Cell-mediated cytotoxicity against ectromelia virus-infected target cells. I. Specificity and kinetics

Spleen cells from mice immunized intravenously with attenuated ectromelia virus were cytotoxic for target cells infected with virulent ectromelia virus in the absence of exogenous complement; cytotoxicity was measured by ⁵¹Cr release from target cells. L929 cells were the most sensitive target cells, but the effect could also be demonstrated with P-815 mastocytoma cells and chick embryo cells; mouse embryo cells were unsatisfactory. Hyperimmune anti-ectromelia serum and complement was not significantly cytotoxic although fluorescein-conjugated antiserum stained virus-infected L929 cells. Release of ⁵¹Cr label from ectromelia-immune spleen cells (at a spleen cell: target cell ratio of 100: 1) increased in a linear manner with time, until a plateau was reached at 20–24 hours. Cytotoxicity appeared to be specific in that ectromelia-immune spleen cells killed ectromelia-infected L929 cells, but not uninfected L929 cells, whereas spleen cells from mice immunized with Listeria monocytogenes (a potent stimulus for cell-mediated immunity) or from normal mice, did not kill ectromelia-infected L929 cells. Spleen cells from mice immunized with vaccinia virus (a poxvirus closely related to ectromelia) were cytotoxic for ectromelia-infected L929 cells. Spleen cells from mice immunized with ectromelia virus had acquired cytotoxic activity by 2 days after immunization. Cytotoxic potency reached a peak at 6 days and had declined to a low level by day 10. The potential significance of the phenomenon in relation to control of viral infection is discussed.     

Cell-mediated cytotoxicity against ectromelia virus-infected target cells. III. Role of the H-2 gene complex

The role of the H-2 gene complex in expression of cytotoxicity exerted by specific ectromelia-immune thymus-derived (T) cells against ectromelia-infected target cells was examined. A repertoire of inbred mouse strains (some congenic) including the H-2 haplotypes k, d, b, s, q, the recombinant H-2^a (k|d) and F₁ hybrids (k/b and d/b) were immunized with virus and their spleen cells tested 6 days later, at the peak of the primary response, against H-2^k, H-2^d and H-2^b target cells. Significant specific cytotoxicity occurred only when the immune cell donors and the target cells shared all or part of the same H-2 gene complex. For example, H-2^a (k|d) immune cells killed both H-2^k and H-2^d target cells. There wasno detectable effect of the non-H-2 genetic background, H-2 public specificities, or the M-locus Target cells infected with ectromelia virus exhibited quantitative or qualitative changes (or both) in expression of normal H-2 antigens as indicated by reduced susceptibility to killing by T cells activated against H-2 antigens in mixed lymphocyte culture. These data are consistent with the hypothesis that T cells in this system are responding to virus-induced, specific changes in antigens on infected cells which are controlled by genes in the H-2 complex; these genes seem likely to be those coding for H-2 private specificities, or genes closely linked to them.     

Cell-mediated cytotoxicity. Characterization of the effector cells.

Isolated human mononuclear cells were fractionated according to their membrane characteristics or physical properties. Adherent cells were depleted by filtration through glass columns; phagocytic cells were removed by iron treatment and cell subpopulations capable of forming rosettes with sheep erythrocytes (E), erythrocyte-antibody-complement (EAC) and chicken erythrocyte-antibody complexes (CEA) were separated by centrifugation of Ficoll-Hypaque gradients. The functional activity of the cell subpopulations obtained was assayed by testing PHA-induced cytoxicity (PIC), antibody-dependent cytoxicity (ADCC) and blast transformation by PHA. The results of this study demonstrate that: (1) cells reacting in PIC and ADCC assays are different, adherent and phagocytic cells being necessary for full expression of PIC and not for ADCC; (2) PHA induces direct blast transformation of purified E-RFC in the absence of PIC cytotoxic cells; (3) cell populations specifically enriched in E or EAC rosette-forming cells are not cytotoxic neither in the PHA nor in antibody mediated cytotoxic assays; (4) cells participating in ADCC can be selectively purified by centrifugation of CEA rosettes.     

Cell-mediated cytotoxicity against Sendai-virus-infected cells.

