singly or in combination. This compound is a promising new candidate in a receptor-based approach to HIV-1 infection in conjunction with currently available combination antiretroviral drug therapies.

Combination of CCR5 and CXCR4 inhibitors in therapy of human immunodeficiency virus type 1 infection: in vitro studies of mixed virus infections.

We studied the combined anti-human immunodeficiency virus type 1 (HIV-1) effects of a derivative of stroma-derived factor 1beta (SDF-1beta), Met-SDF-1beta, and a modified form of RANTES, aminooxypentane (AOP)-RANTES. The antiviral agents were tested singly or in combination at 95 and 99% virus inhibitory concentrations. Clinical R5 and X4 HIV-1 isolates were used. AOP-RANTES inhibited R5 but not X4 viruses, whereas Met-SDF-1beta had the opposite effect. Combinations of these compounds inhibited mixed infections with R5 and X4 viruses (95 to 99%), whereas single drugs were less inhibitory (32 to 61%). Combinations of R5 and X4 inhibitors are promising and deserve further evaluation.

Defensins act as potent adjuvants that promote cellular and humoral immune responses in mice to a lymphoma idiotype and carrier antigens.

Defensins released by neutrophils are able to kill a broad spectrum of microbes. They also induce leukocyte migration in vitro and elicit inflammatory leukocyte responses at s.c. injection sites in mice. In vitro experiments showed that human defensins enhanced concanavalin A-stimulated murine spleen cell proliferation and IFN-gamma production. This led us to examine the effects of human defensins on specific immune responses in vivo. BALB/c mice were immunized with 50 microg of keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide and administered with defensins in aqueous solution. Intraperitoneal administration of defensins significantly increased the production of KLH-specific IgG1, IgG2a and IgG2b antibodies 14 days after immunization. In vitro splenic KLH-specific proliferative responses were higher in mice treated with KLH and defensins than in those treated with KLH alone. Increased IFN-gamma and, to a lesser extent, IL-4 production were also detected in the supernatants of ex vivoKLH-activated spleen cells from mice treated with defensins. Finally, defensins significantly enhanced the antibody response to a syngeneic tumor antigen, lymphoma Ig idiotype and also augmented resistance to tumor challenge. These results indicate that defensins act as potent immune adjuvants by inducing the production of lymphokines, which promote T cell-dependent cellular immunity and antigen-specific Ig production. Thus, defensins appear to function as neutrophil-derived signals that promote adaptive immune responses.

Enhanced inhibition of human immunodeficiency virus type 1 by Met-stromal-derived factor 1beta correlates with down-modulation of CXCR4.

CXCR4 is a chemokine receptor used by some strains of HIV-1 as an entry coreceptor in association with cell surface CD4 on human cells. In human immunodeficiency virus type 1 (HIV-1)-infected individuals, the appearance of viral isolates with a tropism for CXCR4 (T tropic) has been correlated with late disease progression. The presumed natural ligands for CXCR4 are SDF-1alpha and SDF-1beta, which are proposed to play a role in blocking T-tropic HIV-1 cell entry. Here, we demonstrate that addition of an N-terminal methionine residue to SDF-1beta (Met-SDF-1beta) results in a dramatically enhanced functional activity compared to that of native SDF-1beta. Equivalent concentrations of Met-SDF-1beta are markedly more inhibitory for T-tropic HIV-1 replication than SDF-1beta. A comparison of the biological activities of these two forms of SDF-1beta reveals that Met-SDF-1beta induces a more pronounced intracellular calcium flux yet binds with slightly lower affinity to CXCR4 than SDF-1beta. Down-modulation of CXCR4 is similar after exposure of cells to either chemokine form for 2 h. However, after a 48-h incubation, the surface expression of CXCR4 is much lower for cells treated with Met-SDF-1beta. The enhanced blocking of T-tropic HIV-1 by Met-SDF-1beta appears to be related to prolonged CXCR4 down-modulation.

CTL generation in the presence of IL-4 is inhibited by free p40: evidence for early and late IL-12 function.

