Inhibition of cell-mediated cytotoxicity by Corynebacterium parvum

Summary We investigated the effect of altering dose and route of Corynebacterium parvum (C. parvum) administration on the adjuvant's inhibition of cell-mediated cytotoxicity (CMC). Primary in vivo and secondary in vitro CMC of C57B1/6 mice alloimmunized to P815 were depressed if C. parvum was administered systemically (IV or IP) but not when it was given SC. Similarly, only systemic C. parvum generated cells capable of suppressing in vitro CMC. Primary and secondary CMC in spleen was equally inhibited by 700 and 70 g, whereas suppressor cell activity was marked with 700 g and minimal with 70 g. Administration of C. parvum SC admixed with alloantigen resulted in early enhancement and late depression of primary CMC. Secondary CMC was depressed but suppressor activity was absent. Dissociation of CMC depression from suppressor cell generation indicates that these phenomena can be separated under certain conditions.     

Inhibition of memory cell-mediated cytotoxic response by systemic administration of Corynebacterium parvum.

Intravenous administration of Corynebacterium parvum to mice during a developing immune response to alloantigens resulted in the marked inhibition of the generation and expression of memory cell-mediated cytotoxic response in the spleen. The inhibition was observed following rechallenge in vivo or by in vitro culturing with the same alloantigen. The impairment in vitro was due, in part, to the generation of regulatory cells which were non-T phagocytic cells, probably macrophages activated by C. parvum administration. These suppressor macrophages appear to act by inhibiting proliferation and clonal expansion of memory cytotoxic cells.     

Systemic administration of Corynebacterium parvum during sensitization to tumor alloantigen-modified response to rechallenge

Summary Intravenous administration of Corynebacterium parvum (C. parvum) to mice during a primary immune response against tumor alloantigens impairs their ability to generate memory cell-mediated cytotoxicity (CMC) in response to an intraperitoneal rechallenge with the same tumor alloantigens. Decreased CMC was observed in spleen and mesenteric lymph nodes, whereas CMC of lymphoid populations from the peritoneal cavity was merely delayed, reaching comparable levels to those found in control animals by day 5. Serum levels of cytotoxic antibody were unaffected, indicating that C. parvum administered during a primary immune response has selective effects on the cytotoxic memory response.     

Inhibition of NK cell-mediated cytotoxicity by oxysterols.

Some of the oxidation products of cholesterol (oxysterols) have profound effects on plasma membrane structure and function. The present studies were undertaken to determine the effects of oxysterols on NK cell-mediated cytotoxicity. When mouse spleen cells were preincubated with certain oxysterols, NK cell cytotoxicity was inhibited without loss of effector cell viability. The strongest inhibition was observed with oxysterols that are oxidized at the C-5, C-6, or C-7 positions of the sterol nucleus. Among these, 7 beta-hydroxycholesterol caused more inhibition than 7 alpha-hydroxycholesterol suggesting that the spatial orientation of the hydroxyl group in the beta-position results in a greater perturbation in plasma membrane structure than that oriented in the alpha-position. In contrast, oxysterols that are oxidized at the C-20 and C-25 positions that are located on the C-17 acyl chain had little or no inhibitory effect, suggesting that oxidation in the cholesterol nucleus which is situated closer to the phospholipid headgroups at the lipid bilayer-aqueous interface results in a more profound effect on the plasma membrane physical structure. These results suggest that the lytic function of NK cell is sensitive to alterations in the physical state of its plasma membrane induced by oxysterols.     

Inhibition of T cell-mediated cytotoxicity by anti-inflammatory steroids

We have tested the capacity of glucocorticoids to modulate the effector function of splenic cytotoxic T lymphocytes (CTL) obtained after i.p. immunization with allogeneic cells. Although acute exposure to glucocorticoids did not inhibit the activity of freshly obtained splenic CTL, preincubation of these CTL for several hours with subnanomolar concentrations of several different glucocorticoids caused marked inhibition. The relative inhibitory potency of the steroids tested correlated with their reported activity both in glucocorticoid receptor binding assays and in assays of anti-inflammatory potency in man. The inhibitory effects of low concentrations (10(-10) M to 10(-9) M) of dexamethasone were reversed by human or mouse interleukin 2 (IL 2)-containing supernatants, but were not reversed by IL 1-containing supernatants. The inhibitory effects of higher concentrations (10(-8) M to 10(-7) M) of dexamethasone could not be reversed even by very high doses of mouse IL 2. In contrast to previous reports of minimal direct glucocorticoid effects on CTL activity, the present results suggest that after preincubation, splenic CTL from in vivo-immune mice are sensitive to inhibition by glucocorticoids, and that the glucocorticoids may act both indirectly (on IL 2 production) and directly on the CTL.     

