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.     

Anti-thymocyte autoimmunity in BALB/C mice accompanying immunization to a syngeneic lymphoma.

As immunization of BALB/c mice to the syngeneic P1798 lymphoma is effected by administration of iodoacetamide-modified P1798 cells, serum antibodies appear which are reactive with P1798 and normal BALB/c thymocytes, splenocytes, and peripheral blood lymphocytes. Anti-P1798 serum also cross-reacted with thymocytes from AKR, DBA/2, and C3H mice as well as the allogeneic lymphoma 6C3HED. Anti-P1798 serum was unreactive with the Thy-1 deficient L1210 lymphoma. Multiple absorptions of anti-P1798 serum with normal BALB/c thymocytes or brain or P1798 removed antibodies to P1798 and thymocytes commensurately. Normal BALB/c liver and kidney did not absorb antibody activity. Treatment of a BALB/c splenocyte suspension with anti-Thy 1.2 serum and complement removed the population of spleen cells which were capable of reaction with anti-P1798 serum. The data suggest that antibodies to P1798 and thymocytes are the same and that specificity may be directed toward a Thy-1 related structure but without distinguishing Thy-1.1 and Thy-1.2.     

Biochemical and molecular characterization of the glucocorticoid receptor of lymphosarcoma P1798 variants

Three phenotypically distinct isolates from lymphosarcoma P1798 have been compared with respect to properties of the glucocorticoid receptor. Wild type P1798 cells express functional receptors and glucocorticoid treatment of such cells causes cytolysis in vivo. Wild type cells do not undergo cytolysis in culture. Rather, such cells exhibit reversible inhibition of proliferation in the presence of dexamethasone. Two variant populations were selected from this background. One was selected for the ability to form tumors in mice receiving pharmacological doses of glucocorticoids. Cells from such tumors are resistant to the cytolytic effects of glucocorticoids in vivo, but are sensitive to the antiproliferative effects of the hormone in culture. Variants were also selected based upon their ability to proliferate in the presence of dexamethasone in culture. These variants were resistant to glucocorticoid-mediated cytolysis in vivo. Wild type P1798 cells express approximately 20,000 high affinity dexamethasone-binding sites per cell. Dexamethasone-mesylate labeling and immunoblotting experiments indicate that hormone binding is due to a polypeptide of Mr 90-100 K. This polypeptide is encoded in an mRNA species that resolved as a single entity of approximately 7000 nucleotides. Variants selected for resistance to cytolysis in vivo are indistinguishable in any of these respects from wild type cells. The receptors are fully functional, as evidenced by their ability to precipitate growth arrest of dexamethasone-treated cultures. Variants selected for resistance in culture harbor a receptor mutation. They express fewer than 500 dexamethasone-binding sites per cell. Such variants contain neither detectable dexamethasone-mesylate-binding protein nor any protein that is recognized by a receptor antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis

We have previously described a 14,700 m.w. protein (14.7K) encoded by the E3 region of adenovirus that prevents TNF-mediated cytolysis of adenovirus-infected C3HA mouse fibroblasts. In the studies described here we have extended our analysis of TNF cytolysis of C3HA cells and the circumstances under which 14.7K protects these cells from cytolysis. C3HA cells were killed by TNF in the presence of inhibitors of protein synthesis, in the presence of cytochalasin E (which disrupts the microfilaments), and when adenovirus E1A was expressed. As described for other cell types, pretreatment of C3HA cells with TNF prevented cytolysis by TNF plus cycloheximide or TNF plus cytochalasin E, indicating that TNF induces a response that protects against these treatments. Remarkably, when 14.7K was expressed in virus-infected cells, it also prevented TNF-induced lysis whether sensitivity to TNF was induced by inhibition of protein synthesis, disruption of the cytoskeleton by cytochalasin E, or expression of adenovirus E1A. The 14.7K protein also prevented TNF lysis of cells that are spontaneously sensitive to TNF lysis. Thus, 14.7K appears to be a general inhibitor of TNF cytolysis, and as such should be an important tool in unraveling the mechanism of TNF cytolysis. There was one exception; NCTC-929 cells were spontaneously sensitive to TNF lysis and that lysis was not affected by 14.7K even though the protein was made in large quantities and was metabolically stable in these cells. This suggests that there is heterogeneity among TNF-sensitive cell lines. The 14.7K protein was found in both the nuclear and cytosol fractions of TNF resistant as well as all spontaneously sensitive cells suggesting that 14.7K may have more than one site of action within the cell.     

