The tissue distribution of spontaneous cell-mediated cytotoxicity effector lymphocytes in swine

Peripheral blood lymphocytes, intraepithelial lymphocytes from the small intestine and lymphocytes from the thymus, spleen, mesenteric lymph nodes and Peyer's patches from 5 young adult pigs were used as effector cells in a spontaneous cell-mediated cytotoxicity chromium release assay against PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. Both peripheral blood and intraepithelial lymphocytes caused marked specific chromium release, while the lymphocytes from the remaining tissues were inactive in spontaneous cell-mediated cytotoxicity.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) and cell-mediated cytotoxicity (CMC) to HBsAg-coated target cells in patients with hepatitis B and chronic hepatitis (CAH).

A new technique using HBsAg-coated Chang cells as target cells was developed in order to measure cell-mediated immune reactions to HBsAg. The specificity of cytotoxic reactions was tested in experiments using Chang cells conjugated with human serum albumin. Antibody-dependent cell-mediated cytotoxicity (ADCC) specific for the HBsAg-coated target cells was demonstrated up to dilutions of anti-HBsAg serum of 10,000 : 1, when lymphocytes from the peripheral blood of normal individuals were added to the target cells. Spontaneous cell-mediated cytotoxicity (CMC) to HBsAg-coated target cells was demonstrated for lymphocytes from patients with hepatitis B and from patients with chronic active hepatitis (CAH), but not for lymphocytes from healthy controls. The CMC of hepatitis B lymphocytes to HBsAg-coated target cells was inhibited in the presence of antiserum to HBsAg. In experiments using purified lymphocyte populations evidence is presented that the CMC is T-cell dependent. HLA-restriction of the CMC was not observed. The described cytotoxicity test system has the advantage that target cells conjugated with defined antigens are used and that relevant control target cells are available.     

Fas (CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediated cytotoxicity and immune pathology

Members of the tumor necrosis factor (TNF) superfamily are crucially involved in the regulation of T cell activation, homeostasis and cytotoxicity. In particular, Fas ligand (FasL), expressed by activated T lymphocytes, induces cell-mediated cytotoxicity and may also be responsible for apoptotic suicide. Tight regulation of this death-inducing ligand is a prerequisite for proper immune defense and homeostasis. In this review, we will discuss various aspects of FasL regulation in cell-mediated cytotoxicity, immune homeostasis and the immunopathology of diseases.     

The participation of antibody in spontaneous and antibody-dependent cell-mediated cytotoxicity in the pig

Treatment of porcine lymphocytes with trypsin reduced their spontaneous cell-mediated cytotoxicity (SCMC) activity against target cells persistently infected with transmissible gastroenteritis virus (PK15-TGE cells), but had no effect on antibody-dependent cell-mediated cytotoxicity (ADCC). SCMC activity was partially restored to trypsin-treated lymphocytes by incubation in RPMI-1640 medium or in medium containing F(ab')2 fragments of rabbit anti-porcine immunoglobulin, but not by brief incubation in autologous serum. F(ab')2 fragments of anti-porcine immunoglobulin did not block the SCMC reaction, but ADCC was greatly reduced by this reagent. Thus SCMC and ADCC mediated by porcine lymphocytes against PK15-TGE target cells clearly involved two distinct mechanisms in terms of antibody participation and sensitivity to trypsin.     

Characterization of cell-mediated cytolytic mechanisms against human transitional cell carcinoma line, 253J.

We examined the nature of effector cells that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and spontaneous cell-mediated cytotoxicity (SCMC) against the 253J human bladder transitional cell carcinoma cell line. Fractionation of peripheral blood lymphocytes (PBL) showed that ADCC and SCMC against the 253J targets were mediated by non-adherent, surface immunoglobulin-negative lymphocytes lacking or having only weak affinity for sheep erythrocytes. Macrophages were not cytotoxic against 253J cells in 18 h culture, as removal of macrophages by glass adherence or treatment with silica particles did not reduce ADCC or SCMC, and macrophage-enriched effector cell preparations were only weakly cytotoxic.     

