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At least 127 records · Page 7Linked to original sources
PubMed2002

Mechanism of norepinephrine-mediated inhibition of human NK cytotoxic functions: inhibition of cytokine secretion, target binding, and programming for cytotoxicity.

Norepinephrine (NE) has been shown to inhibit human peripheral blood-derived natural-killer (NK) cell cytotoxicity (NKCC) in vitro. We demonstrate in this study that NE not only inhibits IL-2-activated NKCC but antibody-dependent cellular cytotoxicity (ADCC) as well. NK cytotoxicity by purified NK cells against K562 (NKCC) and against Raji cells (ADCC) were inhibited by NE (1-100 microM) by more than 50% in a 4-h (51)Cr release assay. The mechanism underlying the inhibition has been examined. NK cytotoxicity is dependent on target recognition and formation of NK-target conjugates, and activation by IL-2 is dependent on the secretion of cytokines (such as TNF-alpha) by NK cells. We hypothesized that the inhibition of NK functions by NE may be due to disruption of NK-target conjugation, blocking programming for lysis, and/or inhibition of cytokine secretion. Pretreatment of human peripheral blood mononuclear cells (PBMC) with NE for 15 min significantly reduced the binding to K562 cells by CD16(+) NK lymphocytes. In the presence of K562 cells, NE down-regulated the expression of CD16 (FcgammaRIII) by human PBMC, an NK cell receptor responsible and necessary for ADCC and cytokine secretion. We also demonstrate that NE inhibited the IL-2-mediated up-regulation of the activation marker CD69. At concentrations of 10(-6) to 10(-5) M, NE inhibited TNF-alpha, IFN-gamma, and GM-CSF secretion by NK cells, which are essential for IL-2-driven NK maturation and functions. In addition, using single-cell analysis, NE pretreatment of lymphocytes reduced the frequency of killer cells in the NK-K562 conjugate population in a concentration-dependent manner, indicating an inhibition of the programming for lysis by NK cells. In summary, these data demonstrate that NE-induced inhibition of NK cytotoxicity is manifested at multiple levels, including a modification of NK cell receptor ligation to target cells, blockade of NK cytokine secretion necessary for NK maturation and differentiation, and inhibition of the target-induced activation of the cytotoxic mechanism(s) in NK cells. Thus, sympathetic activation, as often induced experimentally, may profoundly impair natural cellular immunity through varied measurable pathways.

Antigens, CD↗
PubMed1997

TNF-mediated cytotoxicity of L929 cells: role of staurosporine in enhancement of cytotoxicity and translocation of protein kinase C isozymes.

The role of protein kinase C (PKC) in tumour necrosis factor (TNF)-mediated cytotoxicity was investigated, using the L929 cell line. The PKC activator phorbol-12-myristate 13-acetate (PMA) increased proliferation and inhibited TNF-induced cytotoxicity of L929 cells. A range of specific PKC inhibitors had no effect on TNF-mediated killing, suggesting that PKC does not play a direct role in this response. However, staurosporine enhanced cytotoxicity by TNF in the presence of actinomycin D, a protein synthesis inhibitor. In view of this finding, the authors investigated the role of specific PKC isozymes in both TNF-mediated cytotoxicity and staurosporine-induced sensitization to killing. PKC-alpha, beta, epsilon and zeta were detected in L929 cells. PKC-beta was only weakly detected in the cytoplasm, PKC alpha, epsilon and zeta were all found in resting cytoplasm and membrane. Stimulation with PMA caused a strong translocation of PKC-alpha but not zeta to the membrane. TNF had no effect on these isozymes but interestingly caused a translocation of PKC-epsilon, which also occurred in response to PMA. Staurosporine caused a translocation of PKC-zeta to the plasma membrane and a loss of PKC-epsilon from the cytosol. Although TNF induced PKC-epsilon translocation, this is unlikely to be involved in cytotoxicity as this effect was also induced by PMA which protected against TNF-mediated cytotoxicity. Staurosporine also induced translocation of PKC-zeta, an isozyme whose activity was previously found to be resistant to inhibition by staurosporine. These findings suggest the possibility that the mechanism by which staurosporine potentiates TNF action does not involve inhibition of PKC, but in contrast may involve modulation of PKC-zeta.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Reduced LAK cytotoxicity of peripheral blood mononuclear cells in patients with bladder cancer: decreased LAK cytotoxicity caused by a low incidence of CD56+ and CD57+ mononuclear blood cells.

