Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity

This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.     

Augmentation of human natural cell-mediated cytotoxicity by recombinant human interleukin 2

Human peripheral blood mononuclear cells (PBMC) demonstrated increased natural cell-mediated cytotoxicity (NCMC) activity after only 5 min of exposure to purified recombinant human IL 2 or interferon (IFN)-gamma. The mechanism of NCMC augmentation by treatment with IL 2 is not entirely dependent on IFN-gamma production because: a) IL 2 was found to augment NCMC activity at levels which did not induce detectable IFN-gamma; b) IL 2 required only 5 min of exposure to PBMC to augment NCMC activity, whereas 3 hr of contact were required to demonstrate detectable IFN-gamma levels; c) the levels of NCMC enhancement by treatment with IL 2 exceeded the amount of NCMC enhancement that could be due to IFN alone; d) anti-recombinant IFN-gamma, which totally eliminated the augmentation of NCMC enhancement by IFN-gamma, only partially reduced the augmentation of NCMC activity by IL 2; and e) combination treatment of PBMC with IL 2 and IFN-gamma resulted in a synergistic enhancement of NCMC. The results strongly support the conclusion that augmentation of NCMC by IL 2 and IFN-gamma involve overlapping mechanisms.     

Augmentation of NKT and NK cell-mediated cytotoxicity by peptidoglycan monomer linked with zinc

BACKGROUND: Peptidoglycan monomer (PGM), which was originally prepared by biosynthesis from culture fluids of penicillin-treated Brevibacterium divaricatum, is an immunostimulator, the activities of which might be improved by addition of zinc (Zn) to the basic molecule. METHODS: To test the possible cytotoxic effects of this new analogue, we analyzed the ability of PGM-Zn and PGM to change the phenotypic profile of hepatic and splenic mononuclear lymphatic cells and to affect the growth of malignant T-cell line YAC-1 and syngeneic thymocytes. RESULTS: Pretreatment of C57BL/6 mice primarily with PGM-Zn over 6 days (10/mg/kg intraperitoneally) significantly enhanced the proportions of NK1.1high+, CD4-CD8-, CD69+, and CD3intermediate/NK1.1+/IL2R-beta+ (NKT) cells in the liver, and major histocompatibility complex class II+, CD69+, and CD8+ cells in the spleen. Both types of cells were highly cytotoxic against YAC-1 and syngeneic thymocytes, increasing the destruction of YAC-1 by 70% on addition of hepatic cells and by 30% on addition of splenic cells. Destruction of thymocytes increased by 10 and 50%, respectively. CONCLUSION: The results point to PGM-Zn as a potent cytotoxicity-inducing agent, which also generates autoreactive NKT cells.     

Augmentation of the generation of lymphokine-activated killer cells after a single dose of mitomycin C in cancer patients

Summary The effect of mitomycin C administration on the generation of cytotoxic cells, induced by in vitro activation of peripheral blood mononuclear cells (PBM) with interleukin-2, was studied in patients with various carcinomas. The ability of PBM to generate lymphokine-activated killer (LAK) cell activity against Raji cell targets was significantly augmented 5 and 7 days after a single intravenous dose of 12 mg/m² mitomycin C, when compared to that of PBM obtained before mitomycin C injection. Further, LAK cell activity against autologous tumor cells was also significantly increased after the drug administration. The distribution of lymphocyte subsets exhibited a significant increase in the percentage of CD3⁺ cells after injection, with the elevation of the CD4/CD8 ratio. Furthermore, the proportion of the CD4⁺ Leu8⁺ subpopulation, which identifies inducers of suppression, was significantly reduced. Thus, the decrease in the proportion of suppressor-inducer subsets of PBM might be at least partially, responsible for the augmented generation of LAK cells after mitomycin C administration.     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Potentiation of antiboty-dependent cell-mediated cytotoxicity by target cell-bound C3b.