Interferon-gamma protects human thyroid epithelial cells against cell-mediated cytotoxicity

Interferons have been shown to have antagonistic effects on cell-mediated cytotoxicity. In the present study, we investigated various types of immunologically mediated cytotoxic reactions with a 51chromium release assay, using human thyroid cells as targets, which were cultured in the presence and absence of interferon-gamma (IFN-gamma). Antibody-dependent cell-mediated cytotoxicity (ADCC), natural killer (NK) cell-mediated cytotoxicity, and autologous lymphocyte-mediated cytotoxicity (ALC) were measured utilizing thyroid tissue, sera and lymphocytes from patients with autoimmune thyroid disease as well as from normal subjects. ADCC, determined with sera from patients with Hashimoto's thyroiditis, was reduced by 70% when the thyroid target cells were cultured in the presence of 1000 U human IFN-gamma/ml culture medium. Similarly, natural killer cell-mediated cytotoxicity, as well as autologous lymphocyte-mediated cytotoxicity, was significantly reduced after pretreatment of thyroid target cells with 500-1000 U IFN-gamma/ml culture medium. Although it is known that IFN-gamma renders target cells resistant to NK cell-mediated lysis, this is the first report on this effect using human epithelial cells, which may have major implications for the suggested role of IFN-gamma in the induction and perpetuation of autoimmune thyroid disease.     

Natural killer cells in normal pregnancy: analysis using monoclonal antibodies and single-cell cytotoxicity assays.

Peripheral blood lymphocytes (nylon wool non-adherent) from healthy pregnant women and normal non-pregnant females were tested for natural killer (NK) cell-mediated cytotoxicity against K562 target cells both by 51Cr-release assay and single-cell cytotoxicity assay in agarose. The results indicated depression of NK cytotoxicity in pregnancy due to a decrease in the proportion of target-binding lymphocytes as well as a reduction in the lytic capacity of target-bound cells. The ability of active pregnancy-associated NK lymphocytes to recycle appeared to be unimpaired. Analysis of lymphocyte populations with monoclonal antibodies recognizing NK cell-associated antigens showed that the number of Leu-11+ lymphocytes was reduced in pregnancy. Enumeration of Leu-7+ cells and correlation of NK cell subpopulation data with cytotoxicity assay data suggest that pregnancy is associated with a reduction in the number of mature NK cells and probably also an inhibition of post-binding lytic activity.     

Activation of human blood lymphocytes and monocytes by the streptococcal preparation OK432: enhanced generation of soluble cytotoxic factors

The streptococcal preparation OK432 augments natural cytotoxicity of human blood lymphocytes and monocytes. It also enhanced the production of natural killer soluble cytotoxic factors (NKCF) when the effector cells interact with K562 cells. There was a good correlation between the OK432-induced enhancement of NK cell-mediated cytotoxicity and the released NKCF activity. OK432-pretreated monocytes secreted higher amounts of monocyte cytotoxic factors (MCF) than the untreated monocytes. With the monocytes the enhanced generation of MCF was not always accompanied by the increase in direct cell-mediated lysis of K562. OK432 treatment alone did not induce NKCF release from lymphocytes, and the presence of K562 in the culture was necessary. In contrast, monocytes generated MCF when exposed to OK432. In the supernatants of cocultures of OK432-activated effectors and K562 the NKCF and MCF activity was elevated two- to ten-fold. The OK432-induced augmentation of natural cytotoxicity exerted by lymphocytes and monocytes may be mediated through an increase in the synthesis, activation and/or release of NKCF and MCF.     

Inhibition of NK and K cell function by phenothiazines

Several phenothiazines have been shown to inhibit NK cell-mediated cytotoxicity (NKC) and antibody-dependent cell-mediated cytotoxicity (ADCC) effected by human peripheral blood lymphocytes. Of those tested, chlorpromazine was found to inhibit at the lowest concentration (5 microM) with a 90% inhibition at 10 microM for NKC and 70% inhibition for ADCC. Pre-incubation of either effector or target cells with chlorpromazine did not provide any evidence of inhibition in subsequent cytotoxicity assays. The data for chlorpromazine in particular may be of clinical significance. In vivo inhibition would presumably require the presence of the drug since preincubation did not produce inhibition of NK or K cell function.     

Accessory Cell Paradox: Monocytes Enhance or Inhibit Lectin-Mediated Human T-Lymphocyte Proliferation Depending on the Choice of Mitogen

Monocytes suppressed the mitogenic response of human T lymphocytes to the lectin from Datura stramonium but enhanced the mitogenic response of the same lymphocytes to wheat germ agglutinin under identical culture conditions. The inhibitory action was probably cell-mediated, but was not the result of cytotoxicity. The stimulative activity could be replaced by cell-free conditioned medium.     

