Passive transfer of tumor immunity with spleen cells from guinea pigs cured of hepatocarcinoma by nonspecific immunotherapy

Summary The purpose of this study was to evaluate cell-mediated tumor immunity in strain-2 guinea pigs cured of line-10 hepatocarcinoma by oil-in-water emulsions containing phenol-water extracts from either BCG or the Re mutant of Salmonella typhimurium (Re ET) admixed with mycobacteria glycolipid (P3). Treatment with these emulsions produced complete regression of established tumor nodules and prevented the growth of lymph node metastases in 25 of the 28 animals inoculated intradermally (ID) with 10⁶ line-10 cells and given intralesional immunotherapy 6 days later. No tumor regression was observed in animals given phenol-water extracts alone. Spleen cells, taken from guinea pigs cured of line-10 by BCG extract + P3 or Re ET + P3, were tested for their influence on tumor growth by means of an in vivo adoptive neutralization test (Winn test). Cell transfer was accomplished by the subcutanous injection of various concentrations of spleen cells admixed with 10⁵ viable line-10 cells. The results showed that as few as 10⁷ immune spleen cells completely inhibited the growth of 10⁵ tumor cells in 46–54% of the animals. The best tumor growth inhibition (77–85%) was observed in animals given 5 × 10⁷ immune cells admixed with 10⁵ tumor cells. The onset of transferrable tumor immunity was earlier in animals treated with the BCG extract + P3 than in those given the Re ET + P3. However, the duration of detectable tumor immunity was longer in the latter group. In contrast, no inhibition of tumor growth was observed in animals given spleen cells from normal or tumor-bearing guinea pigs. Moreover, spleen cells obtained from guinea pigs immunized with BCG extract + P3 or Re ET + P3 emulsions only and admixed with line-10 cells failed to transfer tumor immunity to normal animals. Thus, results from this study clearly demonstrated that cell-mediated tumor immunity was elicited in animals cured of line-10 tumor with combinations of P3 and phenol-water extracts of either BCG or Re mutant of S. typhimurium and that sensitized spleen cells effectively transferred systemic tumor immunity to normal recipients.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Stimulation of human lymphocytes with irradiated cells of the autologous Epstein-Barr virus-transformed cell line. I. Virus-specific and nonspecific components of the cytotoxic response

Peripheral blood lymphocytes were cocultivated with irradiated cells of the autologous EB virus-transformed cell line at different responder:stimulator (R:S) ratios and the cytotoxic response was assayed up to 12 days later. In cocultures set up at a R:S ratio of 4:1, the response from both EB virus antibody-positive (seropositive) and negative donors was dominated by a broad-ranging NK-like cytotoxicity which did not segregate within the E-rosette-forming subpopulation of effector cells. In contrast, cocultures set up at a R:S ratio of 40:1 and harvested after 10 to 12 days gave rise, in the case of seropositive donors only, to effector T-cell preparations which appeared to be both EB virus specific and HLA-A and B antigen restricted. Strong lysis of the autologous virus-transformed cell line and demonstrable activity against certain allogeneic HLA-A and/or B antigen-related virus-transformed lines occurred in the absence of any significant killing either of the corresponding lines from HLA-unrelated donors or of a variety of EB virus genome-negative target cells (K562, HSB2, BJAB) particularly sensitive to NK-like cytotoxicity; furthermore, lysis of the autologous cell line by these effector T cells was specifically inhibited by monoclonal antibodies binding to HLA-A, B, and C antigens on the target cell surface. This work demonstrates that an HLA-restricted EB virus-specific cytotoxic T-cell response can indeed be induced in vitro by stimulation of fresh lymphocytes with autologous EB virus-transformed cells providing cocultures are set up at the correct R:S ratio.     

Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

Methods for amplifying the induction and expression of cytotoxic response in vitro to syngeneic and autologous freshly-isolated solid tumors of mice

Summary Spontaneously arising tumors are frequently poorly immunogenic and exhibit a limited capacity to induce cytotoxic effector lymphocytes. In the present study, various approaches have been used to amplify the induction and expression of cytotoxic responses in vitro toward freshly isolated, autologous, and syngeneic solid neoplasms of spontaneous origin in mice. Cytotoxic lymphocytes were generated in one-way mixed lymphocyte-tumor cell cultures (MLTC) consisting of splenocytes or lymph node cells from normal and from tumor-bearing mice co-cultured with inactivated tumor cells. Optimal culture conditions have been established for the number of responder (R) cells, the method of inactivation of the stimulating (S) tumor cells, the responder/stimulator (R/S) cell ratio, and the duration of sensitization. Under optimal sensitization conditions only weak cytotoxic responses, as measured by the ⁵¹Cr-release assay, were generated. The antitumor cytotoxic activity could be augmented 2- to 12-fold by using each of the following procedures: (a) addition of crude or of partially purified interleukin-2 (IL-2) to the sensitization cultures; (b) depletion of nylon-adherent cells from the responding cell population; (c) enrichment of large lymphoblasts from the sensitized effector cell population by Percoll density gradient; and (d) treatment of mice donating the responder lymphocytes with low doses of either cyclophosphamide, adriamycin, or indomethacin. Although the highly reactive effector cells generated under the improved conditions also reacted appreciably with unrelated tumor target cells, only low levels of cytotoxicity could be demonstrated against normal target cells. The antitumor cytotoxic cells in sensitized splenocyte cultures were exclusively Thy1⁺, Lyt1^–2⁺, whereas in lymph node cell cultures some cytotoxicity was also exerted by Thy1⁺, Lyt1⁺2⁺ cells.     

Study of the immune responses to nucleated cells. I. In vitro functional evaluation of the immune effectors responsible for complement-dependent cellular cytotoxicity

A modification of the ⁵¹Cr cytotoxic test has made it possible to assess under the same conditions not only cytotoxic serum antibodies, and cell-mediated immunity, but also cells releasing cytotoxic antibody. The measurement of these antibody-releasing cells was carried out with nucleated target cells, both normal and leukemic, across θ or H-2 antigenic differences. This test was found to be specific. The release of ⁵¹Cr from the labeled target cells was proportional to the ratio of immune cells to target cells, and for a given ratio to the incubation time, 60 min usually being the optimum time at ratios of 50–100 to 1. The test was not affected by treatment of the effector cells with an anti-θ serum; however, pretreatment of these cells with an anti-IgM serum, even without complement, inhibited the test with cells taken during primary responses. Both cytotoxic IgM and IgG antibodies were detected by the assay directly without the addition of enhancing serum; discrimination between these two γ-globulins can be made by suppressing the cytotoxicity due to either Ig class consequent to the addition of the appropriate specific anti-globulin serum during the incubation.     

Augmentation of human monocyte-mediated cytolysis by interferon

Human monocytes, separated by either plastic adherence or adherence to microexudatecoated surfaces, from the peripheral blood of most normal donors were shown to have significant cytolytic activity against TU5, a mouse SV40-transformed target cell. Spontaneous cytolysis ranged from 0 to 32% at a 40:1 effector:target (E:T) ratio. Augmentation of cytolysis was usually seen when human fibroblast interferon (IF) (10³–10⁴ units/ml) was cultured with the effector and target cells for the duration of the assay. The mean increase in percentage cytolysis at 40:1 and 20:1 E:T ratios was greater with monocytes obtained by a microexudate method (24.1 and 22.4%) than with monocytes obtained by a plastic adherence method (16.0 and 8.1%). Only a slight augmentation of cytotoxicity was observed when the effector cells were pretreated with IF for 1-hr. The increased levels of cytotoxicity observed when IF was present during the assay did not appear to be due to the toxic effects of IF on the target cells or to a stable increase in the susceptibility of the target cells to lysis.     