Purification and properties of mouse thymus thymidylate synthase. Comparison of the enzyme from mammalian normal and tumour tissues

Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells. The enzyme is a dimer composed of 35,000 mol. wt monomers. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34 degrees C, reflecting a change of rate-limiting step in dTMP formation. MgATP at millimolar concentrations inhibits mouse thymus enzyme.     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.     

IFN-gamma differentially modulates the susceptibility of L1210 and P815 tumor targets for macrophage-mediated cytotoxicity. Role of macrophage-target interaction coupled to nitric oxide generation, but independent of tumor necrosis factor production

IFN-gamma primes murine macrophages to render them responsive for triggering by subactivating concentrations of bacterial LPS to mediate nonspecific tumor cytotoxicity. However, IFN-gamma also has direct anti-proliferative effects on transformed cells that serve as sensitive tumor targets for cytotoxic macrophages. We investigated the effects of preexposure of L1210 mouse leukemia and P815 mouse mastocytoma targets to rIFN-gamma on changes in their susceptibility to cytotoxicity by LPS-activated mouse peritoneal macrophages (PM). Co-incubation of inflammatory PM and either L1210 or P815 targets with IFN-gamma and LPS produced a classical synergistic cytotoxicity for both targets over that of IFN-gamma or LPS alone. Similar synergistic augmentation of cytotoxicity occurred when effector PM were preprimed for 24 h with IFN-gamma before testing for cytotoxicity of untreated targets. However, pretreatment of L1210 and P815 targets for 24 h with IFN-gamma (50 U) before assay produced divergent results in that L1210 was more susceptible, whereas P815 was less susceptible to cytotoxicity by LPS-activated macrophages. Similar results were obtained when both macrophages and targets were pretreated separately with IFN-gamma for 24 h before their combined assay for tumor cytotoxicity. Pretreatment of L1210 targets for 1, 4, or 24 h with IFN-gamma produced similar effects on their increased susceptibility to macrophage cytotoxicity. In contrast, P815 pretreated for 1 and 4 h with IFN-gamma showed an early increased susceptibility to macrophage cytotoxicity followed by a decrease after 24 h pretreatment. The pretreatment of L1210 or P815 targets with IFN-gamma before their exposure to LPS-activated macrophages had no effect on the production of TNF. However, there was a corresponding increase in nitric oxide generation by LPS-activated macrophages after their exposure to IFN-gamma pretreated L1210 targets and a decrease in the presence of IFN-gamma-pretreated P815 targets that correlated with their changes in susceptibility to macrophage killing. Nitric oxide generation by macrophages alone in response to LPS was found to be greater than when effector macrophages were exposed to the tumor targets and this was either increased by L1210 or decreased by P815 that had been pretreated with IFN-gamma. Our results indicate that IFN-gamma may act directly and differentially on tumor targets to alter their susceptibility for macrophage cytotoxicity, which was coupled to changes in the generation of cytotoxic nitric oxide, rather than TNF production by the macrophage.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential cytolysis of murine spleen, bone-marrow and leukemia cells by melittin reveals differences in membrane topography

L1210 leukemia cells are 2-4 fold more sensitive to the cytolytic effects of melittin, the membrane-active toxin of bee venom, than normal DBA/2 mouse spleen and bone-marrow cells. Lysis of the normal cells was abolished when either 75 mM galactosamine, glucosamine or 100 microM beta-lactoglobulin was added to the melittin-cell reaction, but lysis of the leukemia cells was unaffected. The amino-groups appeared necessary for blocking melittin-mediated lysis since glucose, galactose and the N-acetyl derivatives were not inhibitory. Bone-marrow cells were more readily protected from lysis than spleen cells. Since melittin-inhibitor complexes were not detected by gel chromatography and the inhibitor could be added to the cell suspension after melittin, the evidence suggests that bone-marrow cells are rich in membrane binding sites for carbohydrates that decrease in mature spleen cells and are virtually absent after neoplastic transformation.     