Determination of the frequency of HLA antibody secreting B-lymphocytes in alloantigen sensitized individuals

Sera from prospective transplant patients are usually screened for HLA antibodies prior to transplantation, but presently available tests do not permit quantification of the humoral alloantigen directed response. We adapted a culture system for isolated human B-lymphocytes to assay the secretion of HLA-antibodies on a single cell basis. B-cell supernatants were screened for HLA antibodies by complement dependent cytotoxicity. The assay assigns precursor frequencies for HLA-alloantibody secreting B-lymphocytes (BCPFs), and simultaneously allows for dissection of the humoral alloantigen directed response into its monoclonal components. The lymphocytes of 15 HLA-seropositive multiparous women that were used to validate the assay, were found to contain HLA-BCPFs ranging from 0 to 123 per 10(6) B-lymphocytes (mean: 43 +/- 45 per 10(6) B-lymphocytes). The HLA-specificities of antibodies in the B-cell supernatants were in agreement with serum specificities. Genuine HLA reactivity of B-cell supernatants was confirmed using an ELISA with purified HLA class I antigens. When applied to lymphocytes of patients on transplant waiting lists, the present assay may enable the unraveling of serum specificities in their components, thus supplementing HLA antibody serum screening data.     

Reconstitution of natural cell-mediated cytotoxicity with specific antibodies.

The apparent nonselective reactions of natural cell-mediated cytotoxicity (NCMC) are selective when tested by inhibition of cytotoxicity with competitor cells indicating a recognition of specificities by the effector cell. N cells that mediate this NCMC in humans have most of the characteristics of K cells that mediate antibody-dependent, cell-mediated cytotoxicity (ADCC) and possess Fc receptors. IgG antibodies attached loosely to N cells through their Fc region, form part of the class of lymphocytes with surface immunoglobulin. We hypothesized that ADCC and NCMS involved similar mechanisms but with the specificity of NCMC directed by the natural IgG antibodies already attached to N cells. Removal of the antibodies with trypsin and reconstitution with specific anti-HLA antibodies produced specific effector cells supporting the role of antibodies on N cells as directors of specificity in NCMC.     

Mitogen-induced lymphocyte-mediated cytotoxicity in vitro: effect of mitogens selectively activating T or B cells

Concanavalin A (Con A) and phytohemagglutinin (PHA) are selective T cell mitogens, whereas lipopolysaccharide (LPS) and purified protein derivative of tuberculin (PPD) are selective B cell mitogens. Con A and PHA induced mouse lymphocyte-mediated cytotoxicity in vitro, which was equally expressed on autologous, allogeneic and heterologous target fibroblasts, as measured by thymidine release from prelabeled targets. The dose response curves of Con A-induced lymphocyte-mediated cytotoxicity and DNA synthesis were parallel, 5 μg/ml being optimal, both higher and lower concentrations being less effective. Con A pretreated mouse lymphocytes failed to express cytotoxicity even though they were irreversibly activated with regard to DNA synthesis and were morphologically transformed. However, when Con A pretreated lymphocytes were added to target fibroblasts in the presence of soluble Con A, the cytotoxic effect markedly exceeded that induced by Con A in non-pretreated lymphocytes. It is suggested that cytotoxicity not only requires close contact between target and aggressor lymphocyte, but actual binding of the lymphocyte to the target, in this case effectuated by Con A. Con A and PHA failed to induce lymphocyte-mediated cytotoxicity in B lymphocytes. LPS and PPD did not induce cytotoxicity in normal or in B lymphccytes. Even LPS or PPD pretreated B lymphocytes failed to exert cytotoxicity when aggregated to the targets by Con A. It is suggested that the lymphocytes responsive to LPS and PPD are not the non-T cells responsible for cell-mediated cytotoxicity on antibody-coated target cells. Con A and PHA, but not LPS and PPD, induced lymphocyte-mediated cytotoxicity in human lymphocytes on allogeneic and heterologous target fibroblasts. The dose response curves for induction of cytotoxicity and DNA synthesis by Con A were not always identical in this case, cytotoxicity persisting at low Con A concentrations, which failed to activate DNA synthesis.     

Expression profiles of TCRbeta and CD8alpha mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp

Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8alpha and TCRbeta genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8alpha and TCRbeta mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8alpha and TCRbeta mRNAs. In addition, mRNA expressions of CD8alpha and TCRbeta in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.     

