Inhibition Of Cytolytic Activity Research Articles

Cancer-associated mucins: role in immune modulation and metastasis.

Mucins (MUC) protect epithelial barriers from environmental insult to maintain homeostasis. However, their aberrant overexpression and glycosylation in various malignancies facilitate oncogenic events from inception to metastasis. Mucin-associated sialyl-Tn (sTn) antigens bind to various receptors present on the dendritic cells (DCs), macrophages, and natural killer (NK) cells, resulting in overall immunosuppression by either receptor masking or inhibition of cytolytic activity. MUC1-mediated interaction of tumor cells with innate immune cells hampers cross-presentation of processed antigens on MHC class I molecules. MUC1 and MUC16 bind siglecs and mask Toll-like receptors (TLRs), respectively, on DCs promoting an immature DC phenotype that in turn reduces T cell effector functions. Mucins, such as MUC1, MUC2, MUC4, and MUC16, interact with or form aggregates with neutrophils, macrophages, and platelets, conferring protection to cancer cells during hematological dissemination and facilitate their spread and colonization to the metastatic sites. On the contrary, poor glycosylation of MUC1 and MUC4 at the tandem repeat region (TR) generates cancer-specific immunodominant epitopes. The presence of MUC16 neo-antigen-specific T cell clones and anti-MUC1 antibodies in cancer patients suggests that mucins can serve as potential targets for developing cancer therapeutics. The present review summarizes the molecular events involved in mucin-mediated immunomodulation, and metastasis, as well as the utility of mucins as targets for cancer immunotherapy and radioimmunotherapy.

Counter-regulation of cytolytic activity and cytokine production in HIV-1-specific murine CD8+ cytotoxic T lymphocytes by free antigenic peptide.

We have reported previously that the cytolytic activity of murine CD8(+) cytotoxic T lymphocytes (CTL) specific for HIV-1 gp160 envelope glycoprotein was markedly inhibited by brief exposure to the free minimal antigenic peptide (I-10: 10mer peptide from gp160) by direct binding to class I MHC molecules of specific CTL in the absence of antigen-presenting cells (APC). Here, we show that treatment of such CTL with the peptide induced not only the inhibition of cytolytic activity but also IL-2Rbeta down-modulation, followed by the inhibition of IL-2-dependent growth. The peptide-mediated inhibition and restoration of expression of IL-2Rbeta were well correlated with changes in both cytolytic activity and IL-2-dependent growth of the CTL. Since enzymatic activity of granzyme B, and mRNA expression of granzyme B and perforin were significantly reduced in peptide-treated CTL, the inhibition of cytolytic activity was mainly caused by the exhaustion of cytolytic molecules. Moreover, treatment of the CTL with the epitopic peptide resulted in production of high levels of IL-2, IFN-gamma, tumor necrosis factor-alpha and MIP-1beta in the culture supernatant. Maximum amounts of cytokines were obtained in the culture supernatant when the level of cytolytic activity was the lowest. Thus, although the CTL temporarily lost their cytolytic activities, they simultaneously gained the abilities to produce cytokines for activation of various cell populations. These changes induced by free antigenic peptide in CD8(+) CTL reveal an interesting counter-regulation between their cytolytic activities and cytokine production.

Inhibitory receptors for HLA class I molecules on cytolytic T lymphocytes. Functional relevance and implications for anti-tumor immune responses.

In recent years, the molecular mechanism by which natural killer cells lyze, or fail to lyze, different target cells has been elucidated. Natural killer cells express receptors which recognize MHC class I molecules on target cells. This interaction leads to inhibition of cytolytic activity, thus preventing lysis of target cells. The receptors belong to two distinct molecular types: (1) the lg superfamily which includes receptors (p58.1, p58.2, p70, and p140) which recognize specific HLA allotypes; (2) CD94 molecules which display a broad specificity for HLA class I molecules. Recently, a subset of cytolytic T lymphocytes has been shown to express the various natural killer cell receptors. Such T cells are detectable in peripheral blood, spleen, tonsils, and lymph nodes, but not in the thymus and cord blood. In some instances, two or more natural killer receptors can be coexpressed at the single cell level. Surface marker analysis has revealed that natural killer cell receptor-positive T cells always express a memory phenotype. Moreover, they are characterized by a skewed T cell receptor V beta repertoire. Further analysis of the T cell receptor VDI sequences revealed that natural killer cell receptor-positive, CD3-positive cells isolated from a given individual are oligoclonal or monoclonal in nature. Crosslinking of natural killer receptors leads to inhibition of different T cell functions, including non-specific lysis of appropriate HLA class I-negative target cells, T cell receptor mediated cytotoxicity, and cytokine production. The inhibitory effect on T cell receptor-mediated function has important implications. Thus, the expression of natural killer cell receptors as a consequence of chronic antigen stimulation may result in functional impairment of specific cytolytic T lymphocytes. Preliminary data indicate that this phenomenon may occur in tumor or virally infected patients. Remarkably, various patients with large granular lymphocyte expansions characterized by a CD3/ natural killer receptor-positive phenotype had chronic viral infections. The fact that antigen-specific cytolytic T lymphocytes may simultaneously express T cell and natural killer cell receptors, both recognizing HLA class I molecules but mediating opposite signals, offers new perspectives in our appreciation of the regulation of T cell responses and offers new clues for understanding the immunopathological events involved in certain diseases.