Suppression of growth of guinea pig line-10 hepatocarcinoma. I. Effect of simultaneous administration of tumor cells and antibodies from normal rabbits.

Suppression of growth of the line-10 hepatocarcinoma in strain-2 guinea pigs occurred when line-10 cells were injected intradermally together with sera or immunoglobulins derived from normal rabbits. A significant number of animals were resistant to subsequent rechallenge with tumor cells. This immunity was specific, depended on contact of immunoglobulins with tumor cells and on the concentration of immunoglobulins. Repeated injections acted as potent vaccines and resulted in the development of immunity in 84.6% of recipients. Fc receptors were not detected on line-10 cells. Antibodies reacting with line-10 cell unique antigens as well as with antigens common to line-10, line-1 and normal guinea pig spleen cells were found in NRS. Injection of line-10 cells together with rabbit immunoglobulins from which antibodies reacting with antigens derived from line-10 cells had been removed did not result in tumor suppression. The specific antigen(s) recognized by antibodies that suppressed growth of the line-10 tumor in vivo was not determined.     

Naturally soluble tumor antigens from guinea pig hepatomas: isolation and partial characterization.

Naturally soluble tumor antigens were detected in the ascites fluid of guinea pigs bearing an ascites tumor and from exhausted tissue culture media of cultured tumor cells. Two antigenically distinct cell lines of diethylnitrosamine-induced strain-2 guinea pig hepatomas (line-10 and line-1) served as the source of tumor antigens. Tumor antigen activity was detected by four different techniques: immunodiffusion, inhibition of complement-mediated cytotoxicity, inhibition of membrane immunofluorescence, and delayed cutaneous hypersensitivity. With syngeneic tumor-specific antiserum, line-10 guinea pig tumor antigens were detected by immunofluorescence in the concentrated ascites and tissue culture fluids. With a xenogenic antiserum, demonstrated to be tumor specific, line-10 tumor antigens were detected not only in the concentrated ascites and tissue culture fluids but also in two of the partially purified fractions of these fluids. When the line-10 concentrated ascites and its fraction I were subjected to ultracentrifugation at 300,000 x G for 1 hr, the antigen activity was retained in the supernatant and thus by this criterion the tumor antigens detected in these samples are soluble. Immunodiffusion data indicate that more than one antigen is present in the line-10 system since three lines of precipitation were detected when line-10 concentrated ascites was reacted with the line-10 tumor-specific antiserum. In contrast to this, the line-10-concentrated tissue culture fluid displayed only one line of precipitation. Although tumor antigens could not be demonstrated in the other antigenically distinct tumor cell line, line-1, by immunodiffusion or inhibition of membrane immunofluorescence, inhibition of complement-mediated cytotoxicity was able to detect tumor antigens in the line-1 concentrated ascites and tissue culture fluids.     

Cell-mediated immunity in interstitial nephritis. II. T lymphocyte effector mechanisms in nephritic guinea pigs: analysis of the renotropic migration and cytotoxic response.

The present studies investigate the effector role of lymphocytes in guinea pigs with an interstitial nephritis. Several observations were made relative to a number of functions expressed by these cells. The results of adoptive cell migration studies suggest that a subpopulation of T cells in nephritic animals traffic renotropically to either normal or damaged kidneys on transfer. Similar lymphocytes were also tested in vitro for direct effector function by utilizing target kidney cell monolayers in a cell-mediated cytotoxicity assay. The kinetics of the observed cytotoxic response were studied over the life span of nephritic animals. Optimal target-cell lysis occurred 12 to 17 days after sensitization, simultaneous with the onset of active histopathologic changes. The cytotoxicity was stoichiometrically titratable and relatively specific for fetal kidney tissue. In addition, cells from the spleen or lymph nodes of diseased animals effectively suppressed this cytotoxic response. These findings demonstrate that a diverse population of T lymphocytes are both capable of damaging the renal interstitium as well as modulating effector-cell functions on a regional basis with the immune system.     

