Cytotoxicity Of Spleen Cells Research Articles

Gamma interferon production and cytotoxicity of spleen cells from mice infected with Semliki Forest virus.

Interferon (IFN) production by spleen cells from normal mice, mice acutely infected with Semliki Forest virus (SFV) or mice immune to SFV was measured after stimulation in vitro with either infectious or inactivated SFV. All three classes of spleen cells made IFN-alpha beta in response to infectious SFV. Spleen cells taken from mice late, but not early, after infection, or from immune mice, made IFN-gamma in response to inactivated SFV. Amounts of INF-gamma and IFN-alpha beta were similar. Normal spleen cells made no IFN (of any type) in response to inactivated SFV. The cell type producing IFN-alpha beta appeared to be the macrophage, whilst both T-lymphocytes and macrophages were necessary for IFN-gamma production. During the acute infection, the ability of spleen cells to lyse both virus-infected and uninfected target cells arose earlier than the ability to produce IFN-gamma. However, cytotoxicity towards uninfected cells fell to near background levels by day 7, whilst cytotoxicity towards infected targets remained high at that time, when IFN-gamma production was at its peak. IFN-gamma production is therefore temporally associated with cytotoxicity specifically directed against virus-infected targets, and the ability to produce IFN-gamma is a late response to SFV infection.

The effects of syngeneic soluble tumor membrane extract on concomitant spleen cell immunity and spontaneous metastases

The effects of a soluble tumor KCl extract, containing membrane antigen on spontaneous metastases and spleen cell immunity were studied in a syngeneic C57BL/6J murine sarcoma model. The extract was shown to be antigenically distinct from normal tissue by tumor immunization rejection experiments. Tumor soluble extract (SE) was administered daily during the growth of a murine sarcoma. The afferent and efferent arc of immunity were monitored by proliferative index (PI) and in vitro cytotoxicity of spleen cells, as well as the incidence of metastases. PI was significantly activated in the group receiving sarcoma SE as compared to the two control groups receiving muscle SE or saline P < 0.05. However, in vitro cytotoxicity was significantly depressed on Days 7 and 14 ( P < 0.05) of tumor growth in the mice receiving sarcoma SE. The incidence of metastases was significantly increased to 70% in the sarcoma SE group as compared to the incidence in the control groups of 50% P < 0.05. This data supports the hypothesis that release of soluble antigen membrane components by growing tumor facilitates the growth of metastases in this model.

Human spleen cells mediating natural killing: altered natural cytotoxicity of spleen effector cells from patients with carcinoma.

Spleen cells from eight patients with abdominal carcinoma and six patients undergoing major surgery for a variety of disease entities were assayed for natural cytotoxicity towards 51Cr-labelled K 562 target cells. Patients with abdominal cancer were shown to have relatively weak splenic natural cytotoxicity compared with the reactivity of effector cells from non-carcinoma patients. Nylon wool non-adherent spleen effector cells from cancer patients showed reduced cytolytic capacity compared with effector cells prepared from the spleens of other patients or peripheral blood mononuclear cells (PBMC) obtained from healthy individuals, whereas the splenic reactivity of non-cancer patients showed the same nylon wool separation profile as PBMC, high cytolytic activity being associated with nylon wool non-adherent effector cells. Splenic effector cell cytotoxicity from cancer and non-cancer patients was enhanced following exposure to human interferon, and inhibited by treatment with cholera toxin and simple sugars. Furthermore, fractionation of spleen cells on Percoll discontinuous density gradients demonstrated natural cytotoxic activity to reside predominantly in the low density cell fractions, similar to that found with NK cells from peripheral blood. Thus the properties described here for human cytotoxic spleen cells are similar to those described for peripheral blood NK cells, suggesting these two effector cell populations to be closely related, if not identical.

Nonspecific cytotoxicity of vaccinia-induced peritoneal exudates in hamsters is mediated by Thy-1.2 homologue-positive cells distinct from NK cells and macrophages.