Lymphokine-activated killer (LAK) cells: I. Age-dependent decline of LAK cell-mediated cytotoxicity

Experiments were designed to assess age-related changes in generation of lymphokine-activated killer (LAK) cells and to test whether these changes can be modified by diets differing in the proportion of polyunsaturated to saturated fatty acids (P/S). Ficoll-Hypaque-isolated spleen lymphocytes of rodent chow-fed, 6-85-week-old C57BL/6 (H-2b), 8-81-week-old C57BL/10 (H-2b) and 6-62-week-old SJL (H-2s) mice were cultured in IL-2-containing medium and examined in 51Cr cytotoxicity assay. Similarly, Ficoll-Hypaque-isolated spleen lymphocytes of 6-36-week-old SJL mice fed diets which differed in the ratio of polyunsaturated/saturated fatty acids were cultured in IL-2-containing medium and assayed for cytotoxicity. Age-related decline of LAK cell-mediated cytolysis was observed in mice of both H-2b and H-2s haplotype. The age-related decline of LAK cell-mediated cytolysis was the consequence of age-related decrease in the rate of LAK cell precursor maturation. SJL mice fed from birth with diets differing in P/S did not differ in LAK cell-mediated cytolysis.     

Anti-CD3-or PHA-induced cytotoxicity in human intraepithelial and lamina propria lymphocytes

Morphological data in humans and rodents and functional data of intraepithelial lymphocytes of mice support the idea that cytotoxic cells are a significant population of the human mucosa. Previously it was shown that human IEL have no spontaneous cell-mediated cytotoxicity and that human LPL cells have anti-CD3-mediated cytotoxicity. We confirm that most individuals have anti-CD3- or PHA-mediated cytotoxicity of LPL. In IEL we do not find cytotoxic function in short-term assays. There is no difference between patients with colon cancer and inflammatory bowel disease.     

Characterization of K-cell activity by use of depletion experiments.

Normal human blood lymphocytes with affinity for ox red cells sensitized with IgG antibody, for normal or papain-treated sheep red cells and for ox red cells coated with mouse complement were used for rosette formation and the rosetting cells separated by density gradient centrifugation on Ficoll/hypaque. The non-rosetting cells at the interface were collected and compared with cell suspensions before treatment for direct and antibody-dependent cytotoxicity of human target cells. Depletion of Fc-receptor-bearing lymphocytes strongly decreased antibody-dependent cell-mediated cytotoxicity; reduction in the number (or depletion) of T cells and cells with C'3 receptor had no effect, showing the same or enhanced K-cell activity. It is concluded that one type of K or killer cell has Fc receptors but lacks C'3 receptors.     

Cell-mediated immune response in vitro: Independent differentiation of thymocytes into cytotoxic lymphocytes

Differentiation of dispersed mouse thymus cells into cytotoxic lymphocytes has been shown for the first time to occur in an in vitro allograft system. It is concluded that the generation of cell-mediated cytotoxicity is a T cell phenomenon with no thymus-bone marrow synergism being required.     

In vitro cell-mediated cytotoxicity to the male specific (H-Y) antigen in man

We have studied the role of HLA antigens in restricting specificity of the cytotoxic lymphocytes (CTL). CTL's developed between female responder/male stimulator combinations, were tested for H-Y antigen killing in cell-mediated lympholysis. Two CTL's demonstrated HLA-restricted H-Y cytotoxicity. In both instances, the responders are married parous females and both are positive for HLA-A2. These CTL's lysed target cells from donors who are either positive for the sensitizing HLA antigen or who are HLA-A2-positive males. On the other hand, one CTL where the HLA-A2-positive responder is not a parous female did not show HLA-restricted H-Y cytotoxicity. Also, CTL's where responders do not carry HLA-A2 showed no H-Y cytotoxicity. The data suggest that pregnancy(ies) is sufficient in itself to induce HLA-restricted H-Y cytotoxicity and that it can be recalled by in vitro stimulation with lymphocytes from an unrelated male donor. Also, in these studies HLA-restricted H-Y cytotoxicity was obtained only with targets that shared HLA-A2 with the effectors.     

The use of antibody-coated liposomes as a target cell model for antibody-dependent cell-mediated cytotoxicity.

A simple model system was developed for antibody-dependent cell-mediated cytotoxicity (ADCC) using antibody-coated synthetic membranes (liposomes) as a target cell model. A synthetic hapten, dinitrophenyl-phosphatidylethanolamine (DNP-PE), was incorporated into fluorophore-quencher-loaded liposomes and the latter were coated with pure anti-DNP antibodies. Normal spleen lymphocytes were capable of binding and subsequently lysing these liposomes. This process is dependent upon the presence of an intact (Fc-containing) IgG molecule and independent of exogenous serum complement. The effector lymphocytes are nylon non-adherent, devoid of Thy-1 antigen and present in nude mice, suggesting an identity with K (Fc receptor positive) lymphocytes. These studies indicate that liposomes may be used as a model to study the requirements for the binding and lysis of target cells in this cell-mediated cytotoxic system.