Enhancement of selective tumor cell binding by activated murine macrophages in response to phorbol myristate acetate

The fundamental biology of how stable cell-cell bonds develop between activated macrophages and tumor cells, although essential to lysis of the neoplastic targets, remains poorly understood. To investigate whether this phenomenon could be pharmacologically manipulated, we analyzed the effect of phorbol diesters on tumor cell binding by macrophages. Activated murine peritoneal macrophages, treated in vitro with as little as 1 ng/ml of phorbol myristate acetate (PMA), bound significantly more tumor cells than did untreated macrophages. The effect was induced rapidly by PMA (i.e., maximum enhancement was seen within 15 min) and resulted in an average approximately twofold increase in the number of targets bound. The interaction between PMA-treated activated macrophages and tumor cells was completed much more rapidly than by untreated macrophages. The enhanced binding was seen only in macrophages treated with biologically active phorbol esters. Only the selective interaction between activated macrophages and tumor cells was affected (i.e., PMA treatment had no effect on nonselective interactions between activated macrophages and non-neoplastic targets or between nonactivated macrophages and any type of target). Pretreatment of activated macrophages with PMA apparently altered the requirements for microfilaments and microtubules in establishing binding, because cytochalasin B and colchicine, which inhibited control binding, as well as phagocytosis, had no effect on PMA-enhanced binding. PMA treatment did not alter energy requirements for binding, however, because low temperature (4 degrees C) or inhibitors of glycolysis and oxidative phosphorylation blocked both control and PMA-enhanced binding. The enhancement of binding apparently was not due to large quantities of secreted oxygen metabolites but did correlate closely with increased spreading and surface area of the macrophages. PMA treatment resulted in enhanced expression of trypsin-sensitive tumor-cell binding sites on the macrophage surface. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of macrophage membrane proteins labeled with 125I by the lactoperoxidase method revealed at least four trypsin-sensitive cell surface proteins that were re-expressed after PMA treatment. The data suggest that rearrangement and/or induced expression of surface binding sites may be an important step in the binding of tumor cells and indicate that PMA is a useful pharmacologic probe in dissecting the establishment of such binding into discrete steps.     

A microassay for the rapid and selective binding of cells from solid tumors to mouse macrophages

Summary A microassay was developed to study the rapid binding characteristics of murine macrophages activated by gamma interferon and muramyl dipeptide to adherent neoplastic or nonneoplastic target cells. The binding of tumor cells to both activated and nonactivated macrophages was time- and temperature-dependent, and independent of tumor cell type. Activated macrophages bound more tumor cells than nonactivated macrophages. The initial binding of macrophages to target cells did not necessarily lead to lysis. First, primed macrophages bound tumor cells but did not lyse them, and second, nonactivated macrophages bound nontumorigenic cells without subsequent lysis. The rapid binding assay described here could prove useful in investigating the recognition mechanism(s) between macrophages and tumor cells derived from solid primary and metastatic cancers.     

Macrophage binding of cells resistant and sensitive to contact-dependent cytotoxicity

We compared macrophage binding and killing of F5b cells to the binding and killing of P815 mastocytoma cells and to several other nontransformed and transformed cell lines. Formalin fixation of elicited or activated macrophages did not affect binding of F5b or 3T3 cells but did abrogate binding of P815 cells. However, formalin fixation abrogated resident macrophage binding of F5b and 3T3 cells. Therefore, depending on the type of macrophage or target cell, formalin fixation may affect binding. Only the binding of P815 cells was dependent upon activation; macrophage binding of target cells F5b and 3T3 was not. Even though macrophages bound F5b and 3T3 cells, macrophages only mediated contact-dependent cytotoxicity against F5b cells. Macrophages did not kill 3T3 cells. Experiments also compared macrophage binding and killing of the uv-light-induced tumor cell lines 1422, 2237, and 2237a46. Only the cell line 2237a46 was susceptible to contact-dependent killing. Both 1422 and 2237 cells were resistant. In contrast, cell lines 2237a46 and 1422 were bound by activated macrophages while 2237 cells were bound poorly.     

Increased binding of tumor cells by macrophages activated in vitro with lymphocyte mediators.

Following incubation in vitro with lymphocyte mediators, activated macrophages become capable of binding more tumor cells than nonactivated macrophages. Increased binding occurs rapidly (within 1 hr), does not require the presence of serum in the medium, and is inhibited by treatment with trypsin. The increased binding by activated macrophages is quantitatively selective for tumor cells. Incubation with lymphocyte mediators of cell types other than macrophages does not increase the binding of tumor cells to such monolayers. These results indicate that the binding of tumor cells by activated macrophages results from the stimulation of a specific macrophage function during the process of macrophage activation.     