After injection of Sendai virus, a parainfluenza virus type I, mice generate cytotoxic lymphocytes which lyse specifically Sendai-virus-infected target cells in vitro. Their action is not inhibited by specific antibody in vitro. Killer cell activity appears 4 days after infection, reaches a maximum on the 7th day and disappears on the 14th to 16th day. Decrease of cytotoxic cell activity is correlated with an increase of haemagglutinating antibodies. The cytotoxic effector cell could be characterized as a thymus-derived cell, there is no specific activity in antibody-dependent cell-mediated cytolysis (ADCC). The degree of cytotoxic effector cell activity is only slightly influenced by the dose of injected infective virus. Using different syngeneic Sendai-virus-infected cells as targets for cell-mediated cytotoxicity, a tumor line was not lysed by cytotoxic lymphocytes in spite of viral surface antigens. Preliminary experiments were performed to demonstrate the H-2 gene restriction of the cytotoxic interaction. Using macrophages and tumor cells as targets only syngeneic infected target cells were lysed.     

Cell-mediated cytotoxicity: ATP as an effector and the role of target cells

Cell-mediated cytotoxicity involves a number of distinct mechanisms as well as the active participation of the target cell. Recently, several investigators have demonstrated that extracellular ATP can act as a cytotoxic effector.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. II. The mouse nonadherent K cell

A cell of lymphoid morphology capable of killing antibody-coated chicken erythrocytes was isolated from nonimmune mouse spleen using a combination of carbonyl iron treatment and glass bead column passage. This nonphagocytic effector cell, which is referred to as the nonadherent K (killer) cell, is distinguished from the non-phagocytic myeloid K cell described earlier (Greenberg, A.H., Shen, L. and Roitt, I.M., Clin. Exp. Immunol. 1973. 15: 251) by its relatively weak surface adherence properties and low concentration within the mouse spleen. The cell is further characterized by its relatively large size, lack of theta or immunoglobulin determinants, the presence of Mg⁺⁺-independent complement receptors, affinity for aggregated IgG₂ myeloma proteins, inhibition by cytochalasin B and good survival in cell culture. The possible lineage of the cell is discussed.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. III. Ultrastructural studies.

On the basis of electron microscopic observations, four types of cells apparently cytotoxic for antibody-coated chicken erythrocytes were identified in non-immune mouse spleen; monocytes, polymorphonuclear leucocytes, immature granulocytes and a type of lymphoid cell. Both phagocytosis and extracellular lysis of target cells were observed. Monocytes and polymorphonuclears were able to interact with the target cell by both mechanisms while the intermediary granulocytes and lymphoid cells were only capable of extracellular lysis. It is argued that these observations provide a morphological basis for the previous classification of antibody-dependent cytotoxic cells into myeloid and lymphoid cells (Greenberg et al., 1973b, 1975).     

Natural cell-mediated cytotoxicity of bovine mononuclear cells against virus-infected cells.

The ability of mononuclear cells (MC) from peripheral blood of normal cattle to lyse a variety of cells was tested in a 51Cr-release microcytotoxicity assay. Several types of bovine cells infected with parainfluenza 3 virus (PI3V) were susceptible to natural cytotoxicity. Bovine cells infected with infectious bovine rhinotracheitis virus or noncytopathogenic bovine viral diarrhea virus, uninfected bovine cells, and human cell lines MOLT-3, HSB-2, K562, and U-937 were not susceptible. The period of time that target cells need to be infected with PI3V to achieve maximal cytotoxicity was determined. Target cells were infected with PI3V and MC, added 1 h later. After the addition of effector cells, significant levels of cytotoxicity were recorded at 17 h. Maximal cytotoxicity occurred 22 to 24 h postinfection. To define the optimal time that MC must be present, cells were infected with PI3V for a total of 24 h, and MC were left in contact with target cells for various time intervals. Maximal cytotoxicity was recorded when effector cells were present for 20 h, suggesting that a period of activation was needed to stimulate effector cell function. Removal of adherent mononuclear cells on Sephadex G-10 columns did not reduce the low level of cytotoxicity against uninfected target cells, but markedly reduced the level of cytotoxicity against PI3V-infected cells. The effector cell was nonphagocytic and nonadherent. These characteristics and the fact that target cell lysis was independent of genetic restriction indicate that effector cells are similar to natural killer cells described in other species.     

Lymphocyte-mediated cytotoxicity against tumor cells II. Characteristics and tissue distribution of concanavalin A-activated cytotoxic effector cells.