Antibody-dependent cytolytic effector lymphocytes are known to possess, in part, receptors for activated C3. Employing a model system consisting of 51Cr-labeled chicken erythrocytes and purified human peripheral lymphocytes, we investigated the effect of target cell bound C3b on antibody-dependent cellular cytotoxicity (ADCC). At concentrations of anti-target cell antibody too low to cause effective ADCC, target cell bound C3b cooperated with antibody to produce marked target cell lysis. In the presence of a 1/6.25 X 10(6) dilution of anti-chicken erythrocyte rabbit IgG, cell lysis increased from 20% to 65% by the attachment of 18,000 C3b molecules per cell. C3b-dependent enhancement of ADCC was dose dependent. It was augmented by attachment of activated properdin (P) to the C3b-bearing target cells.     

Effect of Vibrio cholerae neuraminidase on the generation of cell-mediated cytotoxicity in vitro.

Vibrio cholerae neuraminidase (VCN))12.5 units/2 X 10(6) cells/ml) continuously present for a standard 5-day MLC will significant (p less than 0.02) increase the cytotoxic activity generated by a given number of responding spleen cells without reducing the specificity. Heat-inactiviated VCN produced no such augmentation. This augmented cytotoxicity could be reproduced by preincubating (1 hr) the responding spleen cells with VCN (25 units/2.5 X 10(6) cells/ml) before addition of stimulating spleen cells. Preincubating the stimulator spleen cells with VCN had no effect. VCN preincubation of target cells or presensitized effector cells produced no augmentation. The addition of soluble VCN to the killing assay also did not increase cytotoxicity. Thus, VCN acts only during the generation of specifically sensitized cytotoxic T cells. When the effect of VCN on MLC reactivity, cell recovery and total cytotoxicity (lytic units/10(6) cells) were compared, it became apparent that VCN increases the proliferation of responder cells after stimulation resulting in both an increased number of cells and also an increase in the proportion of specifically sensitized cytotoxic cells in the culture. VCN treatment of responder cell membrane apparently permits a more ready response to allogenic antigens in culture facilitating both increased proliferation and the increased development of specific cytotoxic killers.     

DNA damage, cytotoxicity and free radical formation by mitomycin C in human cells

Mitomycin C (MMC), a quinone-containing antitumor drug, has been shown to alkylate DNA and to form DNA cross-links. The ability of MMC to alkylate O6-guanine and to form interstrand cross-links (ISC) has been studied using Mer+ and Mer- human embryonic cells. Mer+ (IMR-90) cells have been reported to contain an O6-alkylguanine transferase enzyme and are, in general, more resistant to alkylating agents than the Mer- (VA-13) cell line, which is deficient in the repair of O6-lesions in DNA. Studies reported here show that MMC is more cytotoxic to VA-13 cells compared to IMR-90 cells. The alkaline elution technique was used to quantify MMC-induced ISC, and double strand breaks (DSB) in these cells. The drug-dependent formation of DSB was significantly lower in IMR-90 cells than in VA-13 cells. In contrast, no significant difference in cross-linking could be detected at the end of 2-h drug treatment. Although a small increase in cross-link frequency was observed in the VA-13 cell line relative to the IMR-90 cell line 6 h post drug treatment, it is not clear whether monoalkylated adducts at the O6-position are formed, and contribute to cross-link formation for differential cytotoxicity in VA-13 cells. Electron spin resonance and spin-trapping technique were used to detect the formation of hydroxyl radical from MMC-treated cells. Our studies show that MMC significantly stimulated the formation of hydroxyl radical in VA-13 cells, but not in the IMR-90 cells. The formation of the hydroxyl radical was inhibited by superoxide dismutase (SOD) and catalase. In addition, the presence of these enzymes partially protected VA-13 cells from MMC toxicity but not IMR-90 cells. Further studies indicated that the decreased free radical formation and resistance to MMC may be due to the increased activities of catalase and glutathione transferase in the IMR-90 cell line. These results suggest that MMC-dependent DNA damage (alkylation and DNA DSB) and the stimulation of oxy-radical formation may play critical roles in the determination of MMC-induced cell killing.     