Conditions controlling tumor cytotoxicity of rat liver macrophages mediated by liposomal muramyl dipeptide

Activation of rat liver macrophages with free and liposome-encapsulated muramyl dipeptide (MDP) to a tumorcytotoxic state was characterized by employing various experimental conditions. Macrophage-mediated tumor cytotoxicity was determined using two standard assay systems: a [methyl-3H]thymidine release assay to measure the extent of tumor cell lysis and a [methyl-3H]thymidine incorporation assay to measure the combined effects of tumor cell lysis and stasis. The extent of cell lysis was not affected by the ratio of macrophages to tumor cells within the ratio range of 30:1 to 5:1, provided that the macrophages form a confluent monolayer. Tumor cell lysis, however, was significantly influenced by macrophage density; a low macrophage density for example resulted in a low percentage of tumor cell lysis. Tumor target cells used in this study, i.e., C26 adenocarcinoma, B16 melanoma and P815 mastocytoma, differed in their susceptibility towards macrophage-mediated cell lysis, whereas no differences were observed with respect to tumor cell stasis. Non-tumorigenic cell lines such as human fibroblastic cells and LLC monkey kidney cells were not lysed by activated macrophages, although proliferation of these cells was markedly inhibited. Additionally, the effects of liposomal lipid composition on macrophage activation were studied. With a basic composition of phospholipid/cholesterol/dicetylphosphate, we used either egg-yolk, dipalmitoyl-, distearoyl- or dihexadecylphosphatidylcholine as the bulk phospholipid constituent. Although these liposomes display a widely different susceptibility to lysosomal phospholipase activities, we could not detect any significant difference in either the extent or the duration of the tumoricidal activity induced by MDP encapsulated in these different types of liposomes.     

Binding of activated macrophages to tumor cells through a macrophage lectin and its role in macrophage tumoricidal activity

A 45-60 kDa Gal/GalNAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macrophages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/GalNAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.     

The cytolytic function of mononuclear phagocytes. II. Factors that determine the binding and lysis of tumor cells by activated macrophages

The effects of various modifiers upon the interaction of LPS- and BCG-activated macrophages with cells of mastocytoma P815 have been investigated. The efficiency of binding and lysis of the tumor cells is to a great extent determined by activation of the effector-cells, expression of the trypsin-sensitive receptors on the surface of macrophages, and by the type of target-cells. Introduction into the analytical system (effector-target) of unlabeled tumor cells or membrane preparations obtained from them inhibits substantially both binding and lytic activity of cytotoxic macrophages. If nontransformed cells or their membranes are applied, no significant changes in the investigated processes can be detected. Trypsinization of tumor cells as well as of activated but not resident macrophages modifies considerably the interaction of effectors with targets. The quantity of tumor cells bound with macrophages does not depend on the fact, which of the partners is subject to trypsinization, but it is much less than that of target-cells bound in the control. The incubation of activated macrophages with actinomycin D results in a substantial suppression of their lytic activity, whereas treatment of tumor cells with this inhibitor of protein synthesis leads to a considerable decrease in stability of the targets against lytic activity of the factor activated by effectors. The obtained data reveal the ways of selective binding and effective lysis of transformed targets by activated macrophages.     

Evidence that a target-derived soluble factor is necessary for the selective lysis of SV40-transformed fibroblasts by activated mouse macrophages

Our laboratory is investigating the basis for the selective recognition of transformed cells by activated mouse macrophages. As targets we are using a panel of SV40-transformed, C3H.OL fibroblast cell lines (SV-COL) that display widely different levels of sensitivity to lysis, from highly sensitive to completely resistant. Our results show that adding conditioned medium from the macrophage-sensitive target SV-COL-E8 (CM(E8] to a cytolysis assay with the macrophage-resistant target SV-COL-F5f causes the macrophages to kill the resistant targets in a contact dependent fashion. We have termed this activity "macrophage cell lysis factor" (MCLF). MCLF activity was not detected in conditioned media from cells not killed by activated macrophages (i.e., 3T3-like cell lines or embryo fibroblasts) but was present in conditioned media from six other SV-COL cell lines at levels that were directly proportional to the sensitivity of those targets (r = 0.98). These data suggest that MCLF plays a key role in determining the lytic sensitivity of SV40-transformed fibroblasts. Finally, to ask whether the production of MCLF is sufficient to explain the selective recognition of SV40-transformed fibroblasts by activated macrophages we have tested whether CM(E8) will cause macrophages to kill normal cells. Our results show that in the presence of CM(E8) macrophages will kill immortalized, 3T3-like fibroblasts but will not kill normal embryo fibroblasts. These results suggest that the production of MCLF, or a similar activity, is necessary but not sufficient for macrophage cytolysis to occur and that a change in target cell phenotype that occurs during the process of immortalization is also required.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. III. Inhibition by cytochalasins, colchicine, and vinblastine.