The cytokine requirements for the generation of a CTL response were examined using thymocyte responders stimulated with Con A. In agreement with previous reports, IL-4 alone can generate a strong CTL response. To determine whether IL-12 was required for this response, we added either a neutralizing Ab against IL-12 or an antagonist of IL-12 function, p40. Both anti-IL-12 and p40 were found to inhibit the generation of CTL in the presence of IL-4. Consistent with these data, exogenous IL-12 was found to synergize with IL-4, particularly, when IL-4 was added at levels unable to generate a CTL response alone. This synergistic response was observed whether IL-12 was added at the beginning of culture along with IL-4 or after the first day to cultures initiated with IL-4. Likewise, the combination of IL-2 plus IL-12 evoked a strong synergistic response when cultures were initiated with IL-2 and IL-12 was added initially or after the first day of culture. Addition of either IL-2 or IL-12 alone did not generate a response. In contrast to these data showing that IL-12 was able to signal late in culture, p40 inhibited CTL generation in response to IL-4 only when added at the beginning of culture. These results imply that CTL induction involves a crucial early IL-12 signal, which once delivered permits cells to respond to a later IL-12 signal. These data are consistent with the idea that free circulating p40 could inhibit the generation of CTL by antagonizing the early IL-12 "differentiation" signal.

Phenotypic and functional characteristics of in vivo-induced interleukin-12-activated killer cells.

A single i.p. administration of IL-12 (2000 U/mouse) into the mice caused the elevation of serum IFN-gamma activity and the generation of killer cells which can lyse various kinds of tumor cells including both NK-sensitive and -resistant tumor cells. Such in vivo induced killer cells were not detected in the mice treated with the same dose of IL-2. The generation of IL-12-activated killer cells (IL-12AK) peaked at day 1 and sustained their cytotoxicity until day 3 after IL-12 administration. The generation of IL-12AK was inhibited by in vivo administration of anti-asialo GM1 (ASGM1) Ab but not anti-CD4 or anti-CD8 mAbs, suggesting that the precursor cells for IL-12AK were ASGM1+CD4-CD8- NK cells. The phenotypic characterization of in vivo induced effector cells with IL-12AK activity was carried out by separating the cells with FACStar. The IL-12AK activity was highly enriched in ASGM1+CD4-8- or NK1.1+CD4-8- NK cells, but not in CD8+ T cells and CD4+ T cells. The IL-12AK cells were also generated in tumor-inoculated mice. In parallel with the in vivo generation of IL-12AK generation, the growth of i.p. inoculated MBL-2 lymphoma cells was markedly inhibited by the administration with IL-12. The in vivo antitumor activity of IL-12 was blocked by the administration of anti-ASGM1 but not anti-CD4 or anti-CD8 mAbs in concomitant with the decrease of IL-12AK generation. From these results, it was indicated that ASGM1+NK1.1+CD4-8- NK type IL-12AK cells might play an important role in IL-12-induced local therapy of tumor in vivo.

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-gamma production by anti-tumor T cells.

Unfractionated spleen cells taken from tumor-bearing mice 2 weeks after tumor implantation contained tumor-primed T cells which produced cytokines including IL-2 and IFN-gamma when cultured in vitro. With progressive tumor growth this initial lymphokine-producing capacity decreased. Here, we investigated the ability of IL-12 to (i) restore suppressed IFN-gamma production, (ii) cause tumor regression and (ii) induce anti-tumor protective immunity. Addition of rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearing mice resulted in a striking enhancement in the production of IFN-gamma compared with cultures of these cells in the absence of rIL-12 or of normal spleen cells in the presence of rIL-12. Five i.p. injections of rIL-12 into mice bearing s.c. tumors induced complete tumor regression. This was found when rIL-12 was given at early (1-2 weeks), intermediate (4-5 weeks) or even late (7 weeks) stages of tumor growth. Furthermore, IL-12-treated mice which rejected the primary tumor exhibited complete resistance to a rechallenge with the same tumor but did not reject a second syngenetic tumor. Immunohistochemical analyses following IL-12 treatment revealed that CD4+ and CD8+ T cells infiltrate the tumor. More importantly, IFN-gamma mRNA expression was observed in fresh tumor masses from tumor-bearing mice receiving IL-12 treatment. The importance of IFN-gamma was further demonstrated by the observation that the systemic administration of anti-IFN-gamma mAb prior to IL-12 treatment completely abrogated the anti-tumor effect of IL-12. Thus, these results indicate that administration of modest levels of rIL-12 to tumor-bearing mice results in tumor regression through mechanisms involving reversal of suppressed IFN-gamma production by anti-tumor T cells and the establishment of a tumor-specific protective immune response.