In vivo or in vitro activation of cytotoxic effector lymphocytes by concanavalin A (Con A) has been shown to result in a population of effector cells exhibiting a degree of immunological specificity when Con A was present during the cytotoxic assay (Waterfield, J.D. et al., Cell. Immunol. 1975. 17:392). In this communication we have characterized the Con A-activated effector cell by physical criteria and tried to assign it to a given subpopulation of thymus-derived lymphocytes. It was shown that macrophages played no role in target cell lysis in this system, nor was their presence required for Con A activation of the effector cell. The effector cell proved insensitive to anti-theta serum, but was classified as a T cell by the enrichment of cytotoxicity when a purified population of T cells was activated by Con A in vitro and by the failure to activate the effector cell in nude mice. Precursors of the effector cell were shown to reside primarily in the spleen, to be radioresistant up to 400 R, and to be short-lived after adult thymectomy. Thus, the effector cell can be classified with the T1 population of T cells and has similar characteristics as a cytolytically active cell described by Stobo et al. (J. Immunol. 1973. 110: 362, 652).     

Antibody-dependent cellular cytotoxicity against tumor cells. II. The promonocyte identified as effector cell.

Macrophage precursor cells were cultured from the bone marrow of mice in a liquid culture system in the presence of conditioned medium. To separate their different maturation stages, they were passed through a discontinuous Ficoll density gradient, treated with iron plus magnet or passed through glass bead columns. The different maturation stages have been tested for their function in antibody-dependent cellular cytotoxicity (ADCC) against tumor target cells and in lymphokine-induced macrophage-mediated cytotoxicity. It is shown that the promonocyte, a nonadherent, nonphagocytic precursor cell, is a highly potent cytotoxic effector cell against antibody-coated tumor targets but is totally inactive as an effector cell in lymphokine-induced macrophage-mediated cytotoxicity. In contrast, in the lymphokine-induced macrophage-mediated cytotoxicity the cytotoxic effector cell is a mature macrophage. Thus, ADCC seems to be a function of the macrophage precursor promonocytes, whereas lymphokine-induced cytotoxicity is performed by mature macrophages. The relationship of promonocytes and killer (k) cells in ADCC against tumor targets is discussed.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. I. Phagocytic and non-phagocytic effector cell activity against erythrocyte and tumour target cells in a 51Cr release cytotoxicity assay and [125I]IUdR growth inhibition assay.

Both phagocytic and non-phagocytic effector cells were able to kill rabbit antibody-coated chicken erythrocytes (CRBC) while only non-phagocytic effector cells were active against alloantibody-coated SL2 lymphoma. In addition to the variation in susceptibility of erythrocyte and tumour target cells to various effector cell populations, it was found that different tumour cells can vary markedly in their ability to be killed by non-immune spleen cells in the presence of antibody. It is postulated that both the type of antibody and certain characteristics of the cell membrane are important in determining whether target cells are susceptible to antibody-dependent cell-mediated cytotoxicity detected by the 51Cr release assay. It was also demonstrated that alloantibody-coated P-815-Y mastocytoma, which showed very little evidence of cytotoxicity in the 51Cr release assay, was markedly inhibited in its ability to incorporate [125I]IUdR after incubation with antiserum and non-immune spleen cells. This growth inhibition in the absence of cytotoxicity, or cytostasis, is discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations (Plata, Gomard, LeClerc and Levy, 1974; Newlands and Roitt, 1975) that cytotoxicity and growth inhibition assays detect different effector cell populations in tumour-bearing animals.     

In vitro cytotoxicity by human lymphocytes from individuals immunized against histocompatibility antigens. II. Relation to HL-A incompatibility between effector and target cells

Lymphocytes from humans immunized by allogeneic skin grafts destroyed fibroblast monolayer cultures derived from the skin donor. Cytotoxicity also developed on several allogeneic fibroblast monolayers from unrelated persons. HL-A typing showed that all of these allogeneic fibroblasts shared one or more HL-A antigens with the skin donor. The intensity of the cytotoxic reaction increased with the number of these antigens present on the fibroblast targets, whereas no reaction occurred on allogeneic targets lacking these antigens or on the autochthonous fibroblasts. It is suggested, therefore, that the cytotoxic reaction reflects immunization against antigens within the HL-A system. An analogous correlation between the response of immunized lymphocytes and the number of immunizing HL-A antigens present was demonstrated in mixed lymphocyte cultures.

