Suppression of cellular immunity to Listeria monocytogenes by activated macrophages: mediation by prostaglandins.

We previously demonstrated the suppression of cell-mediated immunity to Listeria monocytogenes by Pseudomonas aeruginosa-induced, macrophage-like cells. The present study was undertaken to evaluate the mechanism for this suppression. P. aeruginosa supernatant was shown to activate macrophages by the criteria of increased bactericidal capacities and increased attachment to glass surfaces. Acquired cellular resistance to L. monocytogenes could also be inhibited by macrophages from L. monocytogenes-pretreated mice. The depression of acquired immunity by P. aeruginosa- or L. monocytogenes-activated macrophages did not appear to be due to a reduction of antigenic stimulus after nonspecific macrophage activation. In contrast, our findings suggest that suppression is mediated by activated macrophages through a prostaglandin-dependent mechanism. In vivo administration of aspirin blocked the immunosuppressive effect of P. aeruginosa- or L. monocytogenes-activated cells. Moreover, the suppressive activity of supernatants of macrophages from Listeria-infected mice was reversed when indomethacin was present during supernatant generation. Finally, prostaglandin E1 treatment in vivo profoundly inhibited the induction of cell-mediated immunity to L. monocytogenes. The possible role and mechanism of prostaglandin in suppressing cellular immunity to intracellular bacteria are discussed.     

Concomitant presence of anti-tumor effector cells and suppressor cells in the spleen of tumor-bearing mice : the nature of suppressor cells

In order to investigate the immunological responsiveness of tumor-bearing hosts to tumor cells, splenic suppressor cells from Ehrlich tumor-bearing mice that inhibited anti-tumor effector cell activity were characterized. In vitro cell-mediated cytoxicity and cytostasis assays were performed to test for the existence of anti-tumor immunity. suppressive activity assayed by cell mixture experiments became apparent with decline of anti-tumor immunity and progressive tumor growth. The cells mediating the suppression were found to be nylon wool column adherent T cells and inhibited T cell dependent cytotoxicity rather than non-T cell dependent cytostasis. In vivo cell transfer experiments demonstrated that intravenous injection of suppressor cells to a host already inoculated with tumor cells mixed with antitumor effector cells resulted in significant enhancement of tumor growth. This inhibition of in vivo neutralization assay be suppressor cells was found in not only allogeneic but also syngeneic tumor system. Splenectomy at the time of tumor resection endowed the host with stronger resistance against subsequent reinoculated tumor than sham-splenectomy did, reflected by prolonged survival times. These results suggest that splenectomy combined with surgical removal of the tumor is a useful treatment of clinical malignancies.     

High nonspecific reactivity of normal lymphocytes against mycoplasma-infected target cells in cytotoxicity assays.

Several rat tumor cell cultures were deliberately infected with three species of mycoplasma commonly found as contaminants of cell lines grown in vitro, and the effect of mycoplasma infection on the results of cytotoxicity assays was examined. Lymph node cells and spleen cells from normal animals showed an apparently high spontaneous cytotoxic activity against tumor cells infected with either M. arginini or M. hyorhinis, but the reactivity against cells infected with M. orale was not significantly higher than that against uninfected cells. The high reactivity towards tumor cells infected with M. arginini and M. hyorhinis bore a close resemblence to natural cell-mediated immunity in that spleen cells were much more reactive than lymph node cells, spleen cells from nude mice were as effective as spleen cells from normal mice, and the reaction crossed both strain and species barriers. However, closer examination revealed that the cytotoxic effects were directly caused by depletion of arginine or other essential nutrients from the medium. These findings imply that a cautious approach should be taken when interpreting certain aspects of spontaneous cell-mediated cytotoxicity, and that the greatest care be taken to ensure that the cells used as targets in any cytotoxicity test are mycoplasma-free.     

Normal natural killer cell activity in Hodgkin''s disease patients in remission.

Patients with untreated active Hodgkin's disease (HD) have a defect in cell-mediated immunity. After therapy many HD patients still have long lasting abnormalities in T cell number and function. We examined whether NK activity is also permanently impaired in HD patients in remission. The mean NK activity of peripheral blood lymphocytes from 42 patients who were disease-free for 6-150 months was not different from that of healthy controls. Augmentation of NK activity after treatment of the cells by interferon in vitro was equal for patients and controls. Normal NK activity in HD in remission was independent of disease stage, age, remission duration and mode of therapy. Measuring PHA-induced lymphocyte proliferation and NK activity simultaneously demonstrates that patients with impaired cell-mediated immunity do not have concomitant reduction of NK activity. We conclude that NK activity in HD in remission is independent of decreased T cell mediated immunity. In addition, NK is resistant to long term suppression by the chemotherapy and radiation protocols that are used in HD.     