The cytotoxicity of unstimulated peripheral blood mononuclear cells (US-PBMC), phytohemagglutinin (PHA)-stimulated PBMC (PS-PBMC) and interleukin-2 (IL-2)-activated PBMC (LAK cells) was assessed in patients with noninvasive and invasive transitional-cell bladder cancer and compared with those determined in healthy controls. The differences in the cytotoxicities were correlated with specific changes in the subsets of peripheral blood mononuclear cells (PBMC). PBMC from 37 patients and 13 healthy controls were tested against the bladder cancer cell line T24 in 51Cr-release assays. The PBMC subsets were analyzed using monoclonal antibodies against T cells, natural killer (NK) -cells, monocytes, and activation markers. The cytotoxicities of US-PBMC, PS-PBMC, and LAK cells were all significantly lower in the cancer patients than in the controls (P less than 0.05). The percentages of PBMC positive for the NK-cell markers CD56 and CD57 were lowest in the patients and were correlated to the decrease in cytotoxicity. Depletion of CD56+ or CD57+ cells from PBMC prior to or after 2 days stimulation with IL-2 demonstrated that these cells are the major source of LAK-cell cytotoxicity and showed that the reduced ability of bladder cancer patient PBMC to develop LAK-cell cytotoxicity is a result of a low incidence of CD56+ and CD57+ cells in the blood. These findings indicate that IL-2 therapy alone might not be a sufficient therapy of bladder cancer patients.

Aged↗
PubMed1984

Natural cytotoxicity of human blood monocytes: production of monocyte cytotoxic factors (MCF) during interaction with tumor cells.

Human blood monocytes obtained by EDTA-reversible adherence to autologous serum-coated plastic dishes expressed natural cytotoxicity against NK-sensitive K562 cells in a 4-h 51Cr release assay. These monocytes released soluble cytotoxic factors, termed monocyte cytotoxic factors (MCF), when cultured with target cells. In contrast, blood monocytes obtained by adherence to fetal calf serum-coated plastic surfaces failed to kill K562 cells and to produce MCF. Although some lysis could be detected at 18 h, optimal lysis of K562 cells by MCF was observed after 48 h incubation in a microcytotoxicity assay using trypan blue dye exclusion. The addition of actinomycin D to the cytotoxicity assay enhanced the sensitivity and then NCF activity was detectable in a 18-h Cr release assay. Neither supernatants produced by culture of monocytes alone nor lysates of monocytes were cytotoxic. In addition, cytochalasin A inhibited both direct cell-mediated lysis and generation of MCF. Optimal production of MCF occurred after 6-24 h of interaction with K562 cells, although significant activity was already present by 3 h. Treatment of monocytes with OKM1 monoclonal antibody plus complement abrogated both cell-mediated lysis and MCF generation, whereas Leu-11b plus complement were ineffective. These results indicate that human blood monocytes can release MCF during interaction with tumor cells and that this may be involved in the lytic mechanism of monocyte-mediated natural cytotoxicity.

Cells, Cultured↗
PubMed2005

The role of cytotoxic skin-homing CD8+ lymphocytes in cutaneous cytotoxic T-cell lymphoma and pityriasis lichenoides.

BACKGROUND: Pityriasis lichenoides (PL) is a rare cutaneous lymphoproliferative disorder of unknown origin. Malignant transitions of PL have been described, but are very rare. We recently observed the fatal course of a 26-year-old patient who presented with a clinical picture resembling PL but had cytotoxic CD8+ T-cell lymphoma of the skin (CxCTL). This case prompted us to reinvestigate the role of cytotoxic T lymphocytes in PL and its relationship to antiviral immunity. METHODS: Skin biopsy specimens of 11 patients with PL and two biopsy specimens of CxCTL were included. In all, 5 biopsy specimens taken from healthy skin and 5 samples of varicella-zoster virus (VZV) skin lesions were analyzed for control purposes. The inflammatory infiltrate was characterized by immunohistochemistry using monoclonal antibodies against CD3, CD4, CD8, CD20, cutaneous lymphocyte-associated antigen (CLA), CCR4, CXCR3, Granzyme B, Tia-1, and MxA. Flow cytometry was used to analyze the expression of chemokine receptors on peripheral blood mononuclear cells in CxCTL. RESULTS: The CxCTL skin lesions were dominated by a dense infiltration of CD8+ cytotoxic lymphocytes with a skin-homing CLA+ CCR4+ phenotype. PL and VZV skin lesions were also characterized by a predominantly CD8+ T cellular infiltrate with strong expression of the cytotoxic molecules Granzyme B and Tia-1 and the skin-homing molecules CLA and CCR4. Coexpression analyses confirmed that skin CLA+ CD8+ cytotoxic T cells are present in CxCTL, VZV, and PL skin lesions. Strong lesional production of the antiviral protein MxA, which is specifically induced by type I interferons, could be found in all investigated disorders. The study was based on histologic, immunohistologic, and flow cytometric analyses in a limited number of patients, because of the rareness of the investigated diseases. CONCLUSION: Our results revealed a striking similarity between the immunohistologic picture of malignant CxCTL, benign PL, and VZV skin lesions. Strong expression of the antiviral protein MxA in all disorders supports the view that a common antiviral immune response pattern leads to aberrant skin recruitment of CLA+ CCR4+ cytotoxic T lymphocytes in PL and CxCTL.