Lymphocytes from kidney grafted patients were not cytotoxic to any fibroblasts tested, including those from the kidney donor, not even during periods of clinical rejection. Humoral antibodies directed against the donor cells were demonstrated in one patient, but still no cytotoxicity occurred on the donor fibroblasts. Similarly negative results were obtained with lymphocytes from bone grafted patients.

     

Decreased cell-mediated cytotoxicity against virus-infected cells in systemic lupus erythematosus.

Cell-mediated cytotoxicity, directed against virus-infected tissue culture cells, was studied with peripheral blood mononuclear cells from 11 patients with systemic lupus erythematosus (SLE) and 12 matched, normal subjects in a 51Cr release assay. Baseline (preimmunization) levels of cytotoxicity against target cells infected with influenza A/Victoria, influenza B/Hong Kong, Newcastle disease virus, and herpes simplex virus were significantly decreased in patients with SLE compared to normal subjects (P less than 0.001), although serum antibody levels to the respective viruses were similar in both groups. After intramuscular administration of inactivated influenza A/Victoria vaccine, SLE patients failed to generate elevated levels of cytotoxicity against A/Victoria-infected cells, in contrast to normal subjects. SLE patients responded with levels of serum hemagglutination-inhibition antibody which were similar to those of normal subjects. Thus, SLE patients manifest decreased cell-mediated cytotoxicity against virus-infected target cells, although humoral antibody responses appeared to be intact. Studies of SLE patients with influenza may help to define the role of cell-mediated immunity in the pathogenesis of certain viral infections.     

HLA-restricted T lymphocyte-mediated cytotoxicity against herpes simplex virus-infected cells in humans.

Cytotoxic T lymphocytes (CTL) against herpes simplex virus (HSV) were induced in vitro from human peripheral blood lymphocytes by stimulation with HSV antigen. CTL generated by HSV type 1 (HSV-1) antigen stimulation killed not only HSV-1-infected target cells but also HSV type 2 (HSV-2)-infected target cells, though at a lower level. This evidence suggests that CTL against HSV recognize the HSV type-specific and type-common determinants on HSV-infected target cells. These CTL were generated from high responders against HSV-1 antigen as measured by antigen-specific T lymphocyte proliferation in vitro, but not to such an efficient degree from low responders. The cytotoxic activities of CTL against the allogeneic HSV-infected target cells were high when at least one of the HLA-A or -B antigens was shared. However, the HLA-A and -B nonidentical target cells were not killed effectively. The data presented here suggest the possibility of HLA restriction of HSV-specific CTL in humans.     

Antibody-dependent cell-mediated cytotoxicity in cows: comparison of effector cell activity against heterologous erthrocyte and herpesvirus-infected bovine target cells.

Bovine peripheral blood leukocytes (PBL) and cells collected from the bovine mammary gland were assayed for antibody-dependent cell-mediated cytotoxicity (ADCC) against chicken erythrocyte (CRBC) and bovine herpesvirus-infected bovine kidney cell targets. Bovine antisera were used to sensitize target cells. Both PBL and mammary leukocytes expressed ADCC, with the latter cell population having greater activity against both target cells. Only the CRBC target cells were killed by nonadherent PBL and phagocyte-depleted PBL. Nonadherent mammary leukocytes, rich in monocytes and macrophages, did kill virus-infected target cells. Carbonyl iron-treated mammary leukocytes failed to kill virus-infected targets but could destroy CRBC targets. Antimacrophage serum inhibited lysis of both CRBC and virus-infected targets, but antilymphocyte serum only inhibited CRBC killing. These observations indicated that at least two kinds of cells could mediate ADCC against CRBC but only cells of the mononuclear phagocytic series could kill virus-infected target cells. The herpesvirus-infected target cells became susceptible to ADCC 9 h after virus infection. A case is made for investigating the phenomenon of ADCC using in vitro systems that closely mimic the in vivo situation. The possible role of the ADCC mechanism as instrumental in causing recovery from herpesvirus infections is discussed.     

