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.     

Systemic immunosuppression following a single pharyngeal aspiration of 1,2:5,6-dibenzanthracene in female B6C3F1 mice

1,2:5,6-Dibenzanthracene (DBA) is ubiquitous in our environment as a contaminant produced by incomplete combustion of organics from sources such as forest fires, cigarette smoke, and asphalt paving, and it is more immunosuppressive of the T-dependent antibody-forming cell (AFC) response than the well-studied polycyclic aromatic hydrocarbon, benzo(a)pyrene. The systemic immunosuppressive effects of DBA were investigated following a single pharyngeal aspiration (pa) in female B₆C₃F₁ mice. The immunotoxic effects of DBA were evaluated using numerous assays of varying complexity to evaluate innate (natural killer [NK] cell activity), cell-mediated (T-lymphocyte proliferation, mixed leukocyte response [MLR], cytotoxic T-lymphocyte [CTL] activity, delayed-type hypersensitivity [DTH]), and humoral immunity (B-lymphocyte proliferation, T-dependent antibody responses). A single pa of DBA at doses up to 30?mg/kg had no effect on NK cell activity, anti-CD3 antibody-mediated T-lymphocyte proliferation, the MLR, or B-lymphocyte proliferation. DBA at 30?mg/kg suppressed Concanavalin A (ConA)-stimulated T-lymphocyte proliferation and the CTL response. DBA exposure reduced cytokine production in spleen cell culture supernatants after in vitro stimulation with ConA or lipopolysaccharide (LPS). Immunosuppression was observed at lower doses in the holistic assays. The DTH response to Candida albicans was significantly decreased at 3.0?mg/?kg DBA, while the AFC response was intermittently suppressed at 1.0?mg/kg, with no effect observed at 0.3?mg/kg. These results demonstrate that a single pa of DBA produces systemic immunotoxicity, and of the assays utilized, the holistic assays (i.e., DTH, AFC) appear to be most sensitive to the immunosuppressive effects of DBA.     

An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles

Here, we investigated whether titanium dioxide (TiO?) nanoparticles affect in vivo tumor growth through the modulation of mononuclear leukocytes. In vitro lymphocyte proliferation by lipopolysaccharide (LPS) or concanavalin A (ConA) was reduced by < 25 nm TiO? with a dose-dependent manner. Similarly, TiO? nanoparticles inhibited nitric oxide (NO) production from bone marrow-derived macrophages obtained from na?ve mice. When mice were intraperitoneally (IP) injected with < 25 or < 100 nm TiO? once a day for 7 days, total cell number of splenocytes was reduced in the spleen of TiO? nanoparticle-exposed mice. Both CD4+ and CD8+ T-lymphocyte numbers were significantly decreased and B-lymphocyte development was retarded by host exposure to the TiO? nanoparticles. LPS-stimulated spleen cell proliferation was significantly reduced by host exposure to < 25 or < 100 nm TiO?, but no changes were detected in ConA-stimulated spleen cell proliferation. Further, LPS-stimulated cytokine production by peritoneal macrophages and the percentage of NK1.1+ natural killer cells among splenocytes was reduced by the host exposures to the TiO? nanoparticles. When mice were IP injected with TiO? nanoparticles once a day for 28 days prior to the subcutaneous implantation of B16F10 melanoma cells, tumor growth was subsequently significantly increased. Collectively, these results show that TiO? nanoparticles may damage the development and proliferation of B- and T-lymphocytes, reduce the activity of macrophages, and decrease natural killer (NK) cell population levels, outcomes that appear to lead to an increase in tumor growth in situ. These studies allow us to suggest that TiO? nanoparticles might have the potential to enhance tumor growth through immunomodulation of B- and T-lymphocytes, macrophages, and NK cells.     

Immunological Consequences of In Vitro Exposure to Lysergic Acid Diethylamide (LSD)

The ability of lysergic acid diethylamide (LSD) to alter immune function after direct in vitro exposure was examined. It was demonstrated that LSD is able to suppress the proliferation of B-lymphocytes; the production of the cytokines IL-2, IL-4, and IL-6; and the induction of cytotoxic T-lymphocytes at a concentration of 100 μM. In vitro exposure to LSD had differential effects on natural killer (NK) cell activity, with significant enhancement of both basal and IL-2-augmented NK cell function at concentrations between 0.0001 and 0.1 μM, and suppression of NK response at 100 μM. These results demonstrate that LSD may have a direct effect on components of the immune system at concentrations that may be reached upon human exposure.     