Evidence that rapamycin inhibits interleukin-12-induced proliferation of activated T lymphocytes.

Interleukin 12 is a heterodimeric cytokine involved in the regulation of natural killer cell and T lymphocyte responses. In previous studies, we found that IL-12 induces proliferation of T cells only after co-stimulation with lectin, alloantigen, or anti-CD3 antibody. The IL-2-mediated proliferation of long-term T cell lines generated in this fashion is typically insensitive to the immunosuppressive agent, cyclosporine but sensitive to rapamycin. In this study, we examined the effect of cyclosporine and rapamycin on T cells responsive to IL-12. For long-term cultured T cell lines stimulated with phytohemagglutinin, alloantigen, or solid-phase anti-CD3 antibody, rapamycin blocked IL-12-induced proliferation to background levels. Culture in cyclosporine produced minimal inhibition of IL-12-induced T cell proliferation. Freshly isolated CD3+ cells did not proliferate in response to IL-12, nor did culture of these cells in IL-12 lead to upregulation of IL-2 receptor. These data suggest that the effect of IL-12, an important growth regulator for activated T lymphocytes, may involve late cellular activation events.

Characterization of peptide binding to the murine MHC class I H-2Kk molecule. Sequencing of the bound peptides and direct binding of synthetic peptides to isolated class I molecules.

Peptides that are bound by the murine class I MHC molecule H-2Kk have been isolated and sequenced. The initial step in the fractionation was affinity column isolation of the peptide-class I complex from either RDM-4 or x5563 tumor cell lines. Acid denaturation of the complex followed by HPLC fractionation of the peptides allowed us to sequence individual peptides, as well as pools of peptides. To date, a total of 10 sequences have been characterized, and all were 8 mers. The sequences were variable except for glutamic acid in the second position (P2) and isoleucine in the eighth (P8), which were highly conserved. To further study peptide binding to H-2Kk, a competitive binding assay consisting of the immobilized histocompatibility protein and a biotinylated self-peptide for signal generation was developed. A complete set of single-alanine variants for this one self-peptide was tested in the assay, demonstrating that substitution at P2 and P8 markedly decreased the affinity for the class I molecule; alanine at position 3 had an intermediate effect on binding. A comparison of the identified self-peptides for binding to H-2Kk showed that they differed in affinity by more than one order of magnitude. Influenza virus nucleoprotein peptide, SDY EGR LI, associated with the plate-bound class I molecule, and the resulting MHC-peptide complex could trigger TNF release by influenza-primed CTLs. This result demonstrated the functional activity of the plate-bound H-2Kk-peptide complex.

IL-12 augments antigen-dependent proliferation of activated T lymphocytes.

Ag-dependent T cell activation requires multiple transmembrane signals including activation of Ag-specific T cell receptor in combination with signals delivered through cytokine receptors. IL-12 is a heterodimeric cytokine involved in the regulation of NK and T lymphocyte responses. In examining the role of IL-12 in T cell activation, we found a direct relationship between Ag stimulation and IL-12-induced proliferation. Unlike IL-2, which induced proliferation of CTL either in the presence or absence of a CD3/TCR co-signal, IL-12 mediated proliferation of CTL only when the cells were recently co-stimulated with alloantigen or solid-phase anti-CD3 antibody. After culture in the absence of alloantigen or anti-CD3 for 7 to 14 days, these CTL lost the ability to proliferate to IL-12 alone. Under these conditions, however, IL-12 synergized with low-dose IL-2 to induce CTL proliferation. Restimulation with alloantigen or solid-phase anti-CD3 restored the ability of the CTL to proliferate to IL-12 alone. Not all Ag signals resulted in IL-2 independent proliferation to IL-12. For example, CTL with specificity for influenza matrix peptide proliferated best when co-cultured with peptide Ag presented on self MHC and a combination of IL-2 and IL-12. This evidence suggests that IL-12 may be useful in expanding an Ag-specific T cell population, as the culture of CTL with IL-12 and low-dose IL-2 leads to proliferation only in response to an Ag co-signal.