Inhibition of cell-mediated cytotoxicity by Corynebacterium parvum

Summary We investigated the effect of altering dose and route of Corynebacterium parvum (C. parvum) administration on the adjuvant's inhibition of cell-mediated cytotoxicity (CMC). Primary in vivo and secondary in vitro CMC of C57B1/6 mice alloimmunized to P815 were depressed if C. parvum was administered systemically (IV or IP) but not when it was given SC. Similarly, only systemic C. parvum generated cells capable of suppressing in vitro CMC. Primary and secondary CMC in spleen was equally inhibited by 700 and 70 g, whereas suppressor cell activity was marked with 700 g and minimal with 70 g. Administration of C. parvum SC admixed with alloantigen resulted in early enhancement and late depression of primary CMC. Secondary CMC was depressed but suppressor activity was absent. Dissociation of CMC depression from suppressor cell generation indicates that these phenomena can be separated under certain conditions.     

Bcl10 plays a divergent role in NK cell-mediated cytotoxicity and cytokine generation

Activating receptors such as NKG2D and Ly49D mediate a multitude of effector functions including cytotoxicity and cytokine generation in NK cells. However, specific signaling events that are responsible for the divergence of distinct effector functions have yet to be determined. In this study, we show that lack of caspase recruitment domain-containing protein Bcl10 significantly affected receptor-mediated cytokine and chemokine generation, but not cytotoxicity against tumor cells representing "missing-self" or "induced-self." Lack of Bcl10 completely abrogated the generation of GM-CSF and chemokines and it significantly reduced the generation of IFN-gamma (>75%) in NK cells. Commitment, development, and terminal maturation of NK cells were largely unaffected in the absence of Bcl10. Although IL-2-activated NK cells could mediate cytotoxicity to the full extent, the ability of the freshly isolated NK cells to mediate cytotoxicity was somewhat reduced. Therefore, we conclude that the Carma1-Bcl10-Malt1 signaling axis is critical for cytokine and chemokine generation, although it is dispensable for cytotoxic granule release depending on the activation state of NK cells. These results indicate that Bcl10 represents an exclusive "molecular switch" that links the upstream receptor-mediated signaling to cytokine and chemokine generations.     

Augmentation of generation of human allospecific cytotoxic T lymphocyte by PPD in in vitro sensitization culture

Summary PPD augmented human lymphocyte blastogeneic response to allogeneic lymphocytes in the mixed lymphocyte reaction (MLR) and generation of human cytotoxic lymphocytes against allogeneic human lymphocytes in in vitro sensitization (IVS) culture. The augmenting effect of PPD in the MLR was unequivocally synergistic at its lower concentrations (0.05 and 0.01 g/ml). The augmentation of MLR was observed following addition of a supernatant of culture medium of lymphocytes which had been precultured with PPD for 24 h then washed free of PPD and recultured without PPD for another 24 h. PHA and Con A, in contrast, suppressed both MLR and the generation of alloreative cytotoxic cells. The alloreactive cytotoxic lymphocytes whose generation was augmented by PPD belonged to the SRBC-rosette forming fraction and passed through a nylon-wool column. The NK cell-like activities of the alloreactive cytotoxic lymphocytes were not augmented by PPD. Analysis of the alloreactive cytotoxic lymphocytes whose generation was augmented by PPD by competitive inhibition assay with unlabeled cells indicated that the same allogeneic lymphocytes used as sensitizing cells in IVS culture inhibited the cytotoxicity, while MOLT-4 cells, which are frequently used as target cells for the human NK-cell assay, did not. When lymphocytes with known HLA-A and HLA-B were used in the IVS culture and the cytotoxicity assay, PPD was found to augment the cytotoxicity only against the target lymphocytes that possessed the same HLA as the sensitizing lymphocytes in IVS.     