Inhibition of lymphokine-activated killer cell-mediated cytotoxicity by phorbol ester

As previously reported, the culture of mouse spleen cells in the presence of high amounts of human rIL-2 for 4 days caused proliferation and generation of lymphokine-activated killer (LAK) cells, which could lyse a variety of tumor cells. However, an addition of PMA to the culture resulted in a striking inhibition of the generation of LAK cells. In contrast, IL-2-induced cell proliferation, IL-2R expression, and LFA-1 expression were enhanced by the addition of PMA. Kinetic studies revealed that the addition of PMA during the final 24 h, but not 4 h, of the culture was sufficient to inhibit the generation of LAK cells. The same inhibition of LAK activity was observed when 4-day cultured LAK cells were pretreated with PMA for over 12 h before cytotoxicity assay. Flow cytometry analysis showed that PMA pretreatment had no effect on the binding of LAK cells to target cells. PMA pretreatment of LAK cells caused total disappearance of protein kinase C (PKC) activity from LAK cells concomitant with the loss of LAK activity. However, PMA-pretreated LAK cells cultured for another 24 h in the absence of PMA revealed levels of PKC activity and cytotoxicity identical with untreated LAK cells. These results strongly suggest that PMA-induced down-regulation of LAK cell-mediated cytotoxicity is due to the inactivation of PKC-dependent transduction systems that are essential post LAK cell-target cell binding.     

Rejection of tumor metastases in Fischer 344 rats following the administration of killed Corynebacterium parvum

Summary Fischer 344 rats received subcutaneous grafts of syngeneic 13762 mammary adenocarcinoma cells. Thereafter, at 20 days, each animal of a group received either daily (×2) permeating intratumor injections of killed Corynebacterium parvum (1.0 mg) saline, and weekly (×4) intravenous (i.v.) or intraperitoneal (i.p.) C. parvum or saline. In addition, a group each (4 groups) of rats were treated with surgical extirpation of the growing tumor nodule and i.v. and i.p. C. parvum or saline administered at weekly (×4) intervals. The results revealed that following intratumor injection of C. parvum there was rejection of tumor by all animals which exhibited long-term survival for 29 months (intratumor saline: 0% survival). The groups of rats treated with surgical extirpation of the tumor and parenteral administration of C. parvum exhibited 70–75% survival for 20 months (saline treated: 20–35% survival). The surviving animals exhibited tumor-specific protection to subsequent tumor cell challenges. Macrophages separated from the lung, peritoneum, and spleen of rats from the intratumor C. parvum group exhibited respectively, 46.2%, 62.9%, and 29.4% cytotoxicity towards target 13762 tumor cells as measured by ⁵¹ Cr release. Similar studies using macrophages from the group treated with surgery and parenteral C. parvum revealed similar tumor cytotoxicity (pulmonary: 49.2%; peritoneal: 65.7%; spleen: 34.4%). The splenic lymphocytes from the intratumor and parenteral C. parvum groups exhibited, respectively, 16.9% and 14.7% ⁵¹ Cr release following incubation with target tumor cells.This study was supported in part by Grant No. CA18582-01 from the National Cancer Institute     

Inhibition of mast cell-mediated cytotoxicity by IFN-alpha/beta and -gamma.

Although IFN enhance the cytotoxic activity of NK cells, K cells, and monocytes, IFN-alpha/beta and IFN-gamma did not stimulate the cytotoxic activity of rat peritoneal mast cells (PMC), but had an inhibitory effect. Preincubation for 2 h with 100 and 200 U/ml of IFN-gamma and IFN-alpha/beta, respectively, inhibited PMC cytotoxicity against WEHI-164 target cells. Lower concentrations of IFN-gamma (12.5 U/ml) and IFN-alpha/beta (25 U/ml) inhibited cytotoxicity of PMC after 8 h preincubation. The inhibitory effect of IFN was concentration and time dependent. In contrast to cytotoxicity, the release of histamine by PMC was not stimulated by the target cells WEHI-164 and there was no correlation between histamine release and cytotoxic activity of PMC. Specific antibody to subclasses of IFN prevented the inhibition of PMC cytotoxic activity, but preincubation with antibodies to the alternate subclass of IFN did not affect the observed inhibition. Moreover, the presence of both subclasses of IFN showed an additive inhibition of PMC cytotoxicity. The cytotoxic activity of PMC can be completely inhibited by the addition of anti-TNF during the assay. At high concentrations (400 U/ml), IFN inhibited the release of TNF from PMC. In the presence of RNA or protein synthesis inhibitors, IFN did not inhibit cytotoxicity of PMC further. We postulate that IFN may alter gene expression in mast cells in a manner that down-regulates their functions.     