Human dendritic cell mediated cytotoxicity against breast carcinoma cells in vitro

Dendritic cells (DC) are an important subset of antigen-presenting cells characterized by their potent capacity to activate immunologically na?ve T cells. However, their role in effector function in tumor resistance is less well characterized. We report here that activated human peripheral blood DC acquire a potent antitumor effect against breast cancer cell lines in vitro, leading to growth inhibition and apoptosis of the tumor cell. The antitumor effect of DC was augmented by proinflammatory stimuli induced by lipopolysaccharide (LPS) treatment. Tumor necrosis factor alpha (TNF-alpha) produced after DC activation was responsible for the antitumor activity of DC. Interferon-gamma, interleukin-15, or LPS treatment of DC markedly augmented the effector function of DC against most of the breast cells, indicating heterogeneity of the tumor and its susceptibility to cytokine-mediated damage. Treatment of LPS-activated DC or cell-free supernatant with anti-human TNF-alpha significantly reduces the antitumor effect against the tumor cells tested. These results suggest that in addition to their predominant role as immune regulatory cells, DC could serve as innate effector cells in tumor immunity.     

Innate and adaptive immunity to tumors: IL-12 is required for optimal responses

We have investigated the importance of endogenously produced IL-12 in innate and adaptive immunity to tumor transplants. The immunogenic lymphoma RMA and its TAP-deficient variant RMA-S were tested for rejection responses by normal and IL-12-deficient mice. IL-12 was crucial for the immunity induced by one immunization with irradiated RMA cells, as well as for in vivo priming of a CTL response in mixed lymphocyte tumor cultures against this MHC class I-expressing tumor. The defective in vivo response could be overcome by multiple immunizations. In further studies of in vitro CTL responses, we found that IL-12 production from either the antigen-pulsed dendritic cells or from host cells was necessary to obtain strong CTL responses. In the complete absence of IL-12, no or only very weak responses could be detected. NK cell-mediated innate resistance, as assessed in non-immunized mice inoculated with a threshold dose of RMA-S cells, also required IL-12. However, NK cells with reduced activity were present in IL-12-deficient mice and contributed to innate resistance, as demonstrated with lower cell dose challenges. In conclusion, IL-12 is required for optimal adaptive and innate responses against tumors.     

Deficient tumor-specific immunity in old mice: in vivo mediation by suppressor cells, and correction of the defect by interleukin 2 supplementation in vitro but not in vivo

The capacity of old (18-24 months) C57BL/6 mice to develop an immune reaction against MC-B6-1 fibrosarcoma cells was studied using in vivo adoptive transfer experiments (Winn assay) and in vitro T cell-mediated cytotoxicity test. Anti-tumor immunity was found to decline with age, as indicated by a decreased anti-tumor growth T cell activity. A suppressive activity was also found present in the splenic T cell population of old mice which can inhibit the in vivo generation of immune T cells in young mice. These suppressors, or their precursors, were resistant to cyclophosphamide treatment and were effective only when administered 3 days before the immunization of young mice. These mice developed immune T cells perfectly when the suppressors were administered 3 days after immunization, indicating that suppressors may act at an early phase of T cell activation. The protective activity of T cells in vivo correlated well with the in vitro T cell cytotoxicity for MC-B6-1 tumor cells, as both were depressed in old mice. Exogenous interleukin 2 (IL 2) addition during the 4-day culture period partially restored the low cytotoxic activity of old immunized lymphocytes, suggesting that specific clones were present but that a lack of IL 2 limited their expansion. However, in vivo supplementation with IL 2 administered after immunization did not increase the protection mediated by old immunized T cells but, rather, increased the suppression. This work demonstrates the presence of a T cell suppressive activity in the spleen of old mice but also indicates that precursors of cytotoxic cells are generated by the immunization. It seems that in vitro IL 2 addition increases cytotoxic cells while in vivo IL 2 administration amplifies the development of suppressor cells generated during immunization of aged mice.     

A canine model of induced purine nucleoside phosphorylase deficiency.