Augmentation of human natural cell-mediated cytotoxicity by a soluble factor. I. Production of N-cell-activating factor (NAF).

Natural cell-mediated cytotoxicity (NCMC) like immune T cell-mediated cytotoxicity and antibody production is regulated by a soluble factor released during co-culture of lymphocytes with mitomycin C-treated lymphoblastoid cell lines. This N-cell-activating factor (NAF) enhances the activity of effector N cells and increases natural cytotoxicity. There appears to be no restriction for compatibility at the A and B locus of the major human histocompatibility complex in the production or activity of the factor. NAF was observed in the supernatant as soon as 2 days after initiation of mixed culture with a peak of production at 5 days. A soluble factor produced and released by T cells in response to stimulation by other cells acts by enhancing cytotoxicity of effector cells in NCMC, demonstrating a T-N cell cooperation.     

Fractionation of lymphocytes involved in the generation of cell-mediated cytotoxicity over insolubilized conjugated histamine columns.

Spleen cells from normal BALB/c mice were cultured in vitro with irradiated C57BL/6 stimulating cells. Five days later the T cell-mediated cytotoxic activity of the effector cells was assessed with a 51Cr-release assay that used H-2bEL-4 tumor cells as targets. Before the BALB/c responding lymphocytes were sensitized they were fractionated by passing the spleen cells over insolubilized histamine rabbit serum albumin Sepharose columns (H-RSA-S) or over rabbit serum albumin Sepharose (RSA-S) control columns. Fractionation of cells over the H-RSA-S columns depleted or significantly reduced the cytotoxic potential of the unretained cells. All cytotoxic potential was recovered when the cells that adhered to the H-RSA-S were eluted from the columns. In contrast, no effect on responsiveness was detected after the cells had been fractionated over the control column. The loss of response potential by the cells that did not adhere to H-RSA-S could not be accounted for by removal of macrophages nor by the concentration of cells with suppressor activity in the effluent. These cell fractionation studies raise the possiblity but do not prove that cytotoxic precursor cells may express amine receptors that could be responsible for their retention by insolubilized histamine columns.     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Inhibition of NK cell-mediated cytotoxicity by oxysterols.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.     

Inhibition of T cell-mediated cytotoxicity by anti-inflammatory steroids

We have tested the capacity of glucocorticoids to modulate the effector function of splenic cytotoxic T lymphocytes (CTL) obtained after i.p. immunization with allogeneic cells. Although acute exposure to glucocorticoids did not inhibit the activity of freshly obtained splenic CTL, preincubation of these CTL for several hours with subnanomolar concentrations of several different glucocorticoids caused marked inhibition. The relative inhibitory potency of the steroids tested correlated with their reported activity both in glucocorticoid receptor binding assays and in assays of anti-inflammatory potency in man. The inhibitory effects of low concentrations (10(-10) M to 10(-9) M) of dexamethasone were reversed by human or mouse interleukin 2 (IL 2)-containing supernatants, but were not reversed by IL 1-containing supernatants. The inhibitory effects of higher concentrations (10(-8) M to 10(-7) M) of dexamethasone could not be reversed even by very high doses of mouse IL 2. In contrast to previous reports of minimal direct glucocorticoid effects on CTL activity, the present results suggest that after preincubation, splenic CTL from in vivo-immune mice are sensitive to inhibition by glucocorticoids, and that the glucocorticoids may act both indirectly (on IL 2 production) and directly on the CTL.     

H2O2 generation during the redox cycle of mitomycin C and DNA-bound mitomycin C

Reduction of mitomycin C by NaBH₄ or by NADPH in the presence of a cell extract followed by exposure to air results in the generation of H₂O₂. This phenomenon occurs not only with free mitomycin but also with mitomycin irreversibly bound to DNA. In view of these findings, the antibiotic activity of mitomycin was tested in two bacterial systems: a facultative aerobic bacterium grown in the presence or absence of oxygen and an obligate anaerobic bacterium. No oxygen effect could be demonstrated in either case in the growth-inhibitory and bactericidal activity of the drug. Nevertheless, the H₂O₂ generating capacity of mitomycin-DNA complexes inside the nucleus may play a role in the drug-induced biological damage to the genetic material of cells.     