Adult↗
PubMed1997

Later development of Fas ligand-mediated cytotoxicity as compared with granule-mediated cytotoxicity during the maturation of natural killer cells.

We classified CD56+ CD3- natural killer (NK) cells into CD2- CD56dim (CD2- NK), CD2+ CD56dim (CD2+ NK) and CD2+ CD56bright populations, and investigated mainly functional differences between the former two populations. CD2- and CD2+ NK cells were the same in their morphology and several surface molecules except for CD2. The percentages of CD2- NK cells in total NK cells were higher in the cord blood and marrow than in the peripheral blood of adults or children. Freshly isolated CD2- NK cells had CD2 in the cytoplasm, and gradually expressed it on the surface upon incubation with interleukin-2 (IL-2). These results demonstrated that CD2 is an antigen which appears on the surface during the maturation of NK cells. The granule-mediated cytotoxicities, which are mainly performed by the perforin molecule, of CD2+ NK cells against K562 and Daudi cells were higher than those of CD2- NK cells, and they were inhibited to the levels of CD2- NK cells by the addition of a blocking anti-CD2 monoclonal antibody (mAb). Fas ligand (FasL) mRNA was expressed in freshly isolated CD2+ NK cells but not in the CD2- NK cells. Neither freshly isolated NK populations showed FasL-mediated cytotoxicity, and only CD2+ NK cells lysed Fas-transfected targets after the 24-hr incubation with IL-2. Based on these results, CD2- NK cells have already developed granule-mediated cytotoxicity equal to that of CD2+ NK cells except for the CD2-associated activity, but they, unlike CD2+ NK cells, totally lack FasL-mediated cytotoxicity. These findings suggest that FasL-mediated cytotoxicity may be acquired at more mature stages of NK-cell maturation than granule-mediated cytotoxicity.

CD2 Antigens↗
PubMed1980

Natural cytotoxic cells against solid tumors in mice: blocking of cytotoxicity by D-mannose.

Natural cytotoxic (NC) and natural killer (NK) cells have been defined by their ability to lyse certain solid or lymphoid tumor targets in vitro, without prior sensitization. Our present studies describe an attempt to characterize the structures involved in the effector-target recognition leading to tumor cell lysis. Addition of the monosaccharide D-mannose to the NC cell assay significantly blocked cytotoxicity of the fibrosarcoma Meth A target by the effector cells at 50 mM and lower concentrations. D-Galactose showed blocking activity in one of five experiments, only at 50 mM. L-Fucose, D-glucose, and N-acetyl-D-glucosamine did not affect NC cell cytotoxicity at similar concentrations. All of the sugars tested inhibited NK cell lysis of the lymphoma YAC-I target. None of the sugars affected killing of the appropriate target by allosensitized cytotoxic T lymphocytes. The blocking of NC-mediated cytotoxicity was not due to a direct toxic action of the sugars on the effector cells. These findings suggest that, in the NC system, recognition involves lectin-like structures with a specificity for D-mannose (or D-galactose, or both), whereas, in the NK system, such lectin-like structures are less restricted. Such structures appear not to be involved in the specific cytotoxicity mediated by T cells.

Animals↗
PubMed1973

Pokeweed mitogen-, concanavalin A-, and phytohemagglutinin-induced development of cytotoxic effector lymphocytes. An evaluation of the mechanisms of T cell-mediated cytotoxicity.