Inhibition of systemic TNF-alpha cytotoxicity in cancer patients by D-peptidoglycan

The current study was designed to investigate direct inhibitory effects of N-acetylglucosaminyl-muramyldipeptide (GMDP) over the cytotoxic nature of TNF-α. A lactate dehydrogenase (LDH) assay of the inhibition of TNF-α cytotoxicity was donein vitro on the following cell lines: A549 (human lung carcinoma cells), A431 (human breast cancer cells) and L929 (mouse breast cancer cells). In a double-blind placebo-controlled trial, cancer patients with an elevated activity of all five LDH isoensymes were randomized to receive either a GMDP solution or a placebo; 63 patients were evaluated every third day for the mean daily number of episodes of nausea or vomiting, changes in clinical status, cell blood count and blood chemistry. A 95% inhibition of LDH release was noticed on A549 cells. Other cell lines were less sensitive to GMDP, with an observed 72% dose-dependent reduction in LDH activity.In vivo, LDH activity was decreased by 41% (+/−4%) (mean +/−SD) in all 21 subjects who were given 0.5–1.0 mg of GMDP daily. A lowering of LDH activity by 73.4% (+/−4%) was observed in 23 patients who received GMDP at a dosage of 1.5 mg/kg daily. Correspondingly, a 10% (+/−2%) increase in LDH activity was noticed in 19 patients who were given a placebo (P<0.01). During the follow-up period, the overall clinical condition of all patients treated with GMDP was improved. No side effects were observed. In nine patients who experienced nausea from tumor toxicity before treatment, the symptom subsided. In parallel, an extremely beneficial effect on lipids metabolism was noticed in all patients with elevated cholesterol and trigliceride levels. A dietary supplementation of GMDP has been shown to reduce systemic TNF-α cytotoxicity during tumor shock.     

Immunomodulation by Corynebacterium parvum in normal humans

Four normal human donors were immunized with 2 mg of heat-killed Corynebacterium parvum by subcutaneous and intracutaneous injections, and lymphocyte surface markers, antibody-dependent (ADCC), and spontaneous cell-mediated (SCMC) cytotoxicities followed for a 28-day period. Although no changes were observed in the relative proportions of B, T, and Fc receptor-carrying lymphocytes, two T cell subpopulations, namely, the autologous rosette-forming cells and active rosette-forming cells, both exhibited significant increases. Significant increases were also demonstrated in the proportion of monocytes carrying Fc receptors and in the proportion of monocytes phagocytizing Latex beads. Although no consistent changes could be found in ADCC against the P 815 mastocytoma cell line, the SCMC against both the myeloid leukaemia line K 562 and the lymphoma line RAJI was found to be elevated as early as 6 hr post-vaccination. The possibility that the enhanced SCMC activity was induced in vivo by interferon was supported thus: 1) Enhancement of SCMC in vitro by interferon was abrogated by the vaccination. 2) Serum interferon determinations showed significant increases in parallel with the lack of in vitro SCMC enhancement.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).     

Lysis of dengue virus-infected cells by natural cell-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity

Peripheral blood mononuclear cells (PBMC) from humans without antibodies to dengue 2 virus lysed dengue 2 virus-infected Raji cells to a significantly greater degree than uninfected Raji cells. The addition of mouse anti-dengue antibody increased the lysis of dengue-infected Raji cells by PBMC. Dengue 2 immune human sera also increased lysis of dengue-infected Raji cells by PBMC. These results indicate that both PBMC-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) can cause significant lysis of dengue-infected Raji cells. The lysis of infected Raji cells in the ADCC assay correlated with the dilution of dengue-specific antibody which was added, indicating the dengue virus specificity of the lysis of dengue virus-infected Raji cells. Alpha interferon (IFN alpha) was detected in the culture supernatant of PBMC and dengue-infected Raji cells. However, enhanced lysis of dengue-infected Raji cells by PBMC may not be due to the IFN produced, because neutralization of all IFN activity with anti-IFN alpha antibody did not decrease the lysis of dengue-infected cells, and effector cells pretreated with exogenous IFN alpha also lysed dengue-infected cells to a greater degree than uninfected cells. The effector cells responsible for lysis of dengue virus-infected Raji cells in the natural killer and ADCC assays were analyzed. Nonadherent PBMC caused more lysis than did adherent cells. Characterization of nonadherent cells with monoclonal antibodies showed that the predominant responsible effector cells were contained in OKM1+ and OKT3- fraction in the natural killer and ADCC assays.     