Vaccinia virus-induced peritoneal exudate cells (PEC) in the hamster were characterized with regard to cell type(s), target specificity, and expression of the T cell antigen, Thy 1.2 homologue. Hamsters were immunized intraperitoneally with vaccinia virus and cytotoxicity was measured against 51Cr-labeled targets in a 16-hr assay. PEC collected 4 days after immunization were cytotoxic for both baby hamster kidney cells (BHK) and herpes virus-infected BHK (BHKHSV). Both the nonadherent (lymphocyte) and adherent macrophage (MP) fractions of PEC were cytotoxic. Treatment of cells with a monoclonal anti-murine Thy 1.2 antibody (alpha-Thy 1.2) known to detect a Thy 1.2 homologue on hamster T cells, removed all of the cytotoxicity in both PEC fractions, whereas, cytotoxic spleen cells from the same animals were resistant to antibody treatment. Similarly, the cytotoxic cells in PEC induced by bacillus Calmette-Guérin were exclusively of the Thy 1.2 homologue-positive phenotype. Target specificities of Thy 1.2+ PEC and Thy 1.2- spleen cells were similar as evidenced by comparable activity against hamster BHK and BHKHSV targets and murine SV3T3 and YAC-1 targets. Previous studies have attributed the cytotoxicity of the adherent PEC to MP. However, as determined by immunofluorescence and morphological studies, treatments that enriched for MP decreased cytotoxic activity, whereas, procedures that enriched for lymphocytes enhanced cytotoxic activity suggesting that all cytotoxicity in PEC is mediated by a non-specific Thy 1.2 homologue positive lymphocyte (Thy 1.2+ CL). Thus our data support the conclusion that intraperitoneal inoculation of hamsters with vaccinia induces two distinctly compartmentalized phenotypes with similar cytotoxic characteristics--the Thy 1.2+ CL and the Thy 1.2 homologue-negative natural killer cell (NK) or NK-like cell in the peritoneum and in the spleen, respectively.

Dissociation of natural killer activity and antibody-dependent cellular cytotoxicity in spleen cells of tumor bearing mice.

Antibody-dependent cellular cytotoxicity (ADCC) increased with the development of tumors in C3H/He mice bearing spontaneous breast cancer or the syngeneic hepatoma MH-134 and in C57BL/6 mice bearing the syngeneic Lewis lung carcinoma 3LL. This cytotoxicity decreased after treatment with guinea pig, monoclonal IgM anti-Thy 1.2 serum and complement to the non-cancer level thus indicating that the increased ADCC in mice with cancer seems mainly attributable to cells with the Thy 1 antigen. On the other hand, NK activity decreased greatly when mice had tumors. Treatment with monoclonal IgM anti-Thy 1.2 serum and complement showed no significant influence on the natural killer (NK) activity of spleen cells of mice bearing MH-134 cancer, but in the 3LL-bearing mice the activity decreased significantly.

Effect of quinonyl-N-acetyl muramyl dipeptide on immune responses in tumor-bearing mice.

The efficacy of 6-O-QS-10-N-acetyl muramyl-L-valyl-D-isoglutamine methyl ester (quinonyl-MDP-66) for restoring impaired immune status was examined in mice bearing Lewis lung carcinoma. Quinonyl-MDP-66 suspended in phosphate-buffered saline was shown to restore the depressed allogeneic cell-mediated cytotoxicity of spleen cells from mice with Lewis lung carcinoma when the chemical was injected twice intraperitoneally, intravenously, or intratumorally. However, primary tumor size and the numbers of lung metastases were not affected when quinonyl-MDP-66 was administered under the present experimental conditions. Intraperitoneal injection of quinonyl-MDP-66 in mice with Lewis lung carcinoma enhanced host resistance to Listeria monocytogenes infection.

Cell-mediated cytotoxicity toward measles virus-infected target cells in randomly bred Syrian hamsters.