Differential effect of sanguinarine, chelerythrine and chelidonine on DNA damage and cell viability in primary mouse spleen cells and mouse leukemic cells

Sanguinarine, chelerythrine and chelidonine are isoquinoline alkaloids derived from the greater celandine. They possess a broad spectrum of pharmacological activities. It has been shown that their anti-tumor activity is mediated via different mechanisms, which can be promising targets for anti-cancer therapy. We focused our study on the differential effects of these alkaloids upon cell viability, DNA damage effect and nucleus integrity in mouse primary spleen cells and mouse lymphocytic leukemic cells, L1210. Sanguinarine and chelerythrine produce a dose-dependent increase in DNA damage and cytotoxicity in both primary mouse spleen cells and L1210 cells. Chelidonine did not show a significant cytotoxicity or damage DNA in both cell types, but completely arrested growth of L1210 cells. Examination of nuclear morphology revealed more cells with apoptotic features upon treatment with chelerythrine and sanguinarine, but not chelidonine. In contrast to primary mouse spleen cells, L1210 cells showed slightly higher sensitivity to sanguinarine and chelerythrine treatment. This suggests that cytotoxic and DNA damaging effects of chelerythrine and sanguinarine are more selective against mouse leukemic cells and primary mouse spleen cells, whereas chelidonine blocks proliferation of L1210 cells. The action of chelidonine on normal and tumor cells requires further investigation.     

Leukemia cell lysis by sulphydryl reagents and thiols.

Iodoacetate and other sulfhydryl reagents as well as cysteine and other thiols caused lysis of the mouse leukemic lymphoblasts L5178Y, L1210, and P388 in culture. Lysis by either iodoacetate or cysteine was preceded by ATP depletion. However ATP depletion was not a sufficient explanation for lysis since deoxyglucose depleted ATP to the same extent as did iodoacetate but did not cause lysis. It was concluded that sulfhydryl-disulfide equilibrium was essential to the maintenance of cellular integrity and ATP concentration.     

Expression of P-glycoprotein in L1210 cells is linked with rise in sensitivity to Ca2+

L1210/VCR cell line (R) was obtained by adaptation of the L1210 mouse leukaemia cells (S) to vincristine and showed P-glycoprotein (P-gp) mediated multidrug resistance (MDR). R cells were observed to be more sensitive to high external calcium as parental S. More pronounced calcium uptake was observed for R cells. Moreover, differences in intracellular calcium cell localization between S and R cells were found ultrastructurally following a calcium precipitating cytochemical method. In S cells, calcium precipitates were found to be localized predominantly along the cell surface coat and within mitochondria delineating the cristae. In R cells, precipitates were also found inside nuclei, at the border of heterochromatin clumps, and scattered within the cytoplasm. High extracellular calcium did not influence the P-gp mediated extrusion of calcein/AM as P-gp substrate. These results indicate that calcium enters and consequently damages the MDR cells to a higher extent than parental cells.     

Effect of anti-lymphotoxin on cell-mediated cytotoxicity. Evidence for two pathways, one involving lymphotoxin and the other requiring intimate contact between the plasma membranes of killer and target cells.