The effect of cytochalasin A and B, colchicine and vinblastine on tumor cell killing by macrophages activated in vitro with lymphocyte mediators was examined. Both cytochalasins reversibly inhibited the killing of tumor cells by activated macrophages. Kinetic studies with cytochalasin B suggested that this drug exerts its effect on an early step of the cytotoxic process. Additional studies revealed that the drug inhibited the binding of tumor cells by activated macrophages.Colchicine inhibited both the binding and the killing of tumor cells by activated macrophages, whereas its structural analogue, lumicolchicine, had no effect on either macrophage function.Vinblastine also inhibited the binding and killing of tumor cells. However, this drug no longer inhibited tumor cell binding at low concentrations (<10^?6M) that still inhibited tumor cell killing. Further, vinblastine inhibited tumor cell killing when added late to an ongoing cytolytic reaction.These results suggest that the cytochalasins, colchicine and vinblastine inhibit macrophage mediated cytotoxicity by preventing intimate contact between the effector macrophages and their targets. In addition, vinblastine also appears to inhibit a later step of the cytolytic process, possibly the secretion of a cytotoxic macrophage product.     

Quantification of the strength of cell-cell adhesion: the capture of tumor cells by activated murine macrophages proceeds through two distinct stages

The binding of tumor cells by macrophages activated with Bacillus Calmette-Guerin is a necessary step toward destruction of those cells. Although several characteristics of the interaction have been defined, little is known of how the actual binding process develops. We used a technique to quantify the forces required to disrupt cell-cell interactions. Over a range of applied relative centrifugal forces, the majority of targets that bound to the activated macrophages fell on two distinct plateaus. Approximately 90% of added targets were bound to the monolayers of macrophages over the range of 1 to 100 X G; 25 to 30% remained bound from 1200 X G to 1500 X G. Two strengths of binding, termed weak and strong binding, respectively, were thus defined on the basis of these curves. Strong binding developed only between activated macrophages and tumor cells. By contrast, weak interactions occurred between either activated or nonactivated macrophages and neoplastic or non-neoplastic target cells. The strong binding required time (60 to 90 min), metabolic activity by the macrophages, and trypsin-sensitive surface structures on the macrophages for development, whereas the weak interaction occurred rapidly and required none of these. Additional evidence indicated the weak binding developed into strong when activated macrophages bound neoplastic cells. This stabilization increased the strength of force to separate tumor cells from the macrophages at least approximately 15 fold (i.e., from approximately 16 mu dynes/cell to approximately 240 mu dynes/cell). Of note, the development of strong binding of antibody-coated targets had distinct requirements for establishment. Taken together, the data suggest the stabilization of binding (i.e., the development of weak into strong binding) leading to effective cell-cell interaction is a complex and dynamic process that may vary depending upon the recognition system involved.     

Mechanisms of tumor cell capture by activated macrophages: evidence for involvement of lymphocyte function-associated (LFA)-1 antigen

The lymphocyte function-associated (LFA)-1 molecule is expressed on certain populations of macrophages that have an augmented capacity to capture tumor cells. Accordingly, we analyzed the role of LFA-1 in the establishment of such cell-cell interactions. F(ab')2 fragments of the M17/4, anti-LFA-1 monoclonal antibody (MAb) inhibited the interaction between activated macrophages and tumor cells by up to 80% in a dose-dependent manner. The anti-LFA-1 MAb reduced (between 55 to 79%) the number of P815, LSTRA, or EL-4 tumor cells bound to trypsin-sensitive structures on bacillus Calmette Guerin activated macrophages. The inhibition appeared selective, because a F(ab')2 fragment of anti-Mac-1 did not inhibit such binding. Inhibition of tumor cell capture could be observed as soon as 15 min after the onset of the cell-cell interaction between activated macrophages and tumor cells. Optimal inhibition occurred when both tumor targets and macrophages were precoated with the MAb. Although P815, LSTRA, EL-4, and BW5147 tumor cells all expressed LFA-1, only the first three but not BW5147 cells were bound by activated macrophages. Furthermore, endotoxin-pulsed macrophages elicited by thioglycollate broth expressed the LFA-1 antigen but did not exhibit selective tumor cell capture. Finally, anti-LFA-1 inhibited the development of weak into strong binding. Taken together, the results suggest that LFA-1 molecules can participate in the interaction between activated macrophages and neoplastic cells.     