Augmentation of proliferation and generation of specific cytotoxic cells in human mixed lymphocyte culture reactions by colchicine

Cellular proliferation and generation of cytotoxic lymphocytes in mixed lymphocyte culture reactions (MLC) results from cellular interactions initiated by individual differences in cell surface structures determined by the HLA-D locus. Cellular microtubular assemblies (MTA) modulate cell shape and surface architecture and have also been implicated in mediating stimulatory signals from the lymphocyte plasma membrane to intracellular sites. To assess the role of MTA in alloactivation, we tested the effect of colchicine, a microtubule-disrupting alkaloid, on the MLC. Diametrically opposite results were observed depending upon the colchicine treatment protocol. Brief exposure of stimulating cells to 10^?6M colchicine resulted in an increase in [³H]thymidine incorporation from 30, 694 ± 2787 to 47,345 ± 4361 cpm/culture (mean ± SEM) (P < 0.001), exposure of responding cells to 10^?6M colchicine resulted in an increase from 33,054 ± 4012 to 46,790 ± 5458 cpm/culture (P < 0.01), and exposure of both cells to 10^?6M colchicine resulted in an increase from 33,054 ± 4012 to 52,685 ± 6720 cpm/culture (P < 0.01). However, direct addition of colchicine to a final concentration of 10^?6M to MLCs at different times resulted in complete suppression of proliferation when added as late as 96 hr, and 63% suppression when added at 120 hr. Pretreatment of stimulating, responding, or both cells with lumicolchicine did not enhance proliferation. Pretreatment of cells with 10^?6 and 10^?4 M colchicine enhanced proliferation, while pretreatment with 10^?2M colchicine prevented blastogenesis. The potentiation of proliferation induced by colchicine was evident as early as 48 hr after the initiation of the MLC. Generation of specific cytotoxic cells in the MLC was also enhanced by exposing lymphocytes to colchicine prior to the proliferative phase in six out of eight experiments (specific chromium release = 168% of control) despite constant effector:target ratios. These findings indicate that early disruption of microtubules leads to enhanced cellular proliferation and generation of cytotoxic lymphocytes in allogeneic one-way MLCs and suggest that the state of polymerization of the MTA may modulate immune responses involving cell-cell interactions.     

Augmentation of in vitro lymphocyte blastogenesis and cytotoxicity responses by tumor cells modified with dodecanoyl cytochrome C

In a model testing the immune responsiveness of allogeneic lymphocytes against the human cervical cancer cell line SW756, tumor cells coupled to dodecanoyl cytochrome C (D-cyt C) demonstrated augmented immunogenicity. The coupling was noncovalent; the D-cyt C spontaneously associated with the SW756 cells during mixing. Augmented immunogenicity was shown in both in vitro blastogenesis and cytotoxicity assays. The extent of cell-associated D-cyt C, which influenced the in vitro responsiveness, was easily controlled. Increased lipoprotein concentrations and increased lipid substitution of the cyt Cs led to greater cell uptake. Under the maximum conditions, 60-80 micrograms D-cyt C were bound/10(7) SW756 cells. In the blastogenesis assay, augmented immunogenicity was observed on days 3 and 4 after cocultivation of the allogeneic lymphocytes with the modified SW756 cells. When cells modified by the optimal D-cyt C preparations (as determined in the blastogenesis assay) were tested in the cytotoxicity assay, augmented responses were observed across a range of lymphocyte:tumor cell ratios. Our results demonstrate that the modification of SW756 cells with D-cyt C can augment the immune responses detected in in vitro assays.