A rabbit anti-lymphotoxin serum produced against partially purified, antigeninduced, guinea pig lymphotoxin, was used to study the role of lymphotoxin in lymphocyte-mediated cytotoxicity in vitro. The anti-lymphotoxin serum inhibited cytolysis resulting from incubation of ovalbumin-immune guinea pig spleen cells with either mouse (P815 mastocytoma) or guinea pig (line 10 hepatoma) target cells in the presence of soluble ovalbumin. The antiserum also inhibited the cytolysis of ovalbumin-coupled target cells by ovalbumin-immune guinea pig spleen cells. In contrast, the anti-lymphotoxin serum did not inhibit: (a) the lysis of line 10 (strain 2) hepatoma cells by spleen cells from alloimmunized Hartley or strain 13 animals; (b) the lysis of line 10 hepatoma cells by spleen cells from tumor-bearing syngeneic animals; or (c) the lysis of P815-mastocytoma cells by spleen cells from P815-immune guinea pigs. These results support the hypothesis that there are at least two distinct pathways by which immune lymphocytes can destroy target cells in vitro, one which involves secretion of a nonspecific soluble factor, i.e., lymphotoxin, and another which probably requires intimate contact between the plasma membranes of the target and killer cells.     

Characterization of L1210 S Cells with Low Sensitivity to Mouse Interferon-γ

A mouse leukemic cell line L1210 Sg with a low sensitivity to interferon-γ (IFN-γ) was described. On the nature of the antiviral action and binding of IFN, L1210 Sg cells were compared with L1210m cell line which is sensitive to IFN-γ. For a half reduction of the vesicular stomatitis virus-RNA synthesis, L1210 Sg cells required 500–fold more IFN-γ than L1210m cells did. However, both cell lines were induced to the antiviral state to the same extent with IFN-α or -β. L1210 Sg and L1210m cells were sensitive to the anti-proliferative action of IFN-α and -β, but insensitive to IFN-γ. (2′-5′)Oligoadenylate synthetase was induced in these cell lines by IFN-β, but not by IFN-γ, which suggests that the induction of this synthetase may not be responsible for the antiviral action of IFN-γ. No substantial difference between L1210 Sg and L1210m cells was found in IFN receptors for IFN-γ and IFN-β either in number per cell or in their affinity to corresponding IFN type. However, differences were noted in time course profiles of cell-associated IFN-γ at 37 C: in L1210m cells, a rise-and-decay profile of IFN-γ bound to cells was observed at 37 C, but in L1210 Sg cells, rise and slight decay was observed. On the other hand, a similar rise-and-decay curve of IFN-β bound to these cells was observed. These results indicated that the low sensitivity of L1210 Sg cells to IFN-γ may be due to this slight decay of receptor-bound IFN-γ.     

In vitro secondary generation of cytotoxic T lymphocytes in mice with mumps virus and their mumps-specific cytotoxicity among paramyxoviruses.

Murine cells (L929, MC57G, and P815 mastocytoma) defectively infected with the egg-adapted vaccine strain of mumps virus were found to be susceptible to cytotoxic T-lymphocyte (CTL)-mediated lysis. In vitro secondary, but not in vivo primary, generated CTL caused cytolysis of these targets in an H-2-restricted manner. UV-inactivated-mumps virus-coated murine cells were also found to be susceptible to CTL-mediated lysis. Comparisons of murine CTL-mediated lysis by three paramyxoviruses (mumps, Sendai, and Newcastle disease viruses) indicated that no cross-reactivity occurred. The CTL response with mumps virus exhibited specific unresponsiveness patterns, as influenced by the H-2 K/D regions of the mouse strains, that were partially different from those of Sendai virus and Newcastle disease virus.     

Immunologic heterogeneity of dihydrofolate reductase from methotrexate sensitive and resistant L1210 leukemia cells

Dihydrofolate reductase from L1210 leukemia cells which are sensitive and resistant to methotrexate has the same physical and kinetic properties and immunoreactivity with a guinea pig antiserum raised to the enzyme purified from the methotrexate resistant strain. However, a chicken antiserum to dihydrofolate reductase from methotrexate sensitive L1210 cells has greater affinity for the homologous enzyme than for the enzyme from the MTX resistant cells indicating that there is some antigenic difference in these molecules.     