Antibody-dependent cytolysis (ADCC) of tumor cells by activated murine macrophages is a two-step process: Quantification of target binding and subsequent target lysis

To analyze the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction between tumor cells and activated murine macrophages in detail, it must be first determined if physical binding occurred between the two cell types. Over 15–20 min in vitro, antibody-coated HSB neoplastic targets became so firmly attached to the activated macrophages that they resisted removal with 4 vigorous washes. When a quantitative assay of binding was employed, attachment of tumor cells to activated macrophages was found to depend on the concentration of antibody and on the density of the macrophages. These two variables also determined the subsequent extent of cytolysis. Binding of antibody-coated targets by macrophages elicited with thioglycollate broth or activated by bacillus Calmette-Guerin (BCG) was comparable. Lysis by the activated macrophages, however, was far greater. Binding occurred at 4, 22, or 37 °C, while the subsequent lytic reaction occurred only at 37 °C. Thioglycollate broth effectively inhibited lysis but had no effect on binding. A porous filter placed between activated macrophages and targets resulted in abrogation of binding and lysis, even when antibody-coated targets were placed beneath the filters. When labeled, uncoated targets were added to cultures of macrophages in the presence of unlabeled antibody-coated targets, no lysis of the bystander (i.e., uncoated) targets was seen. The data suggest that ADCC is a multistep reaction, that vigorous physical binding of antibody-coated targets by activated macrophages is an initial and necessary step in ADCC, that such binding is not sufficient for ADCC, that such binding controls the selectivity of lysis in ADCC, and that the second step in ADCC results in target lysis.     

The effects of experimental conditions on the expression of in vitro-mediated tumor cytotoxicity mediated by murine macrophages

Summary The level of target destruction by tumoricidal mouse peritoneal macrophages was studied under various culture conditions. The degree of macrophage homogeneity in the monolayer was affected by the presence of fetal calf serum in the plating medium and the time allowed for cell adherence. Macrophage tumoricidal activity was not dependent on the ratio of macrophages to target cells, but on the densities of the two cell types in the culture vessel. The development of an in vitro microcytotoxicity assay for the evaluation of tumor cell lysis by activated macrophages revealed that serum concentration and the type of target cell used in the assay may affect the degree of tumoricidal response.     

Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages

Different macrophage populations were investigated for their abilities to secrete tumor necrosis factor (TNF) and to lyse TNF-susceptible tumor cells. In this way we could demonstrate that TNF-secretion, although a feature of all activated macrophage populations, is no absolute requirement for the killing of the TNF-sensitive Wehi 164 target. Macrophage cytotoxicity against this cell but not against the TNF-resistant P815 mastocytoma, was completely inhibitable by a specific anti-TNF serum also in the absence of measurable secreted TNF. Moreover the TNF-dependent lysis of tumor cells could also be performed by activated macrophages that had been fixed with paraformaldehyde before the addition of the target cells. In the indirect radioimmunoassay, TNF could be demonstrated on the surface of fixed effector cells. Our results must be interpreted in terms of membrane-associated TNF as the lytic principle for TNF-susceptible tumor cells.     

Interferon-gamma is required in activation of macrophages for tumor cytotoxicity

The participation of interferon-gamma in activation of murine macrophages for tumor cell lysis was investigated. Biochemically macrophage activation factor and interferon-gamma have not been separated. Antiviral titers correlated closely with macrophage activation in antigen- or mitogen-induced spleen cell supernatants. A monoclonal rat antibody that neutralized virus-induced interferon was also found to neutralize interferon-gamma in such supernatants. These monoclonal antibodies were coupled to CH-Sepharose 4B and used for absorption of antiviral activity from mitogen-induced spleen cell supernatants. Absorption of the interferon was paralleled by the reduction of the macrophage-activating capacity of the supernatants. Data from control absorptions supported the specificity of the absorption effect. These results indicate that interferon-gamma is required for activation of macrophages for tumor cell lysis. These results can be interpreted in two ways: (a) the monoclonal antibodies cross-react with interferon-gamma and with a mediator that is required for activation of macrophages for tumor cell lysis or (b) interferon-gamma itself is an essential cofactor for macrophage activation.     