Inhibition of antibody-dependent cell-mediated cytotoxicity (ADCC) by antiserum raised against brain tissue

Treatment of mouse spleen cells with a rabbit anti-mouse brain (RAMB) antiserum markedly suppressed antibody-dependent cell-mediated cytotoxicity (ADCC) on trinitrophenyl-coupled sheep erythrocyte targets. This inhibitory activity of RAMB antiserum was complement independent, absorbable with mouse brain tissue, and appeared to be separable from the anti-Thy-1 activity of this serum. Absorption studies indicated that various T- and B-lymphocyte cell lines as well as macrophage-like cell lines are not able to absorb the inhibitory activity of RAMB antiserum. In contrast, thymocytes and spleen cells, as well as the neural cell line, PC12, a chromocytoma derived from rat adrenal medulla, were capable of absorbing the inhibitory activity to some extent, suggesting that antigens characteristic for ADCC effector cells can be found on these cell populations.     

Enhancement of cell-mediated cytotoxicity by recombinant tumor necrosis factor (TNF alpha)

The effects of recombinant tumor necrosis factor (rTNF alpha) on the immune responses were investigated. A single iv injection of rTNF alpha (6 x 10(3) U) caused regression of sarcoma-180 transplanted into BALB/c nu/+ mice, but failed to regress this tumor in nu/nu mice. A higher dose of rTNF alpha (2 x 10(4) U) was necessary to induce antitumor effect in nu/nu mice. A host-related factor seemed to be involved in mediating tumor regression. Therefore, the effects of rTNF alpha on various T-dependent immune responses, including delayed footpad reaction (DFR), cell mediated cytolysis (CMC), and plaque-forming cells (PFC) were examined in BALB/c mice, immunized ip with chicken erythrocytes (CRBC). A single injection of rTNF alpha, at the time of the antigen administration, induced the augmentation of CMC to CRBC in a dose-dependent manner. DFR and PFC were not affected in optimal immunization procedures. The TNF alpha injection, at or after the time of antigen administration, was more effective in inducing augmentation of CMC. The increase in CMC by TNF alpha was mediated by nonadherent, Thy 1.2, Lyt 2.2 positive cells and neutralization of TNF alpha by the anti-TNF alpha monoclonal antibody abolished the effect on CMC. These results indicated that the human recombinant TNF alpha induced changes in the T-cell-mediated responses.     

Suppression of cell-mediated tumor cell lysis and complement-induced cytotoxicity by trypan blue.

Different forms of cell-mediated cytotoxicity were suppressed in the presence of trypan blue. The systems affected included lysis of antibody-coated tumor cells by normal and C. parvum-stimulated mouse peritoneal cells and lysis of allogeneic targets by immune effector cells. The inhibition, measured in a 4-hr 51Cr release assay, was reversible and did not occur in the presence of 30% fetal calf serum or albumin. Binding between effector and target cells through Fc receptors was not affected, and lysis of allogeneic cells was inhibited at the lytic step rather than at the binding step. In contrast, lysis of sensitized erythrocytes was not inhibited by trypan blue, suggesting that lysis of these targets may not involve the steps required in tumor cell lysis. Trypan blue blocked the function of antibody before binding to target cells and also suppressed complement-induced cytolysis. Most individual complement components were susceptible to the inhibitory action of trypan blue. These results reveal an affinity of trypan blue for proteins in general that may be responsible for many of its biologic actions.     

Suppression of murine NK activity induced by Corynebacterium parvum

Summary Formalin-killed Corynebacterium parvum (CP), given at a dose of 0.4–0.7 mg/mouse IV or IP, induced suppressor cells for NK activity in B6C3F₁ mice. The suppressor cells belong to at least two different populations, plastic adherent and nonadherent, and were not depleted by antibodies specific for Thy-1.2, Ia^k, or NK-1.2 surface markers. Administration of p-I:C, an interferon-inducer, to animals 18 h before the assay did not affect the suppressor activity. Hypotonic shock treatment of splenocytes abrogated the in vitro suppressive activity, and subsequent reconstitution of the shock-treated cells with RBC failed to restore the suppressive activity. SJL/J mice, which have low NK activity, exhibited suppressor activity comparable to B6C3F₁ mice following CP treatment, whereas CP-treated BALB/c athymic and euthymic mice showed a lower ability to generate suppressors for NK as compared to B6C3F₁ mice.