Inhibition of cytolytic T lymphocyte activity by oxysterols

The objective of this study was to investigate the effects of oxysterols (OS), namely 5 alpha-hydroxy-6-ketocholestanol, 6-ketocholestanol and 25-hydroxycholesterol, on specific cell-mediated cytotoxicity by C57BL/6 spleen cells against P815-X2 (a DBA/2 mastocytoma) target cells. Cytolytic T lymphocytes (CTL) were generated by intraperitoneally injecting C57BL/6 mice with P815-X2 tumor cells 10 d prior to the cytotoxicity experiments. Preincubation of CTL with 10(-5) M 5 alpha-hydroxy-6-ketocholestanol and 6-ketocholestanol for 45 min in lipoprotein-depleted medium resulted in an inhibition of cytolytic activity (73 and 43%, respectively) as measured by 4-h 51Cr release. At a concentration of 5 x 10(-6) M, 5 alpha-hydroxy-6-ketocholestanol inhibited CTL activity by 65%, whereas 6-ketocholestanol did not elicit any inhibition. By contrast, 25-hydroxycholesterol did not inhibit CTL at either concentration, although it is known to be a potent inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme in the cholesterol biosynthetic pathway. When CTL were preincubated with OS in lipoprotein-replete medium, there was no inhibition of CTL activity at the respective concentrations. The results suggest that the inhibition of CTL activity upon short-term incubation with OS is not due to the inhibition of cholesterol synthesis, but may be due to the insertion of OS into the plasma membrane to replace cholesterol and alteration of membrane physical properties.

A requirement for membrane-associated phospholipase A2 in platelet cytotoxicity activated by receptors for immunoglobulin G and complement.

Platelets are potent antibody- and complement-dependent cytotoxic effector cells. We showed previously that a single platelet can lyse a target cell sensitized with immunoglobulin G (IgG) and complement components up to C3 (C integral of 3b denotes the target cell-bound fragment of complement up to C3; the precise nature of the bound C3 fragment has not been established), and that the complete cytotoxic system capable of specific recognition and lysis resides in platelet membranes. To define the components of platelet membranes required for cytotoxicity, a set of inhibitors of phospholipase A2 (PLA2) that act by different chemical mechanisms was tested. The lytic reaction is blocked at appropriate concentrations of bromophenacylbromide, mepacrine, and manoalide. When platelets are treated with bromophenacylbromide, inhibition of cytolytic activity and that of PLA2 enzymatic activity occur in parallel. Platelets release arachidonate when incubated with target cells bearing IgG and C integral of 3b, confirming that Fc gamma R and complement receptor trigger both PLA2 action and efficient lysis. Inhibition by thimerosal of a reverse reaction, i.e., reacylation catalyzed by acyltransferase, causes increased target cell lysis, presumably by increasing the products of PLA2 action. Platelet cytotoxicity is increased when platelets are pretreated with some products of PLA2: exogenous lysophospholipids and not free arachidonic acid increase cytotoxicity. Electron microscopy suggests that platelets and target cells may fuse, possibly as a result of the formation of lysophospholipids which are well-known membrane fusogens. Fixation with paraformaldehyde does not affect platelet cytotoxicity, suggesting that the complete cytotoxic system resides as a preformed complex in platelet membranes. The results indicate that platelet membrane-associated PLA2, together with receptors for Fc and complement, are required for platelet cytotoxicity.

Effects of taxol and taxol/hyperthermia treatments on the functional polarization of cytotoxic T lymphocytes.

Immunofluorescence staining, electron microscopy, and (51Cr) cytolytic release assays are used to investigate the effects of taxol and taxol/hyperthermia treatments on the microtubule organization and cytolytic activity of cytotoxic T lymphocytes (CTLs). A 4 h treatment of CTLs with 1 microM taxol results in an extensive reorganization of the microtubule system to form one to a few large microtubule bundles that extend from the centrosome. The Golgi apparatus is not disrupted by this treatment and remains associated with the microtubule organizing centre (MTOC). This microtubule reorganization has no effect on the ability of CTLs to orient their MTOC towards a bound target cell, nor on their cytolytic activity. In control CTLs, not treated with taxol, a mild hyperthermia treatment (42 degrees C, 30 min) results in an aggregation of the pericentriolar material, a loss of MTOC orientation, an inhibition of cytolytic activity, and a disorganization of the microtubule system [Knox et al.: Exp. Cell Res. 194:275-283, 1991]. In contrast, in taxol-treated CTLs the stabilized microtubule bundles are unaffected by such hyperthermia treatment; however, the other effects of hyperthermia appear identical in control and taxol-treated CTLs. These results indicate that a dynamic, radially arranged microtubule array is not required for the functional polarization of CTLs and suggest that a component of the pericentriolar material may play a key role in effecting MTOC orientation.

Influence of monovalent cation concentrations on monocyte-mediated ADCC.

Monocyte-mediated ADCC was inhibited by ouabain, which blocks active transport of Na+ and K+ by the membrane Na+K+-ATPase. Inhibition of ADCC was incomplete, however, even when monovalent cation transport was completely inhibited. On the other hand, increasing the duration of exposure to ouabain did result in complete inhibition of ADCC. ADCC was also depressed by incubation of monocytes in media with low (less than or equal to 1.5 mEq/l) or high (greater than or equal to mEq/l) K+ concentrations. The results suggest that monocyte ADCC is dependent upon monovalent cation concentrations (secondarily altered by ouabain exposure) but not upon active Na+K+ transport per se. Finally, since monovalent cation transport-related glycolysis accounted for essentially all of the previously observed increase in monocyte glycolysis associated with monocyte ADCC, we hypothesize that exposure of monocytes to sensitizing antiserum in the ADCC assay results in increased monocyte membrane permeability to monovalent cations.