Cytolytic activities of activated macrophages versus paraformaldehyde-fixed macrophages; soluble versus membrane-associated TNF.

Membrane-associated tumor necrosis factor (TNF) and soluble TNF were compared as to their lytic activities, and as to the kinetics of their expression by macrophages activated with LPS and/or IFN-gamma in the presence or absence of cycloheximide. EL 4 tumor cells, resistant and sensitive to lysis by recombinant TNF or membrane-associated TNF (paraformaldehyde (PF)-fixed activated macrophages) were used as targets. In the presence of cycloheximide the TNF-resistant S-EL4 cells were lysed by both TNFs. PF-fixed macrophages was cytolytic after 1 hr activation but not after 3 or more hours of activation. Their activity was totally inhibited by anti-TNF antibodies and was a composite of transmembrane (integral) TNF and soluble TNF conjugated to macrophage membrane TNF receptors. Treatment of the macrophages with glycine pH 3.0 buffer dissociated the conjugated TNF without affecting the integral membrane TNF. When macrophages were activated with LPS +/- IFN-gamma in the presence of cycloheximide or activated just with IFN-gamma their activity after fixation with paraformaldehyde was no longer detected. Nonfixed macrophages under these conditions still remained cytotoxic. Tumor cell susceptibility to membrane-associated TNF activity, in contrast to recombinant (soluble) TNF, was greatly reduced in the presence of nicotinamide, an inhibitor of ADP-ribosyltransferase, suggesting that the mechanisms of lysis by these TNFs may be different. The lytic activity of both TNFs was found to be receptor-dependent in that tumor cells, whose TNF binding sites were "down-regulated" by TPA, were rendered resistant to lysis by both membrane-associated and soluble TNFs.     

L-arginine independent macrophage tumor cytotoxicity

We have investigated the role of L-arginine in macrophage tumor cytotoxicity in coculture. L929, EMT-6, MCA-26, and P815 targets were all susceptible to cytolysis by activated macrophages when cocultured in medium containing L-arginine. When cocultured in arginine-free medium, these targets displayed comparable or even higher levels of lysis. L1210 targets were lytically resistant under either condition. However, 59Fe release from this target did reflect strong dependence on the presence of arginine. The structural analogue, NG-monomethyl-L-arginine, was an effective inhibitor of iron-release from L1210 targets cocultured with activated macrophages, whereas it had minimal inhibitory effects on release of 51Cr from cocultured L929 cells. These results suggest that the L-arginine requiring cytotoxic pathway of activated macrophage is independent of major effector mechanisms involved in tumor cell lysis.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

The interaction of Naegleria fowleri amoebae with murine macrophage cell lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D1, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowleri bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

High strength binding of P815 mastocytoma cells is not necessary for their lysis by macrophages which have been primed and triggered in vitro

We have examined the hypothesis that binding of P815 mastocytoma cells is a necessary step in lysis of these cells by macrophages which are both "primed" and "triggered" in vitro, Macrophages "primed" by conditioned media containing IFN-gamma, or by rIFN-gamma have an increased ability to bind P815. However, adding either heat-killed Listeria or endotoxin to trigger the primed macrophages has opposite effects on lysis and binding of P815. Lysis is increased. Binding is dramatically decreased. This is true when centrifugal forces of 200 x g, 400 x g, and 800 x g are used to disrupt P815-macrophage binding. Although 100% of P815 cells bound by cytotoxic macrophages are lysed, a large additional population of unbound P815 is also lysed. Detailed kinetic studies indicate that macrophages do not rapidly bind and lyse several cycles of P815. There is an initial lag period of 4 to 6 h before P815 lysis can be detected, and completion of lytic events then occurs within 12 to 14 h. Lysis of P815 bound to cytotoxic macrophages is slightly slower than lysis of the total population of bound and unbound P815. In contrast, D3.1, a cloned CD4+ T cell line, is tightly bound to macrophages but not lysed efficiently. When macrophages are simultaneously confronted with P815 and macrophage-bound D3.1, only the former are lysed. Altogether, the data indicate that P815-macrophage binding, as operationally defined by our assay, is not a necessary step for lysis. These results, by use of macrophages primed and triggered in vitro, are in contrast to previously reported experiments examining P815 binding and lysis by macrophages activated in vivo by infection with bacillus Calmette-Guérin.