Since the acquired immunodeficiency syndrome (AIDS) is characterized by opportunistic infections and malignancies indicative of a profound suppression in cell-mediated immunity, we investigated the antibody-dependent cell-mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells of patients with AIDS against chicken red blood cells (CRBC). A marked decrease in ADCC-CRBC activity was observed from patients with AIDS as compared to healthy controls. Furthermore, suppression in ADCC activity was seen when mononuclear cells from healthy subjects were assayed using media containing 25% or 40% sera from AIDS patients. Two of two patients with AIDS and impaired ADCC-CRBC activity were also found to have in vivo impaired reticuloendothelial system Fc-specific clearance of 51Cr-labelled, anti-Rho (D) IgG-sensitized autologous erythrocytes. These data provide further evidence of monocyte-macrophage dysfunction in AIDS and help explain the widespread occurrence of opportunistic pathogens in AIDS.     

Protection against reticuloendotheliosis virus-strain T tumours is associated with JMV-1 culture supernatant-enhanced natural killer cell activity

One-day-old chicks were inoculated intraperitoneally with cell-free JMV-1 culture supernatant and were subsequently challenged with a lethal dose of RECC-CU60 tumour cells at either 7 or 12 days of age. A significant reduction in mortality was evidenced in chicks that had been inoculated with JMV-1 culture supernatant prior to RECC-CU60 tumour cell challenge at 12 days of age, compared with all other treatment groups. Reducing the serum content of JMV-1 culture medium or changing the base medium in which the JMV-1 cells were grown did not affect the reduction in mortality, demonstrating that the protective effects against RECC-CU60 tumour cell challenge were a result of factors secreted into the culture medium by JMV-1 cells. Development of natural killer (NK) cell activity correlated with findings in the RECC-CU60 tumour cell challenge studies. Inoculation of 1-day-old chicks with JMV-1 culture supernatant resulted in a significant increase in NK cell activity in spleens taken from JMV-1 inoculated chicks 12 days later, when compared with spleens taken from chicks in all other treatment groups. Inoculation of chicks with RECC-CU60 tumour cells caused a suppression of their NK cell activity. Treatment of chicks with JMV-1 supernatant at day of age, prior to RECC-CU60 tumour cell challenge restored and significantly enhanced their NK cell activity. These results suggest that the JMV-1 cell line secretes lymphokines that protect against RECC-CU60 tumour cell challenge by directly or indirectly stimulating NK cell activity.     

Immunological tolerance to lymphocytic choriomeningitis virus in neonatally infected virus carrier mice: evidence supporting a clonal inactivation mechanism.

Previous studies have shown that no cell-mediated immunity against LCM virus-infected cells can be detected in neonatally established LCM virus carrier mice suggesting that they are immunologically tolerant to virally-altered cell membrane antigens. In this communication experiments are described aimed at analyzing the mechanism. Virus-specific cell-mediated immunity was assessed by 51Cr release and target cell reduction assays. Attempts to demonstrate cells in spleens of CBA/J carrier mice able to suppress in syngeneic recipients the induction or the effector phase of the cytotoxic T-cell response against LCM virus-infected cells were unsuccessful. Also, no factors were detected in CBA/J and C57BL/6J carrier mice, either spleen cell-associated or free in the circulation, which would block the activity of cytotoxic T-lymphocytes against LCM virus-infected syngeneic target cells. The results indicate that inability of LCM virus carrier mice to act immunologically against virus-infected target cells is due to deletion or irreversible inactivation of T lymphocytes carrying receptors for virally altered cell membrane antigens.     

Intrasplenic immunization for the induction of humoral and cell-mediated immunity to nitrocellulose-bound antigen

Intrasplenic immunization of mice has been shown to induce both specific humoral and cell-mediated immunity to minute amounts of nitrocellulose-bound antigen, electroblotted from sodium dodecyl sulfate-polyacrylamide gels. The test antigens used were aberrantly expressed molecules immunoprecipitated from the lysate of highly immunogenic ('xenogenized') murine lymphoma cells, derived by mutagenesis from a parental, nonimmunogenic cell line. The stained bands of nitrocellulose blots corresponding to the appropriate molecular weights were cut out and the resulting strips deposited in the spleens of recipient mice on three occasions at 15 day intervals. 2 weeks later, the antibody response in the serum was analyzed using a standard ELISA procedure. Cell-mediated immunity was investigated in vitro in terms of cytotoxic T lymphocyte (CTL) activity to radiolabeled xenogenized tumor target cells. In vivo, the immunized mice were assayed for their ability to mount a delayed-type hypersensitivity (DTH) response following footpad challenge with the xenogenized tumor. Our results confirm previous data that the intrasplenic deposition of minute amounts of protein immobilized on a solid matrix effectively stimulates production of specific antibodies. In addition, our results demonstrate that this procedure may also result in the development of T cell-dependent responses detectable in in vitro and in vivo assays of cell-mediated immunity.     

T lymphocyte function as the principal target of lymphocytic choriomeningitis virus-induced immunosuppression.

Plaque-forming cell responses against sheep erythrocytes, Escherichia coli lipopolysaccharide, pneumococcal polysaccharide, and polyvinylpyrrolidone were examined in mice infected with lymphocytic choriomeningitis virus. A 92 to 96 percent reduction of the thymus-dependent anti-sheep erythrocyte responses was observed 2 to 4 weeks after infection. However, the thymus-independent responses against the three other antigens were close to normal at all stages of the infetion. Studies on allograft immunity of infected C3H mice against DBA/2 mastocytoma cells revealed a severe suppression of the T cell-mediated cytotoxic response which was temporally related to the impaired humoral responsiveness against sheep erythrocytes. The capacity of spleen cells from infected mice to restore immune responsiveness of lethally irradiated recipients against sheep erythrocytes was significantly reduced. The adoptive responses, however, were clearly improved when normal thymus cells were added to the inferior spleen cells. Moreover, it appeared that the spleen cells from immunosuppressed donor mice could not confer suppression to normal lymphoid cells. The presented findings are consistent with the assumption that a numeric deficiency of T cells, or cells belonging to some T cell subpopulation, is the primary cause of lymphocytic choriomeningitis virus-induced immunosuppression.     

Murine B16 melanoma vaccination-induced tumor immunity: identification of specific immune cells and functions involved.

Vaccination using inactivated B16 melanoma cells that have been treated in vitro for > 2 weeks with interferon-alpha (IFN-alpha) (B16alpha cells) has been shown to elicit a protective host antitumor immunity. In these studies, vaccination with B16alpha cells has been shown to provide protection against primary B16 tumor challenge, established B16 tumors, and metastatic B16 tumors. Specific immune cells and factors that might mediate this tumor immunity have now been evaluated. Macrophage depletion studies suggest that macrophage function is required for expression of tumor immunity either for processing of antigen or for cytokine production but that macrophage function is not involved in direct cytotoxicity against the B16 challenge tumor. CD8(+) T cell depletion studies show that cytotoxic T cell function is required for expression of tumor immunity. Syngeneic knockout mouse experiments offer further insights into the immune cells and factors that mediate the development and expression of tumor immunity. First, interleukin-12 (IL-12) knockout mouse experiments identify IL-12 as an important cytokine in mediating the development of tumor immunity. Second, specific knockout mouse experiments show that tumor immunity requires the function of CD4(+) T cells, CD8(+) T cells, and natural killer (NK) cells. Third, specific knockout mouse experiments show that tumor immunity does not require the function of B cells. The results suggest that vaccination with inactivated B16alpha cells induces an active, cell-mediated immunity to B16 melanoma cells. The tumor vaccination protocol with B16alpha cell vaccinations establishes a potent tumor immunity against B16 melanoma tumors in mice and may serve as a model for induction of tumor immunity against primary or secondary melanoma tumors in humans.     

Correlation of murine susceptibility to tumor, parasite and bacterial challenge with altered cell-mediated immunity following systemic exposure to the tumor promoter phorbol myristate acetate

Phorbol myristate acetate (PMA), the most potent of the tumor promoting phorbol diesters, modulates function in several immunoresponsive cells following in vitro exposure. Since suppression of cellular mechanisms capable of limiting tumors and infections can adversely affect health, these experiments were designed to evaluate relevant components of cell-mediated immunity (CMI) following in vivo PMA exposure, and to determine the biological significance of any alterations utilizing assays of host resistance. Adult, female B6C3F1 mice were administered 0.2, 2.0, 20.0 or 40.0 micrograms PMA/g body weight subcutaneously over a two-week period. Mechanisms of cell-mediated host resistance were assessed by quantitating natural killer (NK), cytotoxic T-lymphocyte (CTL) and macrophage-mediated lysis of radiolabelled tumor target cells, and macrophage-induced cytostasis in tumor cell populations. Macrophages from PMA-treated mice were cytostatic to tumor cells, inhibiting up to 90% of growth in cultured tumor cells, but were not tumoricidal. Furthermore, pyran-elicited (primed) macrophages, which are activated to fully cytotoxic states by in vitro exposure to lipopolysaccharide, were inhibited in tumoricidal activation by in vivo PMA exposure. The induction of responsive but not cytotoxic macrophages by in vivo PMA exposure is consistent with the enhanced resistance to Listeria bacterial challenge, and increased susceptibility to B16F10 tumor and Trichinella parasitic challenges observed in these mice. Furthermore, previous reports of decreased in vitro NK activity following in vivo PMA exposure and present observations of correlative decreases of in vivo NK activity (55% decrease in mice exposed to 20 micrograms PMA/g) suggest an important role for NK activity in limiting in vivo B16F10 melanoma growth. CTL effector function was less susceptible to PMA-induced suppression than NK function at similar dosages, further supporting a predominant role of macrophages and NK cells or possibly other effector functions in the resistance to Listeria, Trichinella, or B16F10 challenge. Nevertheless, significant suppressive effects of PMA on CTL function at higher dosages cannot be excluded as contributing to altered host resistance to these agents. These studies demonstrate that in vivo exposure to PMA can modify cell-mediated mechanisms of host resistance with coincident alterations in the incidence of infections and tumors.     

Suppression of cell-mediated immunity after infection with attenuated rubella virus.

The effects of attenuated rubella virus infection upon cell-mediated immunity of human volunteers were studied. The volunteers received the vaccine either by nose drops or by the subcutaneous route. Changes in cell-mediated immunity in terms of delayed cutaneous sensitivity to recall antigens, phytohemagglutination stimulation, and spontaneous migration inhibitory factor-like activity were studied at various time periods after infection. Spontaneous migration inhibitory factor-like activity was studied on supernatants of the lymphocytes obtained from the volunteers and incubated for 72 h in the absence of any antigens. A significant proportion of the volunteers showed suppression of one or more parameters of cell-medicated immunity tested by week 2 of infection compared to the control; however, there was no correlation between suppression of the various parameters studied. No difference was noticed in the incidence of cell-mediated immunity suppression between nose drops and subcutaneous route groups.     

Blood transfusion-induced suppression of cellular immunity in man

The effect of planned blood transfusions on cell-mediated immunity was studied in previously nontransfused prospective kidney graft recipients. Following transfusion of washed erythrocytes a marked suppression of cellular immunity was found, indicated by reduced response to mitogenic (PHA, Con A, PWM) and antigenic stimulation (Ag-C containing PPD, tetanus toxoid, streptolysin, mumps, vaccinia antigen). A second transfusion led to a more pronounced and prolonged immunosuppression. No suppression was found when autologous blood was applied to volunteers. Preliminary results show autologous and allogeneic MLR suppression when mitomycin-C treated patient cells taken after transfusion are added. Our findings indicate that blood transfusion-induced suppression of cell-mediated immunity might be caused by an unspecific suppressor cell.     

Characterization of immunoglobulins eluted from hamster SV40 tumors.

In order to determine if progressively growing tumors specifically bind antitumor antibody, which might play a role in blocking tumor specific cell-mediated immunity, low pH elutions were performed on autochthonous and transplantable SV40 tumors growing in situ for various lengths of time. Ouchterlony analysis revealed that IgG2 was present in all tumor eluates, while IgG1 was present only in eluates from tumors that grew in situ for more than sixty days. IgA and IgM were also found in most eluates but their presence did not correlate with duration of in situ growth. While no antibody to cell surface antigens could be detected, the eluates did possess antibody activity directed to the SV40 nuclear T-antigen. The tumor eluates did not block in vitro lymphocyte dependent microcytotoxicity of immune lymphocytes against cultured SV40 tumor cells. The biological role and significance of different classes of immunoglobulins bound to the surface of growing tumor cells is still poorly understood and requires further study.     

Pathogenesis of Rinderpest Virus Infection in Rabbits II. Effect of Rinderpest Virus on the Immune Functions of Rabbits

Rinderpest virus infection was shown to induce marked suppression of both humoral antibody response and cell-mediated immunity in rabbits. The virus exhibited a suppressive effect on primary antibody response as indicated by a decrease in numbers of plaque-forming cells (immunoglobulin [Ig]M) and hemagglutinating antibody titers of both IgM and IgG types to sheep red blood cells, whereas there was no detectable effect of the virus on the production of memory cells. Virus-induced suppression of cell-mediated immunity was demonstrated by a decreased rate of proliferative response of peripheral lymphocytes to phytohemagglutinin stimulus and by a depression of delayed-type skin reactions to purified protein derivative. Such suppressive effects were indicated to persist for 14 days or longer. Alteration in phagocytic activity of the reticuloendothelial system was not observed. The relevance of the virus-induced histological lesions in the lymphoid tissues to the virus-induced immunosuppression was discussed.