Cultures of chicken lymphoid tissues were tested for their capacity to lyse (51)Cr-labeled chicken, burro (BRC), and human red blood cells (HRC) in the presence of phytomitogens. PHA-stimulated cultures lysed all three types of targets, while PWM and Con A showed a "target cell specificity" for HRC and BRC, respectively. In mixtures of target cells only the appropriate targets were lysed by lymphocytes activated by either Con A or PWM indicating that soluble lymphotoxins do not play a major role in these reactions. Preincubation experiments suggested that there may be a population of pre-existing aggressor cells which only require linking to the targets by the mitogens for activation of their cytotoxic potential. Strong cytotoxic reactions were found with spleen cells, peripheral blood leucocytes, and bone marrow cells. Thymocytes were less active but could be stimulated for significant cytotoxicity, while bursal cells were generally unreactive. Spleen cells from agammaglobulinemic chickens totally lacking serum immunoglobulins and B cells with surface-bound immunoglobulins were as active as cells from normal chickens. The activity of spleen cells, from which phagocytic cells were removed was also unimpaired. These results indicate that the development of cytotoxic effector lymphocytes in mitogen-treated leucocyte cultures is a property of T lymphocytes. Although bone marrow cells fail to proliferate in response to these phytomitogens, they do have strong cytotoxic reactivity suggesting that different subsets of thymic-derived lymphocytes are responsible for mitogen-induced transformation and mitogen-induced cytotoxicity.

Agammaglobulinemia↗
PubMed1991

Soluble CD4-PE40 is cytotoxic for a transfected mammalian cell line stably expressing the envelope protein of human immunodeficiency virus (HIV-1), and cytotoxicity is variably inhibited by the sera of HIV-1-infected patients.

Sera were obtained from 50 individuals infected with human immunodeficiency virus type 1 or from HIV-1-uninfected individuals before or after vaccination with recombinant gp160. These sera were evaluated for activity antagonistic to the cell-killing activity of the chimeric Pseudomonas exotoxin hybrid protein, sCD4-PE40. For these studies, Chinese hamster ovary (CHO) cells were transfected with a chimeric plasmid encoding the tat, rev, and envelope genes of HIV-1 and a cell line was selected for stable expression of the envelope glycoproteins at the cell surface (CHO-env). Cytotoxicity of sCD4-PE40 for CHO-env in the presence or absence of added human serum was quantitated spectrophometrically following enzymatic reduction of a tetrazolium bromide within the mitochondria of viable cells (MTT assay). Several HIV+ sera inhibited the cytotoxic activity of sCD4-PE40; the antagonist had properties consistent with those of immunoglobulins in that it was heat stable, absorbed by protein A, and reversible by increasing the concentration of sCD4-PE40. Of 15 HIV+ sera which strongly reacted with gp120, 11 (73%) also potently inhibited sCD4-PE40 cytotoxicity, and cytotoxicity was inhibited by sera from some HIV- individuals after, but not before, immunization with gp160. These data suggested a role for antibody to gp120 in the antagonistic activity. However, not all sera with antibody to gp120 antagonized sCD4-PE40 cytotoxicity and high levels of antagonist activity were frequently (40%) found in HIV+ sera lacking immunoblot-detectable antibody to gp120, or antibody to either CD4 or PE40. Grouping of the HIV+ sera according to the patients' absolute number of CD4+ cells revealed that the degree of inhibition of sCD4-PE40 cytotoxicity approached a Gaussian distribution, suggesting that persons with CD4+ cell counts between 200 and 700/mm3 may be more likely to possess significant levels of serum antagonist. This data have implications for the clinical development of sCD4-PE40 or other sCD4-based therapeutics in the management of HIV-1 infection.

ADP Ribose Transferases↗
PubMed2003

Mesenchymal stem cells inhibit the formation of cytotoxic T lymphocytes, but not activated cytotoxic T lymphocytes or natural killer cells.