Induction of chemokine secretion and enhancement of contact-dependent macrophage cytotoxicity by engineered expression of granulocyte-macrophage colony-stimulating factor in human colon cancer cells

We investigated the role of tumor cell-derived GM-CSF in recruitment and tumoricidal activation of tissue macrophages. Transfection of the murine GM-CSF gene into KM12SM human colon cancer cells decreased the tumorigenicity of transfected cells and nontransfected bystander colon cancer cells in nude mice. Sequential tissue sections from sites injected with high GM-CSF-producing tumor cells (but not from nontransfected or low GM-CSF-producing cells) demonstrated a dense infiltration of polymorphonuclear cells (PMN), followed by infiltration of macrophages, which correlated with expression of the macrophage-inflammatory protein-1alpha and the monocyte chemoattractant protein-1 (MCP-1) in mouse PMN and macrophages. GM-CSF-producing KM12SM cells were highly sensitive to lysis by mouse macrophages and also increased macrophage-mediated lysis of bystander nontransfected KM12SM cells. The incubation of macrophages with GM-CSF induced expression of the CD11b surface adhesion molecule, which was associated with increased attachment to tumor cells. All KM12SM cells were sensitive to macrophage-mediated lysis in the presence of rGM-CSF and recombinant MCP-1. Collectively, the results demonstrate that tumor cell-derived GM-CSF stimulates PMN and macrophages to secrete macrophage-inflammatory protein-1alpha and MCP-1, which triggers recruitment of mononuclear cells, induces expression of adhesion molecules on macrophages, and enhances contact-dependent cytolysis of tumor cells.     

DNA-protein cross-linking in L1210 cells sensitive and resistant to cis-diamminedichloroplatinum (II)

The role of restricted cellular accumulation of cis-diamminedichloroplatinum(II) (cis-DDP) and altered repair of DNA-Pt-protein cross-links in the mechanism of L1210 murine leukemia cell resistance was examined. An immunochemical method was used to analyze the formation and removal of DNA-Pt-protein complexes in L1210 cells sensitive and resistant to cis-DDP. The accumulation of Pt into the cells and the binding of Pt to the DNA was measured by atomic absorption spectroscopy. The results demonstrated that both decreased accumulation of the drug and the rate of DNA-Pt protein cross-link removal may be important factors in L1210 cell resistance to cis-DDP.Abbreviations AAS atomic absorption spectroscopy - cis-DDP cis-diamminedichloroplatinum(II)     

Adriamycin transport and sensitivity in fatty acid-modified leukemia cells

The membrane phospholipids of L1210 murine leukemia cells were modified by supplementing the growth medium with micromolar concentrations of polyunsaturated or monounsaturated fatty acids. This procedure results in enrichment of cellular phospholipids by the supplemented fatty acid. Enrichment with polyunsaturated fatty acids resulted in a marked increase in sensitivity to adriamycin as compared to enrichment with monounsaturated fatty acids. The increased cytotoxicity was directly proportional to the extent of unsaturation of the inserted fatty acid, but there was no difference in cells enriched with n-3 compared with n-6 family fatty acids. To explore the mechanism of this observation, we examined whether augmented uptake of the drug might explain the increased cytotoxicity. The uptake of [14C]adriamycin, which was approximately linear at later time points, was only partially temperature dependent and never reached a steady state. Initial uptake at time points prior to 60 s could not be measured due to high and variable rapid membrane adsorption. Cellular accumulation of drug was greater in the docosahexaenoate 22:6-enriched L1210 cells as compared to oleate 18:1-enriched cells and was about 32% greater after 20 min. When L1210 cells were enriched with six fatty acids of variable degrees of unsaturation, the accumulation of adriamycin was directly correlated with the average number of double bonds in the fatty acids contained in cellular phospholipids. There was no difference in efflux of drug from cells pre-loaded with adriamycin. We conclude that the greater accumulation of adriamycin by the polyunsaturated fatty acid-enriched L1210 cells likely explains the increased sensitivity of these cells to adriamycin compared to 18:1-enriched cells.     