Cell-mediated cytotoxicity (CMC) toward measles virus-infected cells was studied by a (51)Cr release assay with spleen cells from hamsters inoculated with measles virus (strain Lec) or control antigen and with spleen cells from normal hamsters. Spleen cells from measles virus-inoculated hamsters showed greater CMC toward infected than toward noninfected target cells (designated specific CMC). Specific CMC was maximal 7 days after virus inoculation and was declining by 9 to 10 days. Effector cells were present in a nonadherent cell population. Specific CMC was reduced after treatments of effector cells with antithymocyte serum plus complement. The decrease in cytotoxicity was greater toward infected target cells than toward noninfected target cells. Treatment of infected target cells with antimeasles serum did not increase specific CMC by effector cells from the majority of virus-inoculated hamsters. CMC toward infected target cells by normal spleen cells (natural killer cells) or spleen cells from hamsters inoculated with control antigen was approximately the same as, or more often less than, CMC toward noninfected target cells. Natural killer cells were present in a nonadherent cell population. Treatment of natural killer cells with antithymocyte serum plus complement caused a similar decrease in cytotoxicity toward both infected and noninfected target cells. This study demonstrated virus-specific cellular cytotoxicity of effector spleen cells from measles virus-inoculated hamsters. Although the data were compatible with T cells as the source of effector cells in the virus-specific CMC, definitive identification could not be made. Additional membrane markers for better characterization of hamster lymphocyte subpopulations are required.

Generation of cytotoxic lymphocytes by SV40-induced antigens.

In order to study the correlation of in vivo tumor transplantation immunity and in vitro immunologic assays, cell-mediated cytotoxicity against SV40-transformed cells was studied in AL/N strain mice by using 51Cr-release assay. Killing of SV40-transformed AL/N fibroblast cells was observed by spleen cells of AL/N mice immunized with syngeneic SV40-transformed cells. Immunization with the solubilized SV40 tumor-specific transplantation antigen (TSTA) that induced transplantation immunity in vivo did not elicit cytotoxic spleen cells in vitro. However, the spleen cells from mice immunized with solubilized TSTA and then sensitized in vitro with SV40-transformed cells became cytotoxic against SV40-transformed fibroblasts. Similarly, SV40 TSTA (T antigen) purified by immunoprecipitation was able to prime the lymphocytes in AL/N mice: the primed lymphocytes could differentiate into cytotoxic lymphocytes upon in vitro stimulation by SV40-transformed cells. These data indicate that SV40 TSTA (T antigen) plays a role in the induction of cytotoxic lymphocytes.

Studies on natural killer (NK) cells. III. The effects of in vitro culture on spontaneous cytotoxicity of murine spleen cells.

In vitro culture of normal lymphohemopoietic cells is a complex event leading to the generation of a diverse group of effector cells. When murine spleen cells are cultured by themselves in fetal calf serum containing medium for up to 9 days, at least 3 distinct subpopulations of cytotoxic effectors can be distinguished on the basis of tumor target cell preference and expression of cell surface alloantigens. Fresh spleen cell suspensions contain 2 distinct NK cell types: NK-1.2+ NKA cells, which preferentially lyse lymphoma targets, and NK-1.2- NKB cells, which lyse nonlymphoma, mainly solid tumor targets. These NK cells account for all the cytolytic activity on day 0 of culture. NK-1.2+ NKA cells are very labile, and greater than or equal to 80% of their activity is lost within 48 hr of culture. By contrast, the activity of NKB cells increases in culture, up to 4-fold by day 6, and solid tumor target cells resistant to lysis by fresh spleen cells now become susceptible. In addition, an NK-1.2-, H-2+, Thy-1.2+, Ly-1.2+, Ly-2.2+ cytotoxic effector cell arises in culture from, or is dependent upon the presence of, NK-1.2-, Thy-1.2+ cells. This spontaneously arising "Tc" has a peak of activity between day 3 and 6 of culture, and kills a variety of lymphohemopoietic tumor cell types.

In vitro stimulation of mouse NK cell activity by phorbol esters: A pathway different from interferon-induced activation

Tumor promoters of the phorbol diester type were tested in vitro for their effect on mouse natural killer (NK) activity. The 12- O-tetradecanoyl-phorbol-13-acetate (TPA) significantly increased the spontaneous cytotoxicity of spleen cells preincubated with this reagent, with a maximal effect at 10–20 ng/ml. Higher concentrations of the weak tumor promoter phorbol-12, 13-dibenzoate (PDB) were required to display the same NK-enhancing property, while the inactive tumor promoter 4-αphorboldidecanoate was ineffective at any concentration. TPA-responding spleen cells displayed the same characteristics as classical NK cells: they were present in nude mice, did not adhere to nylon wool, bore the Thy-1 antigen, and displayed the same target cell specificity as interferon-activated NK cells. A major difference with the interferon-induced NK activation resides in the fact that the TPA-inducible increase in lytic activity does not require RNA and protein synthesis. Our results suggest two different NK activation pathways for IFN and phorbol esters.

Modulation of natural cytotoxicity by alloantibodies: IV. A comparative study of the activation of mouse spleen cell cytotoxicity by anti H-2 antisera, interferon, and mitogens

Comparisons were made between the characteristics of cytotoxicity activation in mouse spleen cells, induced by specific anti-H-2 antiserum, interferon (IF), interferon inducer (poly (I:C)), and mitogens (concanavalin A (Con A), pokeweed mitogen (PWM)). Important differences were found in the cytotoxicity activation associated with these agents: (a) The cytotoxic enhancing effect of anti-H-2 antiserum was comparatively rapid and was more pronounced in the first 4 hr of the assay, whereas, IF, poly (I:C), and the mitogens were most effective at 20 hr. (b) Anti-IF antiserum could neutrilize the stimulatory effects of IF and poly (I:C) on the cytotoxic activity of mouse spleen cells but had no influence on the stimulatory effects of anti-H-2 antiserum, PWM, and succinyl Con A. (c) The stimulatory effect of anti-H-2 antiserum was target specific and was observed when the K562 cell line was used as the target but not when YAC, P815, or WFu/G1 target cell lines were used. Stimulatory effects of all the other agents were nonspecific and did not depend on the target cells employed. (c) Cellular fractionation studies suggested that the alloantisera and interferon directly activated the natural killer (NK) cells without any participation of T-cells, B-cells, or macrophages. Both T-cells and NK cells, however, contributed to the mitogen-enhanced cytotoxicity of mouse spleen cells. These results indicated that the mechanism by which anti-H-2 antisera induce NK augmentation in mouse spleen cells is distinct from that of interferon and mitogens.

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The cytotoxic sensitivity of murine leukemia virus (MuLV)-infected and noninfected fibrosarcoma cells in syngeneic inbred WKA/Hok rats was compared by in vitro cell-mediated 51Cr release cytotoxicity assay. A highly significant increase in cytotoxic sensitivity of target cells was observe in MuLV-infected tumor cells as compared with noninfected cells when spleen cells from syngeneic tumor-bearing hosts (TBH) were used as a source of effector lymphocytes. The cytotoxicity of spleen cells against MuLV-infected tumor cells was specifically directed to the tumor-associated antigen (TAA), but not to the virus-associated antigen. However, there was no quantitative difference in the amount of TAA on the cell membranes between virus-infected and noninfected tumor cells as measured by a quantitative absorption test of anti-TAA serum. The cytotoxic activity of spleen cells from TBH against MuLV-infected tumor cells was abrogated by the treatment of anti-T-serum plus complement and significantly decreased after trypsin treatment. Spleen cells from normal rats given injections of immune sera from TBH acquired the cytotoxic activity against MuLV-infected tumor cells.

Sex-related differences in the pattern of coxsackievirus B-3-induced immune spleen cell cytotoxicity against virus-infected myofibers.

Spleen cells from adult BALB/c mice injected intraperitoneally with purified coxsackievirus B-3 were tested for cytotoxicity against 51Cr-labeled syngeneic infected and uninfected myofibers. Both male and female immune cells were active against uninfected targets; this reactivity was evident by day 3 of infection and persisted throughout the first week. However, we observed marked sex-related differences in immune cell cytotoxicities against infected myofibers. Males exhibited a strong T-lymphocyte response 4 to 7 days after infection. In contrast, females exhibited a weak response, and only infrequently were the immune spleen cells of females significantly more reactive against infected myofibers than against uninfected myofibers. The demonstration of a stronger effector cell response against infected myocardial cells in male mice correlates with the observation that clinical adult coxsackievirus B myocarditis and pericarditis occur predominantly in males.