BACKGROUND: Mesenchymal stem cells (MSCs) can reduce the incidence of graft-versus-host disease because of their ability to inhibit T-lymphocyte proliferation. There are no publications on the effect that MSCs have on cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, effector cells vital for the graft-versus-leukemia effect. METHODS: Cytotoxic T cells were primed in mixed lymphocyte culture (MLC) against irradiated stimulator lymphocytes, and irradiated third-party MSCs were added at different time points. The CTLs were collected, and their cytotoxic potential was analyzed in a chromium-release assay against the same stimulator cells as in the MLC. Purified NK cells were mixed with irradiated MSCs, and the lysis was measured in chromium-release assay against K562 target cells. RESULTS: We found that MSCs inhibited CTL-mediated lysis by 70% if added at the beginning of the 6-day MLC. The lysis was not affected on day 3 or in the cytotoxic phase. Furthermore, MSCs inhibited the formation of cytotoxic lymphocytes when the cells were separated in a transwell system, which indicates that the effect is mediated by a soluble factor. NK cell-mediated lysis of K562 cells was not inhibited by MSCs. MSCs did not induce proliferation of allogeneic lymphocytes, and they were not lysed by allogeneic CTLs or NK cells. CONCLUSION: Our findings indicate that MSCs escape recognition by CTLs and alloreactive NK cells, and inhibit the formation of cytotoxic T cells by secreting a soluble factor, but that they do not interfere with CTLs and NK cell lysis.

Cell Division↗
PubMed1990

Heterogeneity in direct cytotoxic function of L3T4 T cells. TH1 clones express higher cytotoxic activity to antigen-presenting cells than TH2 clones.

In the process of generating culture supernatant from T cell clones, with anti-CD3 antibodies and the B lymphoma A20 as APC, a striking difference in the stimulation of TH1 and TH2 clones was observed, i.e., TH2 clones produced higher levels of lymphokines than TH1 clones. This prompted us to test the hypothesis that differential killing of APC (thus the removal of stimuli) by T cells led to differential T cell activation. By studying a panel of five TH1 and seven TH2 clones, it was demonstrated that TH1 clones mediated significantly higher levels of cytotoxicity toward A20 cells in the presence of soluble anti-CD3 antibody (as opposed to immobilized anti-CD3). Although T cell clones could, when activated with immobilized anti-CD3, produce lymphokines cytotoxic to A20 cells, experiments in which lymphokine production was blocked indicated that T cell clones, in the presence of soluble anti-CD3, mediated killing of A20 through direct cytotoxicity. A higher level of cytotoxicity, by TH1 compared with TH2 clones, was not restricted to anti-CD3 or a particular target cell type, because it also occurred with Con A- or Ag-dependent killing (a monocyte-macrophage cell line), and LPS blasts. Furthermore, the higher cytotoxic activity of TH1 clones compared with TH2 clones was independent of the stage of T cell activation and was unlikely a result of the length of in vitro culture. High levels of killing of APC led to low levels of T cell activation, the significance of which may be as a negative feedback mechanism in the immune response. Other biologic relevancies of higher cytotoxic activity in TH1 vs TH2 cells were also discussed.

Animals↗
PubMed1991

Presence of Ca2+ is obligatory for the cytotoxic activity of dengue virus-induced cytotoxic factor.

The present study was undertaken to investigate the role of calcium ions (Ca2+) in the cytotoxic activity of the cytotoxic factor (CF) produced by T lymphocytes of the dengue type 2 virus (DV)-infected mouse spleen. It was observed that CF prepared in Ca2(+)-free medium had no cytotoxic activity on normal mouse spleen cells suspended in Ca2(+)-free medium but had activity on cells suspended in medium having Ca2+. The cytotoxic activity of CF was restored by substitution with calcium chloride, the optimal dose being 10(-7) M. CF induced influx of Ca2+ as measured by uptake of radiolabelled calcium chloride (45Ca), in the susceptible target cells, macrophages (M phi) and T lymphocytes, but had no effect on CF-resistant B lymphocytes. Calcium channel blocking drugs, like verapamil, nifedipine and diltiazem, inhibited the cytotoxic activity of CF and also the CF-induced influx of 45Ca in M phi and T cells. Thus, presence of Ca2+ is obligatory for the cytotoxic activity of CF and the cell death is associated with increased intracellular Ca2+.

Animals↗
PubMed1977

Generation of cytotoxic T lymphocytes during coxsackievirus tb-3 infection. II. Characterization of effector cells and demonstration cytotoxicity against viral-infected myofibers1.