Factors responsible for immune resistance to L1210 murine leukemia in hyperimmune mice

Summary The serum of mice hyperimmune to L1210 leukemia was cytotoxic to L1210 cells and, to a much lesser extent, to P388 cells in the presence of complement. However, it did not suppress in vitro growth of L1210 cells, nor did it endow a recipient mouse with immunity to inoculated L1210 cells. This indicates that the serum did not play a significant role, if any, in immune protection of hyperimmune mice.Spleen and peritoneal exudate cells of hyperimmune mice suppressed the in vitro growth of L1210 but not of P388 cells. This is consistent with the fact that hyperimmune mice did not survive the inoculation of P388 cells. The immunocytes failed to suppress the in vitro growth of L1210 cells when preincubated with anti-Thy-1.2 antisera and complement. This, together with the finding that cell populations not adherent to a plastic dish suppressed in vitro growth of L1210 cells, indicates that T cells of immune spleen and peritoneal exudate cell populations were the effectors that suppressed in vitro growth of L1210 cells. Hyperimmune mice lost their immune protection in vivo following the administration of anti-thymocyte antisera, but not with carrageenan or silica, which resulted in the lethal growth of the inoculated L1210 cells. This indicates that T cells were in vivo effectors in immune protection.Hyperimmune spleen T cells endowed a recipient with immunity to L1210 leukemia when transferred in vivo. This confirmed the above results and suggests the applicability of immune cells in an adoptive immunotherapy approach.     

Induction and blocking of cytolysis in CD2+, CD3- NK and CD2+, CD3+ cytotoxic T lymphocytes via CD2 50 KD sheep erythrocyte receptor

The 50 KD sheep red blood cell antigen receptor CD2 is the earliest T cell differentiation marker and is present on all blood-derived T cells, including natural killer (NK) cells. The CD2 antigen is also known to serve as an important activation site regulating various T cell functions. We report that anti-CD2 monoclonal antibodies (MAb) block MHC-restricted class I- and class II-specific cytolysis by CD2+, CD3+ clones of the relevant target cells, irrespective of whether lysis by these clones is blocked by anti-CD3 or anti-CD8 MAb. Moreover, anti-CD2 MAb (but not anti-CD3 MAb) are able to reduce MHC-nonrestricted, nonspecific cytolysis: a) by CD2+, CD3+ clones of K562 target cells; and b) by CD2+, CD3 NK clones of K562 as well as Daudi cells. Different preparations of anti-CD2 MAb vary in their capacity to inhibit cytolysis. For cloned effector cells, the percent inhibition of lysis by CLB-T11 greater than Lyt-3 MAb, whereas with "fresh" NK cells, the lysis inhibitory ability of Lyt-3 greater than CLB-T11. The antibody-dependent cellular cytotoxicity by "fresh" and cloned NK cells is not inhibited by anti-CD2 MAb. Anti-CD2 MAb also prevent the induction of lysis by cross-linked anti-CD3 MAb, e.g., by CD2+, CD3+ cloned cloned cells against (IgG-FcR+) Daudi cells. Anti-CD2 MAb can also induce cytolysis in some, but not all, CD2+, CD3- NK clones against xenogeneic P815 mouse mastocytoma cells. Anti-CD2 MAb, in combination with lectins (PHA or Con A: pretreatment of effector cells), can also induce cytolytic activity by CD2+, CD3+ clones against Daudi cells. Our data therefore support the concept that the CD2 antigen is an important activation site regulating a wide variety of T cell functions including cytolysis. Whether ligand interaction with the CD2 antigens results in augmentation or inhibition of T cell functions may very well depend on the type of CD2 antigen-ligand interaction, e.g., cross-linked ligand-receptor interaction may, in general, enhance the various T cell functions, whereas noncross-linked ligand-receptor interactions may inhibit such functions, as we and other investigators demonstrated earlier for the CD3/Ti antigen-receptor complex activation site.