Effects of L2C leukemia on macrophage-mediated responses

Summary Preliminary experiments have suggested that guinea pig L₂C B-cell leukemia cells were able to evade macrophage-mediated lysis. To determine whether the L₂C cells were resistant to macrophage cytotoxic activity or whether factors associated with the L₂C leukemia contributed to a generalized inhibition of macrophage cytotoxic activity, pulmonary macrophages from strain 2 guinea pigs with L₂C leukemia were tested for their ability to lyse the susceptible K562 cell line after activation by lipopolysaccharide (LPS) or lymphokines. In addition, the potential presence of soluble inhibitors of macrophage tumoricidal activity in serum-free culture supernatants and in serum from strain 2 guinea pigs terminally ill with the leukemia was tested by determining the effects of leukemic guinea pig serum (LGPS) or L₂C-conditioned medium (CM) on the tumoricidal activity of normal pulmonary macrophages. Macrophages from guinea pigs terminally ill with L₂C leukemia were demonstrated to be depressed in their cytotoxic activity against the K562 cell after stimulation by either LPS or lymphokines when compared to normal macrophages. The lymphokine-stimulated cytotoxic activity of normal macrophages was inhibited in the presence of LGPS or CM. Oxidative burst activity of normal macrophages, as measured by zymosan-stimulated production of superoxide and hydrogen peroxide, was also inhibited under these conditions. The data presented here suggests that soluble factors associated with L₂C leukemia cells can suppress oxidative burst activity of macrophages in vitro and that this effect may contribute to the ability of the leukemia cells to evade macrophage-mediated cytotoxicity.     

Inhibition of an vitro cell-mediated response: further experiments on the cell lineage and target of suppressor cells

The contribution of lymphotoxin to guinea pig leukocyte natural cytotoxicity was evaluated with [³H]TdR release and colony-inhibition assays of 104C1 benzo(a)pyrene in vitro-transformed and tumorigenic, tumor-specific transplantation antigen-negative, syngeneic strain 2/ N fibroblasts. Cytolethal ³H-release activities of mitogen (PHA)¹-stimulated nonimmune and ovalbumin (OA) immune as well as OA-stimulated OA immune unfractionated, adherent (macrophage-enriched) and nonadherent peritoneal leukocytes are qualitatively similar. ³H release is maximal by 48 hr, increases with antigen or mitogen concentration, is greatest with unfractionated leukocytes, and is least with adherent macrophages. Lymphotoxin produced by peritoneal leukocytes, alone or in combination with the leukocytes does not or only minimally induces ³H release even after 6 days of incubation with guinea pig target cells although guinea pig lymphotoxin possesses cytolytic activity as indicated by ³H release from αL929 mouse tumor cells. In contrast to the absent or very weak cytolytic activity of guinea pig lymphotoxin for the guinea pig target cells nonimmune macrophages, nonadherent leukocytes, and lymphotoxin all exhibit readily detectable colony-inhibitory (CI) activity for the syngeneic tumor cells. Macrophage and lymphotoxin CI, moreover, are additive, whereas nonadherent leukocyte and lymphotoxin CI are synergistic. The latter may be due to additional lymphotoxin induced by target cell antigens or other mechanisms of target cell stimulation of effector lymphoid cells and result from very high local levels of lymphotoxin released by the effector cells. Lymphotoxin CI, furthermore, can be cytostatic or cytolethal as indicated by resumption of 104C1 but not αL929 colony growth following removal of lymphotoxin, indicating that natural cell-mediated cytotoxicity consists of lymphotoxin-dependent and -independent cytostatic and cytolethal effector mechanisms.     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Cytotoxicity of adherent cells associated with some human tumours and lung tissues