Response of human natural killer (NK) cells to NK cell stimulatory factor (NKSF): cytolytic activity and proliferation of NK cells are differentially regulated by NKSF.

Natural killer cell stimulatory factor (NKSF) is a 70-kD heterodimeric cytokine that was initially isolated from conditioned medium of human B lymphoblastoid cell lines. The effects of recombinant NKSF on the function of human peripheral blood NK cells were examined. NKSF directly augmented the cytolytic activity of freshly isolated NK cells. Both CD56dim and CD56bright NK cells demonstrated enhanced cytotoxicity after brief exposure to NKSF. In contrast, highly purified T lymphocytes did not exhibit major histocompatibility complex-unrestricted cytotoxicity after short-term culture with NKSF. Like interleukin 2 (IL-2), NKSF augmented the lysis of NK-sensitive, NK-resistant, and antibody-coated targets. Both NKSF and IL-2 induced marked upregulation of several NK cell adhesion molecules known to participate in cytolysis, including CD2, CD11a, and CD54. However, NKSF activates NK cells through a pathway distinct from that of IL-2, since the presence of anti-IL-2 receptor (anti-IL-2R) antibodies or IL-4 did not inhibit the effects of NKSF. NKSF by itself induced very little proliferation of resting NK cells. NK cells preactivated in vitro with IL-2 demonstrated enhanced proliferation to NKSF, but the degree of proliferation was always inferior to that induced by IL-2 alone. Moreover, NKSF strongly inhibited IL-2-induced proliferation of either resting or preactivated NK cells. This inhibition was not the result of decreased IL-2R expression, because NKSF-activated NK cells expressed higher levels of both IL-2Rs p75 and p55. Furthermore, NKSF did not inhibit the proliferation of mitogen-activated T cells, indicating a selective effect on NK cell proliferation. Human NK cells expanded in vivo by prolonged continuous infusions of IL-2 remained fully responsive to NKSF. Picomolar concentrations of NKSF were as effective as nanomolar concentrations of IL-2 in augmenting the cytolytic activity of NK cells expanded in vivo by IL-2. NKSF may play an important role in the regulation of human NK cell function, and its possible use as a therapeutic cytokine deserves further investigation.

Approaches to immunotherapy of cancer: characterization of lymphokines as second signals for cytotoxic T-cell generation.

Lymphokines, the soluble molecules produced by cells of the immune system, regulate cell-cell interactions and, consequently, the functional status of the immune system. Altering immunoregulatory pathways with lymphokines in vivo may provide a mechanism for controlling a variety of immunologic disorders. Although normally produced in vivo in very small quantities, the widespread availability of recombinant lymphokines has made it possible to study the molecular signals involved in production of lymphocyte effectors with activity against tumor. For example, interleukin-2-based cancer immunotherapy programs have, in certain clinical situations, suggested that immunologic intervention can influence the regression of metastatic cancer. Ultimately the successful application of these biologic agents requires an understanding of the interaction between the immune system and tumor on a molecular level. To induce a given biologic effect, it is necessary both to classify the required lymphokines and to identify the relevant effector cell populations. This review will examine the progress made in identifying the requirements for lymphokine-induced cytotoxic T-lymphocyte function.

IL-4-supported induction of cytolytic T lymphocytes requires IL-2 and IL-6.