Cytoreductive conditioning for severe combined immunodeficiency – help or hindrance?

Use of chemotherapy-based conditioning-facilitated engraftment in patients with severe combined immunodeficiency (SCID) is contentious. In T- and NK lymphocyte-negative, B-lymphocyte-positive (T-B+NK+) and T-B-NK+ SCID, the osteo-medullary space is occupied by recipient hematopoietic stem cells and mature B-lymphocytes. The thymic niche is empty in T-B+NK+ SCID but fully occupied by developmentally arrested T-lymphocyte precursors in T-B-NK+ SCID. The outcome of infusion of donor stem cells differs and is dependent on genetic defect and the lymphocyte developmental arrest stage. At best, donor hematopoietic stem cell osteo-medullary engraftment induces normal B-lymphocyte function and long-term thymopoiesis; at worst, peripheral expansion of donor T-lymphocytes from the stem cell source results in a restricted T-lymphocyte receptor repertoire with possible B-lymphocyte failure. Conditioning improves immunoreconstitution but causes short- and long-term toxicities, and increased mortality. Newborn screening for SCID will propel the search for safe, effective methods of achieving donor cell engraftment and full immunoreconstitution without toxic sequalae.     

Tumor associated antigen and interleukin-12 mRNA transfected dendritic cells enhance effector function of natural killer cells and antigen specific T-cells

Immunotherapy aiming at the combined activation of tumor associated antigen (TAA) specific cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells may be crucial to eradicate both MHC-I positive and negative tumors. Vaccination with mature dendritic cells (DC) transfected with mRNA encoding for TAA and the pro-inflammatory cytokines interleukin (IL)-12 and IL-18 may increase NK cell and TAA specific CTL activity. We demonstrate here that IL-12 over-expressing human DC induces increased NK cell activation and effector function and confirm the increase in TAA specific CTL by TAA/IL-12 double transfected DC. The effects of IL-18 transfection were limited to phenotypic activation and down-regulation of tissue homing receptors and did not add to the effect of IL-12 on NK cell effector function. In conclusion, co-transfection of TAA and IL-12 mRNA into mature DCs offers a vaccine for the induction of an anti-tumor immune response mediated by CTL and NK effector cells.     

Differential effect of beta-endorphin on three human cytotoxic cell populations

We have examined in vitro the effect of the proopiomelanocortin gene product, beta-endorphin (bE), on the cytotoxic activity of natural killer (NK) cells, lymphokine activated killer (LAK) cells and cytotoxic T-lymphocytes (CTL). Our studies show that bE reproducibly suppressed LAK cytotoxic activity in all donors tested. The effect of bE on the generation of CTL varied, and was negligible on CTL cytotoxic function. Our study also confirms the variable nature of the effects of bE on NK cytotoxicity. In all instances, the effects of bE were generally small, but could be blocked by opioid receptor antagonists, or by prior heat-inactivation of the peptide. The magnitude of the effects was greatest at low effector:target ratios in all of the three systems studied. These results support the emerging body of evidence that the neuroendocrine system may influence host defense mechanisms mediated by cytotoxic cells.     

T-Lymphocyte and B-lymphocyte dichotomy in anuran amphibians: III. Assessment and identification of inducible killer T-lymphocytes (IKTL) and spontaneous killer T-lymphocytes (SKTL)

We have established the existence of alloreactive inducible killer (IK) T-lymphocytes in $\text{Rana}$$\text{pipiens}$ by injecting immunogenic concentrations of allogeneic frog erythrocytes (RBC). Assessment of specific IK activity was determined microscopically, observing effector-target conjugate formation, and spectrophotometrically as released hemoglobin (Hb) from lysed targets (RBC). The presence of spontaneous killer (SK) T-lymphocyte activity was also determined using unimmunized frogs and similar assay conditions. Assays using rabbit anti-frog Thy-1.1 antiserum inhibition, but not E-rosetted T-lymphocyte depletion, confirmed the T-lymphocyte category of both effector cell populations in $\text{Rana}$$\text{pipiens}$. For IK activity, we determined the 1) best priming doses, 2) best effector cell source (peripheral blood), 3) best priming route (intraperitoneal), 4) kinetics of immunity development, and 5) kinetics of lysis. Kinetics of lysis and organ distribution for spontaneous killer cells were also determined. Our results may assist 1) in establishing the evolutionary origin of cytotoxic T-lymphocytes (CTL) and natural killer (NK) cells, and 2) in predicting where the capacity of immuno-surveillance against modified-self appeared in phylogeny. The implications are important for understanding origins of mechanisms of resistance against neoplastic conditions.     