Summary Cells were isolated by adherence from human tumours, and the majority of them showed the capacity to phagocytose latex particles, incorporate neutral red, and form rosettes with antibody-coated ox erythrocytes, and were positive for nonspecific esterase activity. The monolayers lyse antibody-coated human erythrocytes at low effector: target ratios. In 17 of 25 cases cytotoxicity for autologous tumour cells was apparent in an 18-h ⁵¹Cr release assay. Cytotoxicity was dose-dependent and nonspecific for tumour targets, but cells isolated from tumour-free lung areas appeared to be resistant to damage. Adherent cells from tumour-free lung areas also killed autologous tumour targets but not normal lung cells. The problems of the interpretation of these data are discussed.     

Elucidation of cellular population and nature of anti-amoebic antibodies in cytotoxicity to Entamoeba histolytica (NIH:200)

Interactions between trophozoites of Entamoeba histolytica and cells of the immune system were studied in vitro by mixing effector cells obtained from normal and immunized guinea pigs with trophozoites in a ratio of 10:1. Crude amoebic extract (CAE) and its highest molecular weight (MW 650,000) fraction (F-I) were used for priming the effector cells in vivo. The effector cells were collected from the spleens of normal and immunized animals. Lymphocytes were prepared by allowing splenic mononuclear cells to adhere to plastic, followed by passage through nylon wool column. There was no significant difference between the killing capacity of mononuclear cells, monocyte depleted mononuclear cells or nylon wool fractionated lymphocytes, indicating that probably monocytes and B-cells were not involved in the cytotoxicity against E. histolytica. The data suggest that effector cells probably belonged to the T-cytotoxic and K-cell category. Both CAE and F-I sensitization could induce cytotoxicity of lymphocytes against trophozoites. Similarly, anti-CAE and anti-F-I sera were found to enhance the killing capacity of effector cells in vitro. The ability of anti-amoebic serum to induce cytotoxicity was found to be independent of complement involvement and resided in IgG but not in IgM.     

Natural killer cell activity correlates with the amount of beta 2-microglobulin on human peripheral blood lymphocytes

Two cytotoxic assays, lectin-dependent cytotoxicity and natural killer (NK) cytotoxicity, were used to assess the competence of cord blood and neonatal peripheral blood mononuclear cell (PBMC) and T-cell cytotoxic reactions. The effect of exogenous interferon was also studied. Results were compared with cytotoxic capabilities of adult cells and cells from patients with primary immunodeficiency syndromes. Lectin-dependent cytotoxicity (LDCC), a property of both T and non-T cells, was assessed by lysis of chromium-labeled EL4 tumor target cells in the presence or absence of exogenous fibroblast interferon (IFN-β). Natural killer cytotoxicity was assessed by lysis of two different chromium-labeled tumor target cells, Molt 4f and K562 in the presence or absence of IFN-β. Lectin-dependent cytotoxicity (LDCC) of PBMC of cord blood (32 ± 4% SEM) and adult cells (36 ± 2% SEM) were equivalent but neonatal cells had slightly decreased LDCC (22 ± 3% lysis). T-depleted cells from cord or neonatal blood had increased LDCC but T-enriched (>95% sheep erythrocyte rosette-forming cells) from both cord (22 ± 3%) and neonatal blood (18 ± 5%) had significantly reduced LDCC compared to 55 ± 2% for adult T cells. This deficiency corrects with age and is near normal after age 2. Preincubation with IFN-β did not enhance LDCC of newborn or adult cells. The LDCC of some cord T cells was markedly reduced and was in the same low range as patients with severe combined immunodeficiency. Natural killer (NK) cytotoxicity of PBMC from cord and adult cells was equivalent at three effector:target ratios against the Molt 4f target but against the K562 target, cord PBMC had significantly less NK activity (22 ± 11 SD) compared to adult NK activity (50.5 ± 22.2 SD) at a 50:1 effector:target ratio. Similar differences were noted at 25:1 and 10:1 target:effector ratios. NK cytotoxicity against Molt 4f targets of adult cells was significantly enhanced by preincubation with IFN-β but NK of cord cells was only variably enhanced. By contrast, IFN-β enhanced NK against K562 targets of both adult and cord cells, adult greater (67.7 ± 20) than cord cells (37.8 ± 2.0). These T-cell effector deficiencies are in marked contrast to the vigorous proliferative responses of newborn T cells, and parallel deficiencies of certain neonatal lymphokines. These defects may explain the newborns' enhanced susceptibility to intracellular viruses and to congenital viral infections.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.     