This report describes studies characterizing the virus-specific cytotoxic effector cells which are present in the spleens of mice 7 days after infection with Coxsackievirus B-3. An in vitro 51Cr assay employing eyngeneic virus-infected neonatal fibroblasts was used to measure cytotoxic activity. Treatment of immune cells with (anti-thy-1.2) and complement abolished dtheir cytotoxic activity, but no reduction occurred when B cells were removed by incubation with anti-Ig and complement or macrophages eliminated by adherence depletion. The findings therefore imply that the cytotoxic reaction was mediated by sensitized T cells and that B cells and macrophages did not play an important role. Reciprocal assays performed with BALB/c and CBA/J cells showed that Coxsackievirus-immune spleen cells lysed infected syngeneic targets but not allogeneic targets, providing further evidence that cytotoxicity was mediated by effector T cells. In addition and in vitro assay system employing neonatal myocardial cells was developed and used to demonstrate that Coxsackievirus-infected myofibers were susceptible to destruction by immune spleen cells. The evidence suggests that mice infected with Coxsackie B viruses are able to mount a cell-mediated immune response with production of cytotoxic T cells which have the capacity to damage tissues infected with these agents.

Animals↗
PubMed1979

Direct cytotoxicity against chicken erythrocytes in mice. III. Degrees of radiosensitivity and cross-reactivity of cytotoxicity, delayed hypersensitivity and antibody production.

Exposure to 600 rad of X-irradiation 3 h after primary immunization with chicken erythrocytes (CRBC) abolished the production of antibodies and the generation of killer T cells, but scarcely affected the induction of delayed footpad reactions. Exposure to irradiation 3 h after secondary immunization reduced only slightly the generation of killer T cells and reduced slightly or substantially the production of antibodies. Delayed reactions persisted for long periods after elicitation in irradiated, boosted mice. Cross-reactivity between CRBC and quail erythrocytes (QRBC) was very weak with respect to the cytotoxicity, antibody and delayed reaction raised after primary immunization. Those raised after secondary immunization with the homologous antigen showed some degrees of cross-reactivity to another antigen. The booster with QRBC in CRBC-primed mice augmented the response to CRBC with respsect to the induction of cytotoxicity and delayed footpad reaction, but only weakly affected the response with respect to the production of antibody. Therefore, effector cells of cytotoxicity and delayed footpad reaction showed greater degrees of cross-reactivity than antibody-producing cells during an anamnestic response. The cross-reactive response raised after secondary immunization was radioresistant with respect to cytotoxicity and delayed footpad reaction but radiosensitive with respect to antibody production. Cytoxicity to immunizing antigen was inhibited by the unlabelled homologous antigen. Cytotoxicity to a cross-reacting antigen was inhibited by the same cross-reacting antigen and also by an immunizing antigen. There may exist heterogeneous populations of cytotoxic lymphocytes with different abilities to recognize antigens.

Animals↗
PubMed1981

Lymphocyte-mediated cytotoxicity against allogeneic tumour cells. IV. Fine specificity mapping and characterization of concanavalin A-activated cytotoxic effector lymphocytes.

Concanavalin A (Con A)-activated cytotoxic lymphocytes have been investigated, mapping the genetic differences between the P815 target and the effector cells required for cell-mediated lympholysis to occur. The target antigens recognized during the effector phase and the phenotype of the killer cell population(s) were also determined. It was found that Con A could activate a population of primed cytotoxic lymphocytes capable of killing target cells that were identical at the major histocompatibility complex (MHC) but differed at other background genes. Thus, after in vivo priming with DBA/2, B10.D2 lymphocytes cultured with Con A were capable of killing the P815 target. Unprimed B10.D2 cells, however, would not. Studies on the involvement of the MHC indicated that differences in the H-2K through H-2S, as well as differences in H-2D and H-2L alone could cause lysis. This killing could not be accounted for by additional differences at Qa-2, a MHC-linked locus. However, the contribution of other similar non-MHC linked loci could not be excluded. Cold target competition experiments indicated that MHC encoded alloantigens were involved as recognition structures on the target cell surface. Antisera plus complement depletion of cytotoxic effector function demonstrated that the cytotoxic cells had the cell surface phenotypes Thy 1.2+, Lyt 2.2+ and natural killer (NK) 1.1-. We conclude that Con A polyclonally activates population(s) of T cells that express antigen-specific cytotoxicity through clonally distributed recognition receptors intrinsic to their membranes when lectin is omitted from the cytotoxic assay.

Animals↗
PubMed1979

Spontaneous cytotoxicity. III. Inhibition of human monocyte mediated cytotoxicity by pokeweed mitogen.