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn's neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.     

Immunological studies of NK cell-deficient beige mice. II. Analysis of T-lymphocyte functions in beige mice

Lymphocytes from natural killer (NK) cell-deficient beige mice have a poor ability to induce many different forms of graft-versus-host reaction (GvHR). In this study, we have examined whether this defect could reflect an associated abnormality in beige T-lymphocyte function. Compared with normal, congenic C57Bl/6 (B6) mice, beige mice had similar numbers and proportions of T-cell subsets and generated normal allospecific delayed-type hypersensitivity (DTH) responses in vivo. In addition, beige mice developed normal or enhanced DTH responses to the protein antigen, ovalbumin (OVA), and were fully susceptible to the induction of tolerance by feeding OVA. Beige lymphocytes recirculated normally in vivo and showed enhanced proliferative responses to mitogens and alloantigens in vitro. In contrast, beige responder cells generated poor cytotoxic T-lymphocyte (CTL) responses in vitro and had quantitatively identical defects in CTL and NK cell activation after alloimmunization in vivo. These results suggest that although many effector and regulatory T-cell functions are normal in beige mice, the NK-cell defect in these animals is paralleled by impaired CTL activity. We suggest that abnormal T-cell function accounts for the inability of beige lymphocytes to induce GvHR.     

An increase in mouse tumor growth by an in vivo immunomodulating effect of titanium dioxide nanoparticles

Here, we investigated whether titanium dioxide (TiO₂) nanoparticles affect in vivo tumor growth through the modulation of mononuclear leukocytes. In vitro lymphocyte proliferation by lipopolysaccharide (LPS) or concanavalin A (ConA) was reduced by < 25?nm TiO₂ with a dose-dependent manner. Similarly, TiO₂ nanoparticles inhibited nitric oxide (NO) production from bone marrow-derived macrophages obtained from naïve mice. When mice were intraperitoneally (IP) injected with < 25 or < 100?nm TiO₂ once a day for 7 days, total cell number of splenocytes was reduced in the spleen of TiO₂ nanoparticle-exposed mice. Both CD4⁺ and CD8⁺ T-lymphocyte numbers were significantly decreased and B-lymphocyte development was retarded by host exposure to the TiO₂ nanoparticles. LPS-stimulated spleen cell proliferation was significantly reduced by host exposure to < 25 or < 100?nm TiO₂, but no changes were detected in ConA-stimulated spleen cell proliferation. Further, LPS-stimulated cytokine production by peritoneal macrophages and the percentage of NK1.1⁺ natural killer cells among splenocytes was reduced by the host exposures to the TiO₂ nanoparticles. When mice were IP injected with TiO₂ nanoparticles once a day for 28 days prior to the subcutaneous implantation of B16F10 melanoma cells, tumor growth was subsequently significantly increased. Collectively, these results show that TiO₂ nanoparticles may damage the development and proliferation of B- and T-lymphocytes, reduce the activity of macrophages, and decrease natural killer (NK) cell population levels, outcomes that appear to lead to an increase in tumor growth in situ. These studies allow us to suggest that TiO₂ nanoparticles might have the potential to enhance tumor growth through immunomodulation of B- and T-lymphocytes, macrophages, and NK cells.     