Previous work indicated that a CTL response can be generated by the combination of IL-2 plus IL-6 or IL-4 alone. Because of the ubiquitous production of IL-6 and its apparent ability to induce IL-2, we explored the interdependence of these lymphokines in supporting a CTL response from murine thymocytes. For thymocytes cultured in IL-4, further addition of IL-6 enhanced thymocyte proliferation. In addition, a role for IL-6 in thymocyte activation was indicated by the ability of anti-IL-6 mAb to block both IL-4-directed proliferation and the cytotoxic response found in the presence of IL-4. The addition of IL-2 to limiting doses of IL-4 augmented the CTL response; however, the response to high levels of IL-4 was not augmented by addition of IL-2. Consistent with this apparent involvement of IL-2 in the IL-4-mediated response we found: (a) that mAb to IL-2 significantly reduced the CTL response generated in the presence of IL-4; (b) that IL-2 activity was present in culture supernatant following incubation of thymocytes with high levels of IL-4; and (c) that enhanced IL-2 receptor expression found in the presence of IL-4 was blocked with the addition of anti-IL-2 antibody to the thymocyte culture. In contrast to the data for proliferation, anti-IL-4 mAb had no effect on the generation of CTL in the presence of IL-2 + IL-6 but readily blocked the CTL response to IL-4. These results indicate that, for thymocyte responders, the CD8+ CTL generated in the presence of IL-4 require both IL-2 and IL-6.

HIV-gp120 can block CD4-class II MHC-mediated adhesion.

A possible component of the immune dysfunction associated with infection by HIV is the inhibition of CD4 function resulting from the avid binding of soluble HIV envelope glycoprotein (gp120) to cell surface CD4. We assessed CD4 function by measuring the ability of CD4+ T cells to form conjugates with cell size lipid vesicles, artificial target cells (ATC), bearing the natural ligand for CD4, MHC class II proteins. Conjugate formation was a transient process with the greatest number of specific cell to ATC conjugates found after approximately 30 min of incubation at 37 degrees C. Addition of gp120 specifically blocked conjugates between CD4+ cells and class II ATC in a concentration-dependent manner. These data indicate that T lymphocyte adhesion mediated by CD4 is a dynamic event and that binding of gp120 to CD4 is able to disrupt the normal progression of the interaction between CD4+ T lymphocytes and class II+ APC.

Production of interleukin-6 in vitro parallels development of cytotoxic T lymphocytes from murine thymocytes.

In an attempt to provide information useful for improving tumor immunotherapy, we examined the lymphokine requirements for generation of cytotoxic T lymphocytes (CTL) from C57BL/6 murine thymocytes. Our previous work indicated that interleukin-6 (IL-6) is involved in the maturation of CTL in vitro. Using a standard chromium 51 release assay and P815 mastocytoma tumor cells as targets, we found that after 66 hours of in vitro culture, a much greater CTL response was generated in the presence of interleukin-2 (IL-2) plus IL-6 (70.5% +/- 10.6%) compared with that generated in the presence of IL-2 only (25.2% +/- 1.0%). After 72 hours of culture, however, this difference was no longer significant, with cultures incubated with both IL-2 and IL-6 yielding 70.6% +/- 1.8% lysis versus 64.5% +/- 3.4% for cultures incubated with IL-2 only. To attempt to understand this difference, we examined the production of IL-6 in thymocyte cultures using a cell line, PC-6, that proliferates in the presence of IL-6. We found that the CTL response generated from unfractionated murine thymocytes in the presence of concanavalin A plus IL-2 correlated with production of IL-6 by cells within the thymic population. These data suggest that the generation of a CTL response in the absence of added IL-6 is due to the production of this ubiquitous lymphokine by thymocytes on in vitro culture. We present this as further evidence that IL-6 is necessary for the development of functional CTL from murine thymocytes and may therefore play a role in the development of effective tumor immunotherapy.

Temperature requirements and kinetics of T cell signaling. Velocity of triggering correlates with functional effects of anti-CD3 mAb.