Sex-related differences in the rapid production of cytotoxic spleen cells active against uninfected myofibers during Coxsackievirus B-3 infection.

Spleen cells from BALB/c mice immunized with a cardiotropic strain of Coxsackie B-3 virus were more cytolytic to uninfected myofibers than were spleen cells from mice immune to a noncardiotropic strain of the virus. Spleen cells immune to either virus were equally cytolytic to endothelial cells. Cytotoxicity was greater in female mice than in males. Analysis of individual reactivities showed that the male response was heterogeneous with only half of the animals developing cytolytic activity. All females responded. Early (day 3) cytolytic cells in both male and female mice appear to be natural killer (NK) cells, since they are not sensitive to anti-thy 1.2 or anti-Ig serum and complement, and lyse allogeneic (CBA) as well as syngeneic (BALB/c) targets. Later (days 4 to 6), the cytolytic cells in males become sensitive to anti-thy 1.2 serum and are restricted to lysis of syngeneic targets, while the cytolytic cells in females maintain the characteristics of NK cells.

Antibody-induced augmentation of murine natural killer cell activity.

Antibodies reactive with effector cells were shown to augment the cytotoxicity of spleen cells from athymic nude and euthymic mice. The addition of alloantibody to the assay or pretreatment of the effector cells with alloantibody resulted in increased cytotoxicity against the human cell K562, a relatively poor target for spontaneous mouse NK activity. When monoclonal antibodies were tested, cytotoxicity was markedly increased by some antibodies, such as anti-H-2, anti-la, and anti-Thy 1.2, while others had no effect. The degree of augmentation of cytotoxicity was dependent on the concentration of antibody added. Nylon-wool-nonadherent nu/nu splenic effector cells mediated the antibody-induced cytotoxicity and anti-asialo GMI plus complement abolished activity, indicating that the cells mediating the cytotoxicity were NK cells and not mature T cells, B cells or macrophages. When spleen cells from mice having different levels of NK activity were evaluated in this system, the magnitude of augmentation by antibody correlated with the level of spontaneous NK activity and no increased cytotoxicity was found with cell populations that had low spontaneous NK activity. Testing a panel of target cells, showed that certain human and mouse cell lines, with low to moderate susceptibility to spontaneous NK activity, were sensitive to antibody-induced NK-cell-mediated cytotoxicity whereas others were completely resistant. Both Fc-IgG receptor-positive and -negative cell lines were susceptible target cells. These results indicate that antibodies reactive with murine NK cells can increase their cytolytic activity.

Suppression of in vitro lymphocyte reactivity by cyclosporin A: existence of a population of drug-resistant cytotoxic lymphocytes.

Cyclosporin A (CS-A) is an unusual endecapeptide isolated from the fungi Cylindrocarpon lucidum Booth and Trichoderma polysporum. It is a potent immunosuppressive drug that prevents rejection of kidney and heart allografts whilst having a low myelotoxicity. Its mode of action is still unclear but its main target appears to be the T lymphocyte. The mixed lymphocyte reaction (MLR) and cell-mediated lymphocytoxicity (CML) used here are generally regarded as in vitro correlates of allograft rejection. Our data show that CS-A is a powerful inhibitor of MLR reactivity, regardless of whether the cells were obtained from normal or presensitized donors; that the CML of lymph node cells is likewise totally inhibited if the drug is added early during cell culture; and that, by contrast, the cytotoxic response of spleen cells from presensitized but not from normal mice is only partially inhibited, even with a tenfold increase in dose. It is therefore suggested that there exists a population of cytotoxic spleen cells that is relatively resistant to the action of this drug.

Augmentation of cell-mediated cytotoxicity to a rat lymphoma. III. In vitro stimulation of natural killer cells by a soluble factor.