In Vitro Inhibitory Effect of Human Syncytiotrophoblast Plasma Membranes on the Cytolytic Activities of CTL and NK Cells

ABSTRACT: We have previously described the purification procedure for syncytiotrophoblast plasma membranes (STPM) and have now studied their immunomodulatory properties on the in vitro cytotoxic assays of generated cytotoxic T-lymphocytes (CTL) and natural killer cells (NK). STPM inhibited the cytolytic activities of CTL and NK against their target cells, whereas RBC ghosts, even at the highest protein concentration used, were ineffective. This inhibitory effect was dose-dependent upon the STPM-protein concentration and appeared to be particularly distinct at low effector/ target ratios. It is hypothesized that the inhibitory activity of STPM may be exerted by blocking the effector cells or by masking their targets. Regardless of the mode of action, since cytotoxic cell activities are known to play an important role in the allograft rejection process, this suppressive inhibitory effect of STPM might be a crucial mechanism in the tolerance of the semiallogenic fetal graft.     

Cytotoxic effector cells of the immune system

Summary The organism contains several types of cytotoxic cells which are able to lyse host and foreign cells. Cytotoxic T-lymphocytes (CTL) appear to play the most important role among the killer cells but other lymphatic cells, such as natural killer (NK) cells and lymphokine-activated killer (LAK) cells as well as macrophages are also highly effective in the lysis of appropriate targets. The various cytotoxic effector cells differ distinctly concerning origin, phenotype, morphology and target cell specificity, but they bear the common feature that they destroy the target cells in a contact-dependent non-phagocytotic process.CTL are characterized by typical lysosomal granules and by the expression of a characteristic pattern of surface molecules. They recognize specific antigens which are presented in context with molecules of class I major histocompatibility complex (MHC). NK cells, on the other hand, kill the appropriate targets without prior immunisation and without requiring recognition of MHC molecules at the target cells. They also bear a typical pattern of surface markers which differ in several aspects from that of CTL. Human NK cells are further characterized by peculiar cytoplasmic granules with parallel tubular arrays which are not present in other cytotoxic cells. LAK cells constitute an additional, only recently described, killer cell population which arise from lymphatic cells in the presence of interleukin-2. They appear to represent a functional unique cytotoxic effector cell system with an exceptionally wide target cell spectrum including normal and malignant cells of different origin. LAK cells, however, show a profound heterogeneity concerning the expression of phenotype surface markers and it is not yet clear whether they are a unique cell line. By electron microscopy they display peculiar intranuclear inclusion bodies which may be associated with prolonged stimulation by interleukin-2. CTL, NK and LAK cells appear to possess similar mechanisms for cytolysis including secretion of pore-forming proteins, serine proteases and other proteins. Furthermore, they are able to trigger the cleavage of DNA in the target cell nucleus by a hitherto unknown pathway.Macrophages differ substantially from other cytotoxic effector cells concerning morphology, phenotype, kinetic of activation and target cell spectrum. They perform a variety of functions whereby contact-dependent target cell lysis represents only one of their properties. After target cell binding they release over 20 different molecules such as interleukin-1 and tumor necrosis-factor-alpha as mediators for cytolysis. Thus, macrophages appear like other cytotoxic effector cells to destroy their appropriate targets by different factors.Taken together the data obtained hitherto suggest that cellular cytotoxicity is mediated by various effector cells which may make a major contribution in the defence against infections and malignancies.Abbreviations ADCC antibody dependent cellular cytotoxicity - CD clone of differentiation - CTL cytotoxic T-lymphocyte - HTL helper T-lymphocyte - IL-2 interleukin-2 - LAK lymphokine-activated killer cell - LGL large granular lymphocytes - MHC major histocompatibility complex - NK natural killer cell - P1 perforin 1 - PTA parallel tubular arrays - TCR T-cell receptor complex     

rIL-4 differentially regulates rIL-2-induced murine NK and LAK killing in CD8+ and CD8- precursor cell subsets

Interleukin 4 (IL-4) and IL-2 have complementary or synergistic roles in many aspects of lymphocyte development. IL-2 supports the induction of cytolytic activity in cytotoxic T lymphocyte (CTL), natural killer (NK), and lymphokine-activated killer (LAK) cells. IL-4 has also been shown to support CTL and LAK in primary murine spleen cell culture. This report demonstrates that IL-4 selectively down-regulates IL-2 inducible murine CD8- precursors of NK cells. For maximal regulatory effect it is necessary to add IL-4 to cultures before 40 h. Enrichment for NK1.1+ cells failed to recover precursor cells which are down-regulated in overnight cultures or can be cultivated in vitro to yield NK cytolytic activity. Furthermore, phenotypic analysis of effector cells demonstrated a marked inhibition of development of NK1.1+ cells in cultures containing IL-4 plus IL-2 versus IL-2 alone. Thus, it appears that IL-4 down-regulates the precursors of murine NK cells by inhibiting proliferation and/or development. In addition, we show that IL-2-induced murine LAK activity mediated by CD8- precursor cells is unaffected by IL-4, while CD8(+)-derived LAK cells are up-regulated by co-culture with IL-4 and IL-2. Analysis of these data relative to reports documenting down-regulation of human LAK by IL-4 suggests that in vitro cultured, IL-2-activated murine NK cells are the correlates to what are commonly described as human LAK cells. The discrepancy may stem from differences in the characteristics of target cells used in the murine versus the human systems. These results clarify the conflicting reports on the effect of IL-4 on killing activity.     