Differential effects of ouabain on human cell-mediated cytotoxicity. II. Stimulation of monocyte-mediated, spontaneous cytotoxicity against chicken red cell targets.

We have previously shown that ouabain inhibits mitogen-induced cellular cytotoxicity (MICC) and antibody-dependent cellular cytotoxicity (ADCC) against chicken red cell (CRC) targets. We now report that ouabain increases spontaneous killing of CRC targets in the absence of mitogen or antibody. Spontaneous cytotoxicity by fresh mononuclear leukocytes (MNL) was enhanced by ouabain in a dose-dependent fashion and was maximal at a ouabain concentration of 5 × 10^?5M. Removal of phagocytic cells from the MNL effector cell population abrogated ouabain-induced spontaneous cytotoxicity, suggesting that the effector cell activated by ouabain was a monocyte. Ouabain-induced spontaneous cytotoxicity was relatively inefficient compared to MICC or ADCC and was only demonstrated consistently at effector:target cell ratios higher than those routinely employed for MICC and ADCC. Very low concentrations of ouabain (5 × 10^?9M) also enhanced spontaneous cytotoxicity of MNL precultured for 7 days, when added at either Day 0 or Day 6 of preculture. The cell effecting spontaneous cytotoxicity after 7 days of culture has been previously shown to be a monocyte. Thus, ouabain has opposing effects on cell-mediated cytotoxic functions: it inhibits MICC and ADCC against CRC targets, but stimulates spontaneous, monocyte-mediated cytotoxicity against the same targets.     

The efficacy of BCG-induced tumor immunity in guinea pigs with regional and systemic malignancy

Summary It has been previously demonstrated that transplanted syngeneic line-10 hepatocarcinoma established in the skin of inbred guinea pigs (strain 2) regressed and regional lymph node metastases were eliminated after intratumoral injection of viable Mycobacterium bovis strain BCG. During the course of this reaction there is the development of systemic tumor immunity. The purpose of this study was to determine the relative efficacy of the induced tumor immunity to eliminate regional as well as systemic tumor burden. The approach to evaluate the efficacy of BCG-induced systemic tumor immunity in vivo, for regional as well as systemic tumor, was to develop a competition assay using increasing doses of intravascular disseminated line-10 tumor cells in animals with established regional tumors. The results clearly show that the efficacy of intratumoral BCG injection in producing regression of regional tumor is abrogated by initial intravascular doses of 10³–10⁶ line-10 cells. That the vascular systemic tumor burden diminished the effective systemic tumor immunity was demonstrated by the inability of animals with systemic tumor burdens to reject contralateral challenge of line-10 tumor cells. The capability of BCG-treated animals to reject contralateral line-10 challenge was inversely proportional to the initial intravascular tumor dose. Survival studies clearly demonstrate that a significant therapeutic effect could be achieved in guinea pigs with regional skin tumors and limited vascular metastases when the modality of therapy included BCG-intratumoral injection, followed 6 weeks later by surgery of the established skin tumor and regional lymph node. These results suggest that the development of tumor immunity after BCG-intratumoral injection is not impaired by the systemic tumor burden, but rather that it is preempted at distant sites. Abbreviations used in this paper: BCG, Bacillus Calmette-Guérin; i.a., intraarterially; i.d., intradermally; i.v., intravenously; SDA, superficial distal axillary.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.