Antibody-dependent cell-mediated cytotoxicity against varicella-zoster virus-infected targets.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against cryopreserved varicella-zoster virus-infected human foreskin fibroblasts was detected in a 51Cr release assay. Target cells, samples of seropositive or seronegative sera, and mononuclear cells obtained by Ficoll-Hypaque centrifugation of human peripheral blood were added to microtiter plate wells and allowed to incubate at 37 degrees C for 4 h. Fibroblasts infected for 48 to 96 h were susceptible to ADCC. Effector cells from seropositive and seronegative normal children were equally active in the assay. Antibody titers were determined by testing serial dilutions of sera in the ADCC assay. Zoster immune globulin had a titer of 204,800. Sera from 40 naturally seropositive individuals were compared by assays for ADCC and fluorescent antibody to membrane antigen. All sera that were negative by fluorescent antibody to membrane antigen (less than 2) were also negative by ADCC (less than 20). All sera that were positive by fluorescent antibody to membrane antigen were also positive by ADCC, but titers of individual sera were frequently 5 to 20 times higher in the ADCC assay.     

Protective activity of secondary effector T cells generated in vitro against ectromelia virus infection in vivo.

The anti-viral activity of secondary effector cells generated in vitro against ectromelia virus infection was investigated. Depending upon the order of administration of cells and virus, 2 X 10(6) cells significantly reduce virus titres in recipient mice. Mice injected with lymphocytic choriomeningitis (LCM) virus are not protected by secondary effector cells against ectromelia virus infection and vice versa. The cells conferring anti-viral activity are sensitive to anti-theta and complement treatment, and must share H-2K or H-2D region genes with the recipients in order for significant reduction of virus titres to occur. The possibility of exploiting this approach in clinical medicine by using T cell-mediated mechanisms against certain viral infections is briefly discussed.     

Lymphocyte-mediated cytotoxicity against tumor cells. III Differentiation of concanavalin A-activated cytotoxic effector cells.

The maturation of selected T cell responses in the lymphoid organs of irradiated CBA mice was followed after adoptive transfer of syngeneic fetal liver cells. Mitogenic responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A) was found to reach control values 3 weeks after reconstitution in the thymus, spleen, and lymph node, of fetal liver repopulated animals. Spleen and lymph node cell reactivity in mixed lymphocyte culture reactions required 6 weeks to reach significant values. However, the ability of spleen cell suspensions to be activated by Con A into cytotoxic effector lymphocytes appeared after only 2 to 3 weeks. It is concluded that two functionally distinct T cell subpopulations exist in the spleen, one which can be activated into cytotoxic effector lymphocytes by Con A, and one which responds to alloantigens by DNA synthesis.     

Antibody dependent cell mediated cytotoxicity against anti-D sensitized human erythrocytes. Influence of various effector cells and enzyme treatment of the target cells

Antibody dependent cell mediated cytotoxicity (ADCC) was measured against anti-D sensitized erythrocytes using various mononuclear cell populations from peripheral blood in a chromium release assay. Adherent effector cells gave stronger cytotoxicity than unseparated and non-adherent cells, and were less dependent on pretreatment of the target cells with papain. However, close correlations were found between the cytotoxicity obtained with the various effector cell populations, indicating that either of them might be used for the purpose of establishing in vitro methods to determine the clinical significance of erythrocyte antibodies. The optimal choice of effector cells is discussed.     

Cell-mediated cytotoxicity against rat fibroblasts induced by Actinomyces viscosus.

Cell-mediated cytotoxicity against syngeneic fetal rat fibroblasts that require in vitro exposure of effector cells to Actinomyces viscosus Ny1 fractions was investigated by measuring the uptake of radioactivity by fibroblasts during a 2-h pulse with [14C]aminoisobutyric acid after 1 to 3 days of coculture with splenic effector cells. By using splenocytes from inbred RIC-Sprague-Dawley rats as effector cells and syngeneic embryonic rat fibroblasts as target cells, strong cell-mediated cytotoxicity dependent on the in vitro exposure to an A. viscosus Ny1 fraction was observed, but only within a small range of effector-to-target cell ratios (3:1 to 10:1). Concanavalin A and lipopolysaccharide from Escherichia coli induced a comparable cytotoxicity, indicating that the effect might be connected with the mitogenic activity of the A. viscosus NY1 fraction. Splenocytes from rats immunized with A. viscosus Ny1 and from control rats induced similar levels of cytotoxicity in 72-h cytotoxicity assays. In shorter assays (24 h), however, splenocytes from immune animals induced low cytotoxicity, which was, however, significantly higher than that induced by splenocytes from control animals. We conclude that both antigen- and mitogen-dependent cell mediated effector mechanisms are operative in this system and that the two normally overlapping effects can be experimentally separated. This new system describes a fibroblast impairment in the presence of splenocytes and bacterial components and may provide a useful model for studying pathogenic mechanisms operative in periodontal disease.