Antibody reactive with the CD3 complex on the surface of T lymphocytes can either: inhibit CTL lysis of target cells expressing Ag; or redirect CTL to lyse target cells expressing FcR in the absence of Ag expression. To investigate these phenomena we examined the effect of anti-CD3 mAb on two indicators of CTL activation, the release of esterase and target cell lysis. Esterase release by long term allo-reactive human CTL in response to target cells (JY or HLA transfected K562 cells) was found to be Ag specific and correlate with target cell lysis. Addition of anti-CD3 to either JY targets or K562 cells expressing FcR resulted in a high level of esterase release. Triggering of esterase release was found with both soluble intact and Fab fragment of anti-CD3 in the absence of cells expressing measurable FcR. This apparent FcR-independent triggering of esterase release occurred at 37 degrees C but not at 24 degrees C. In contrast esterase activity was released from CTL at both 24 and 37 degrees C in response to intact target cells, JY or K562 cells plus intact anti-CD3 mAb. Addition of anti-CD3 mAb, at a level capable of blocking target cell lysis by greater than 50%, resulted in an initial velocity of esterase release almost twice that found in response to JY target cells. With a low level of anti-CD3 mAb, able to block JY lysis by approximately 10%, the initial rate of esterase release was much slower than that found in response to target cells. In contrast when FcR+ cells, K562, were added along with a low level of anti-CD3 the initial velocity of esterase release was about twofold more than the velocity of esterase release triggered by soluble anti-CD3 alone. These results indicate that soluble antibody can trigger long term active CTL and the velocity of this triggering correlates with anti-CD3-mediated inhibition as well as redirected lysis.

A monoclonal antibody to LFA-3, the CD2 ligand, specifically immobilizes major histocompatibility complex proteins.

T cells are activated when the antigen-specific T cell receptor recognizes antigen in association with major histocompatibility complex (MHC) proteins. The T cell surface protein CD2 (T11, LFA-2, the T erythrocyte receptor) and its target or stimulator cell ligand, lymphocyte function-associated antigen-3 (LFA-3), are also involved in T cell adhesion and activation. The molecular mechanisms by which the CD2/LFA-3 interaction affects T cell adhesion and activation are unclear. The CD2/LFA-3 interaction may be modeled by the interaction between LFA-3 and anti-LFA-3 monoclonal antibody (mAb). We used the fluorescence photobleaching recovery technique to investigate the effect of anti-LFA-3 mAb on the lateral mobility of MHC proteins in plasma membranes of JY, a human Epstein-Barr virus-transformed B cell line. Anti-LFA-3 mAb induced immobilization of class I MHC proteins labeled with bivalent but not monovalent fluorescein-conjugated W6/32 mAb. Anti-LFA-3 mAb also caused immobilization of class II MHC proteins labeled with bivalent fluoresceinated LB3.1 mAb. In contrast, anti-LFA-3 mAb did not affect the mobilities of either a B cell membrane protein labeled with bivalent fluoresceinated anti-CD45 (human leukocyte antigen) mAb or a membrane lipid analogue. Unlike anti-LFA-3 mAb, anti-LFA-1 mAb did not affect class I MHC protein mobility. These results suggest that CD2 binding to LFA-3 may trigger a physiological response in which target cell MHC proteins, cross-linked by receptors on the T cell surface, are immobilized at and thereby localized to the T cell-target cell interface.

Inhibition of lymphokine-activated killer cell-mediated cytotoxicity by phorbol ester.

As previously reported, the culture of mouse spleen cells in the presence of high amounts of human rIL-2 for 4 days caused proliferation and generation of lymphokine-activated killer (LAK) cells, which could lyse a variety of tumor cells. However, an addition of PMA to the culture resulted in a striking inhibition of the generation of LAK cells. In contrast, IL-2-induced cell proliferation, IL-2R expression, and LFA-1 expression were enhanced by the addition of PMA. Kinetic studies revealed that the addition of PMA during the final 24 h, but not 4 h, of the culture was sufficient to inhibit the generation of LAK cells. The same inhibition of LAK activity was observed when 4-day cultured LAK cells were pretreated with PMA for over 12 h before cytotoxicity assay. Flow cytometry analysis showed that PMA pretreatment had no effect on the binding of LAK cells to target cells. PMA pretreatment of LAK cells caused total disappearance of protein kinase C (PKC) activity from LAK cells concomitant with the loss of LAK activity. However, PMA-pretreated LAK cells cultured for another 24 h in the absence of PMA revealed levels of PKC activity and cytotoxicity identical with untreated LAK cells. These results strongly suggest that PMA-induced down-regulation of LAK cell-mediated cytotoxicity is due to the inactivation of PKC-dependent transduction systems that are essential post LAK cell-target cell binding.