Pathogenesis of hemophagocytic syndrome (HPS)

Hemophagocytic syndrome (HPS) is a clinicopathologic entity characterized by increased proliferation and activation of benign macrophages with hemophagocytosis throughout the reticuloendothelial system. Uncontrolled T-lymphocyte activation is responsible for increased T(H)1 cytokines secretion such as IFN-gamma, IL-12 and IL-18 that promotes macrophage activation. Genetic defects specific for cytotoxic T lymphocytes (CTL) and natural killer (NK) cells have been identified in patients with primary HPS that are responsible for altered cell death and apoptosis induction or target killing. HPS may be secondary to malignancy, infection or autoimmune disease, and mechanisms involved are poorly understood. However, in adult-onset Still's disease, juvenile chronic arthritis and probably systemic lupus erythematosus, IL-18 might play a role in initiating macrophage activation.     

Modulation of host-immune responses by ticks (Acari: Ixodidae): effect of salivary gland extracts on host macrophages and lymphocyte cytokine production.

Ixodid tick infestation induces host acquired resistance, which involves immunoglobulin cell-mediated and complement-dependent effector pathways. Ticks have developed countermeasures to modulate host antiarthropod responses. Ixodid-mediated host immunomodulation results in vitro in reduced responsiveness to T-lymphocyte mitogens for cells obtained from infested hosts and impaired antibody responses to a thymic dependent antigen. Salivary gland extracts from days 0-9 of engorgement from unmated, female Dermacentor andersoni Stiles suppressed lymphocyte proliferative responses (LPS) to the T-cell mitogen Con A up to 68.4%, whereas responsiveness to E. coli LPS was enhanced. Cytokines assessed in this study included interleukin-1, IL-1, and tumor necrosis factor (TNF) alpha produced by macrophages, and interleukin-2, IL-2, and gamma interferon (IFN-G) secreted by T-lymphocytes. Salivary gland extracts prepared from tissues obtained on days 0-5 of engorgement suppressed IL-1 elaboration from 89.8% on day 0 through 37.5% on day 6. Levels of TNF were reduced from 40.7 to 94.6% throughout the course of the study. Production of IL-2 was suppressed by 14.1-31.9%, and IFN-G was reduced by 8.7-57.0%. Reduced IL-1 levels during the early phases of feeding indicated reduced host ability to activate T-lymphocytes and provide costimulatory, differentiation, and development signals for B-cells. Both IL-1 and TNF are endogenous pyrogens and activate polymorphonuclear leukocytes. Activities of TNF and IFN-G include antiviral properties and induction of expression of class I and II major histocompatibility complex molecules, which are critical components in the recognition of antigen by T-lymphocytes. The autocrine role of IL-2 in proliferation of T-lymphocytes is central to the development of immune reactivity involving T-cell regulation or effector functions or both. Reductions in cytokine levels would suppress immune responses directed toward immunogens introduced into the host during the course of tick feeding. These results indicates that immunomodulation of the host during tick feeding facilitates engorgement and pathogen transmission.     