We have shown that human mononuclear cells become spontaneously cytotoxic to a variety of erythrocyte targets after 7 days of in vitro culture. This phenomenon of spontaneous cytotoxicity occurs in low concentration of either autologous serum of FCS and is independent of any known stimulant. It does not require exogenous antibody, mitogen, or antigen. Once expressed, the cytotoxic cell has little, if any, specificity. Monocytes appear to be responsible for this observed in vitro cytotoxicity. We now present evidence that the development of spontaneous monocyte-mediated cytotoxicity in vitro is completely reversed by the addition of optimally mitogenic doses of PWM. PWM appears to reverse monocyte-mediated killing by stimulating a potent cell-mediated suppressor. This suppressor cell is radiosensitive and its action is blocked by inhibitors of protein synthesis. In contrast to other mitogen-driven suppressor models, PWM will inhibit spontaneous monocyte-mediated cytotoxicity even when added as late as 6 hr before the end of a 7-day culture.

Cell Separation↗
PubMed2002

Involvement of CD27/CD70 interactions in antigen-specific cytotoxic T-lymphocyte (CTL) activity by perforin-mediated cytotoxicity.

CD27 molecules are shown to be essential in the regulation of the death, activation and differentiation of T and B cells. However, the influence of CD27 on cytotoxic T-cell function remains obscure. Autologous EBV transformed B-cell lines (LCL), which highly express CD27 ligand CD70, here stimulated T cells and induced the cytotoxic T-lymphocyte (CTL) activity via T-cell antigen receptors (TCR). The cytotoxicity against LCL was diminished when anti-CD70 blocking MoAb was added initially in the culture. Resting T cells killed more CD70-transfected P815 cells than wild type P815 cells in the presence of anti-CD3 MoAb as measured by a 4-h 51Cr release assay, and the cytotoxicity of both of the cell populations completely disappeared in the presence of concanamycin A (CMA). The expression of the perforin by the LCL-induced CTL in the presence of anti-CD70 blocking MoAb was diminished as compared with that without the blockage of CD27/CD70 interactions. The CTL induced by LCL did not kill Fas-transfected WR cells. CD27 signalling in the T cells did not affect Fas ligand (FasL) mRNA expression, LAK activity and IFN-gamma synthesis in humans. Our data demonstrate that CD27/CD70 interactions enhance the cytotoxicity of CTL in the induction phase through enhancement of killing activity induced via the perforin-dependent mechanism, but not via the Fas/FasL system.

Antigens, CD↗
PubMed2000

Interleukin-10 increases Th1 cytokine production and cytotoxic potential in human papillomavirus-specific CD8(+) cytotoxic T lymphocytes.

Interleukin-10 (IL-10) is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage-dependent antigen presentation, T-cell proliferation, and Th1 cytokine secretion. However, several studies have challenged the perception of IL-10 solely as an immunosuppressive cytokine. As part of an investigation on potentiation of the cytotoxic activity of human papillomavirus E7-specific CD8(+) cytotoxic T lymphocytes (CTL) for adoptive transfusions to cervical cancer patients, we found that IL-10 in combination with IL-2, unlike several other combinations, including IL-2 with IL-12, gamma interferon (IFN-gamma), tumor necrosis factor alpha, and transforming growth factor beta, was able to consistently increase cytotoxicity. This augmentation in cytotoxic activity correlated with a significant increase in the cytoplasmic accumulation of perforin as detected by fluorescence-activated cell sorter. Surface expression of both the alpha and beta chains of the CD8 heterodimeric coreceptor and CD56 molecules was increased by exposure of CTL to IL-10. More importantly, we found that administration of IL-10 in combination with IL-2 after antigen stimulation consistently increased the intracellular expression of Th1 cytokines (i.e., IFN-gamma and IL-2) compared to results for control CD8(+) T cells cultured in IL-2 alone. In kinetic studies, proliferation, intracellular perforin levels, cytotoxic activity, and IFN-gamma expression were consistently elevated in CTL cultures containing IL-10 compared to control cultures, both at early and late time points following stimulation. In contrast, intracellular IL-2 expression was consistently increased only at early time points following stimulation with autologous tumor cells or solid-phase anti-CD3 antibody. Taken together, these data support the use of IL-10 in combination with IL-2 for the in vitro expansion and potentiation of tumor-specific CTL for clinical use in the therapy of cancer.

Cells, Cultured↗