The in vitro cytotoxicity of rat natural killer cells was augmented by the incubation of normal spleen cells with a soluble factor derived from the 24-h culture of spleen cells and various tumour cells. Cytotoxicity was augmented approximately 4-, 3- or 2-fold depending on whether the spleen cells were incubated with the factor for 18 or 3 h or during the course of the cytotoxicity assay respectively. The factor was produced in cultures containing syngeneic, allogeneic or xenogeneic tumours and no correlation was observed between the ability of tumour cells to induce the production of the factor and their susceptibility to lysis in the 4-h cytotoxicity assay. The factor was produced in cultures of spleen cells of several rat strains, and it stimulated the cytotoxicity of syngeneic and allogeneic spleen cells. The cells responsive to stimulation closely resembled NK cells of normal rats. Culture supernatants containing the stimulatory factor possessed anti-viral activity, and evidence suggested that NK cells were stimulated by a factor resembling a type-I interferon. The relevance of these findings to the stimulation of NK cells by tumours in vivo is discussed.

Dissociation of biological activities of Corynebacterium parvum by chemical fractionation

Fractionation of Corynebacterium parvum (C. parvum) by phenol-water extraction resulted in components which differentially affected the cell-mediated cytotoxic response to tumor alloantigens in mice. The residue of the extraction (Fraction C) and its light component (Fraction D) retained all of the activities of the whole micro-organism, i.e. inhibition of primary spleen cell cytotoxicity, inhibition of in vitro memory cell-mediated cytotoxicity (CMC) and generation of macrophage-like suppressor cells. Administration of the phenol phase extracted material (Fraction A) inhibited both the primary and memory cytotoxic responses, but did not elicit suppressor macrophages. Destruction of carbohydrate components of Fraction D significantly reduced its ability to inhibit the memory response and to elicit suppressor macrophages. In contrast, its ability to inhibit the primary cytotoxic reponse was unaffected. This study clearly demonstrates that the several effects of C. parvum administration on the cell-mediated cytotoxic response to tumor alloantigens can be associated with different components of the organism. Furthermore, the dissociation of the effects indicate that the generation of primary cytotoxic lymphocytes, the priming of memory cytotoxic lymphocytes, and the induction of regulatory macrophages are not necessarily linked during the generation of a normal cytotoxic response.

Sensitivity of rat heart endothelial and myocardial cells to alloimmune lymphocytes and to alloantibody-dependent cellular cytotoxicity

We have attempted to define the structural target components for the two cytotoxic mechanisms in rejecting rat heart allograft: antibody-independent (direct) and antibody-dependent (ADCC) cellular cytotoxicity. Immune spleen cells and alloantibody were obtained at 1 and 2 weeks after heart allotransplantation, respectively. The cytotoxicity of immune spleen cells, with and without alloantibody, was tested against endothelial- and myocardial-enriched heart cell populations. We found endothelial cells to be sensitive to direct cellular cytotoxicity, most likely mediated by T lymphocytes, while myocardial cells were sensitive to ADCC. Both reactions were shown to be immunologically specific.

In Vitro Heterologous Cytotoxicity by T Effector Cells from Mice Immunized with Sindbis Virus

Abstract An in vitro correlate of cell-mediated cross-protection among α-viruses was demonstrated by cytotoxicity of Sindbis-immune spleen cells from mice to both Sindbis and Semliki Forest virus (SFV)-infected target cells. This cytotoxicity was shown to be mediated by the T cell population of the spleen and was independent of the presence of macrophages or B cells. The time when the level of the lymphocyte-mediated cytotoxicity (LMC) to SFV-infected cells was maximal coincides with the time when immunity to SFV is maximal in vivo, as reported previously, and when adoptive immunity to SFV can be transferred. After one i.p. injection of Sindbis virus, the level of homologous LMC was higher than the level of heterologous LMC. However, following a second injection of Sindbis virus as immunogen, at a time when the mice are cross-protected to SFV, the heterologous LMC was considerably higher than homologous LMC. We propose that there is suppression of the effector T cells specific for Sindbis-infected cells after the second immunizing injection, probably by homologous antibody. In contrast, there appears to be an anamnestic cell-mediated response to SFV.