Persistent suppression of humoral and cell-mediated immunity in mice following exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene

Carcinogen-induced immunosuppression has been implicated as an epigenetic mechanism in promoting the outgrowth and metastasis of neoplastic cells. It has previously been reported that the complete carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) suppresses both humoral immunity (HI) and cell-mediated immunity (CMI) 3-5 days following exposure. Since persistent systemic immunosuppression may be more relevant in tumor outgrowth, assays quantitating HI and CMI, including those functions involved in tumor resistance, were performed 4 and 8 weeks following exposure to tumorigenic doses of DMBA. Adult B6C3F1 female mice were administered DMBA dissolved in corn oil subcutaneously at 5, 50 and 100 micrograms/g body weight in ten equal doses over 2 weeks (corn oil = vehicle control). The number of splenocytes producing IgM antibody to the T-dependent antigen, sheep erythrocytes, was suppressed up to 95% and 98% at 4 and 8 weeks, respectively. The IgG response was similarly depressed 75% and 98% at 4 and 8 weeks, respectively. Lymphoproliferation of splenocytes in response to the mitogens LPS, PHA and Con A were depressed up to 88%, 78% and 83% at 4 weeks and 63%, 63% and 67% respectively, at 8 weeks. In addition, alloantigen-induced proliferation of splenocytes in a one-way mixed lymphocyte culture was suppressed up to 90% at 8 weeks. The ability to generate cytotoxic T-lymphocytes (CTL) in vitro against P815 tumor cells was depressed at both time periods (88% and 60%, respectively) as was natural killer (NK) cell cytolysis of YAC-1 tumor targets (84% and 55%, respectively). The immunosuppression noted in these parameters was similar to that observed within 3-5 days following DMBA dosing. The persistent immunosuppression induced by the PAH carcinogen DMBA, including CTL and NK cell tumoricidal functions, may represent an important epigenetic mechanism contributing to tumor outgrowth or metastasis by this class of agents.     

Effect of Aerva lanata on cell-mediated immune responses and cytotoxic T-lymphocyte generation in normal and tumor-bearing mice

Cell-mediated immunity offers protection against virus-infected cells and tumor cells, involves activation of natural killer (NK) cells, production of antigen-specific cytotoxic T-lymphocytes, and release of various cytokines in response to an antigen. Administration of an ethanolic extract of Aerva lanata was found to stimulate cell-mediated immunological responses in normal and tumor-bearing BALB/c mice. A significant enhancement in NK cell activity in both normal and tumor-bearing hosts was observed after administration of A. lanata. Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement-mediated cytotoxicity (ACC) were significantly enhanced as well in both sets of treated hosts. In addition, in vivo production of IL-2 and IFNg were each significantly enhanced by extract treatment. The stimulatory effect of A. lanata on cytotoxic T-lymphocyte (CTL) production was determined by Winn’s neutralization assay using CTL-sensitive EL4 thymoma cells. A. lanata treatment caused a significant increase in CTL production in both in vivo and in vitro models, in each case as indicated by a significant increase in the life-spans of tumor-injected mice. Taken together, all of these results in the murine model indicate that administration of an ethanolic extract of A. lanata could enhance the cell-mediated anti-tumor response.     

A comparison of membrane markers on rat cytotoxic cells.

The antigenic characteristics of cytotoxic T cells (CTL) and cells mediating antibody-dependent cellular cytotoxicity (ADCC) were defined using monoclonal antibodies W3/13, W3/25 and MRC OX8, and compared with the phenotype of natural killer (NK) cells previously defined by these antibodies. CTL, ADCC effector cells and NK cells were also tested for expression of Ia antigen using MRC OX6 monoclonal antibody. The fluorescence-activated cell sorter was used to separate effector populations into antigen-positive and antigen-negative subsets, and the cytotoxicity of the resultant lymphocyte fractions was then assessed using 6 hr 51Cr release assay and a quantitative method of analysis based on consideration of target cell lysis as an enzyme substrate reaction. CTL, ADCC effector cells and NK cells were W3/25 negative and OX6 negative. CTL were positive for W3/13 and OX8 antibody with no cytotoxicity in the W3/13 negative and OX8 negative fractions. In contrast, ADCC effector cells showed heterogeneity of staining with W3/13 and OX8 antibodies, with 50% cytotoxic activity in the W3/13 negative fraction, and 20-30% cytotoxic activity in the OX8 negative fraction. In parallel experiments, NK cells and ADCC effector cells showed identical marker heterogeneity when tested with W3/13 and OX8 antibodies.     

Helper role of NK cells during the induction of anticancer responses by dendritic cells

Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other's activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-gamma and TNF-alpha, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of "helper" function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic "effector" function of NK cells, the induction of "NK cell help" requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than "exhausted" ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma.