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.

     

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.     

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.     

Measurement of the cytotoxicity of human lymphocytes using HeLa cells as target

A simple and convenient method is described for the determination of spontaneous, PHA-induced, and antigen-induced cytotoxic activities of human peripheral blood lymphocytes. It involves measuring [³H]thymidine incorporation in HeLa cells as target. This methodology presents some advantages over the more commonly used ⁵¹Cr-release test.     

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).     

Ability of human cord blood lymphocytes to mediate antibody-dependent cellular cytotoxicity against influenza virus-infected cells

Cord blood lymphocytes, monocytes, and neutrophils from newborns were shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against influenza virus-infected cells. Antibody mediating ADCC was detectable in cord plasma, indicating that all components necessary for ADCC against influenza virus-infected cells are present in newborns. Among adult lymphocytes, two effector cell populations of influenza ADCC are recognized: non-T and T gamma cells. Each of these cell types expresses an antigen recognized by monoclonal HNK-1 antibody. The proportion of HNK-1 antigen-positive lymphocytes in cord blood was markedly lower than in adult blood; furthermore, ADCC was mediated by cord blood lymphocytes which were HNK-1 negative. By lymphocyte fractionation, the effector lymphocytes in cord blood were, as in adults, non-T and T gamma cells, suggesting that HNK-1 antigen is not expressed on these cell lineages in newborns.     

Cell-mediated cytotoxicity against ectromelia virus-infected target cells. II. Identification of effector cells and analysis of mechanisms

Cell-mediated cytotoxicity of ectromelia-immune spleen cells against ectromelia-infected L929 target cells was abrogated by treatment of the spleen cells with anti-Θ antibody and complement. Treatment of spleen cells with anti-immunoglobulin serum and complement, or removal of macrophages had little or no effect on cytotoxicity. Specific or nonspecific soluble cytotoxic factors were not detected, and exogenous interferon did not enhance cytotoxicity. No blocking of cytotoxicity was detected with either hyperimmune or 9-day immune anti-ectromelia serum. By varying the spleen cell-target cell ratio from 3: 1 to 400: 1, it was shown that the efficiency of cytotoxicity was inversely related to spleen cell density. These results were interpreted to mean that thymus-derived (T) cells, probably acting alone, were responsible for cytotoxicity and that the mechanism involved contact between T cell and target cell.     

Correlation between effector lymphocytes in natural and antibody-mediated cytotoxicity

Human sera enhanced spontaneous cell-mediated cytotoxicity (SCMC), while anti-IgG (Fab') 2 treatment decreased this cytotoxic activity of human lymphocytes for an in vitro growing cell line (K--562). Trypsin treatment of the effector cells considerably decreased the cytotoxic potential. However, a significant cytotoxic activity could always be found in serum-free medium. While these findings suggest the involvement of antibodies in the SCMC, they also reflect the existence of serum-indpendent (sui generis) SCMC activity of lymphocytes. Removal of SCMC of Fc receptor bearing effector cells was performed by target cell adherence (rosetting). Separation of the target cell-bound lymphocytes was done by centrifugation on special Ficoll gradient. The depletion of SCMC effector cells resulted in a 62% reduction of SCMC and in a 39% reduction of ADCC. On the other hand, removal of Fc bearing effector cells showed a similar reduction in both ADCC (66%) and SCMC (78%). Our results suggest that SCMC represents a complex activity, arising partly from the interactions of certain serum-derived or lymphocytes surface-bound antibodies and partly from a spontaneous cytotoxic function of the effector cells. It is possible that the effector cells involved in both SCMC and ADCC derive from the same lymphocyte population and the differences are due mainly to the lower number of SCMC effector cells.     

Somatostatin receptors on human lymphocytes and leukaemia cells

Receptors for somatostatin were identified on mitogen-activated human peripheral blood lymphocytes (PBL) and human leukaemic cells in 87.5% of lymphoblastic leukaemia and in 12.5% of non-lymphocytic leukaemia, using a somatostatin radiobinding assay. The specific binding of 125I-somatostatin of these cells increased linearly with the cell numbers and was suppressed by non-iodinated somatostatin. We investigated the distribution of fluorescent somatostatin to mitogen-activated PBL by using a fluorescence-activated cell sorter (FACS). Over 95% of the cell populations bound fluorescent somatostatin and no distinct predilection was found among certain lymphocyte subpopulations and somatostatin receptor-positive cells. Scatchard analysis showed a single class (low affinity) of binding site on mitogen-activated PBL and two classes (high and low affinity) of specific binding sites on lymphoblastic leukaemia cells.     

Enrichment by counterpart centrifugal elutriation of human lymphocytes cytotoxic to human tumour cells.

The enriched fractions of cytotoxic cells responsible for natural killer (NK) activity against both human sarcoma and neuroblastoma (LA-N2) cell lines were readily obtained by countercurrent centrifugal elutriation (CCE). The NK cells were obtained in the larger lymphocyte fractions (fraction 6 +/- 1), having a mean cell volume of 180 u3. The cytotoxic-enriched fraction contained 51% large lymphocytes having cytoplasmic granules. On the other hand, monocytes were purified to greater than 90% and isolated in another fraction (final fraction) and these cells had the lowest NK activity against both human tumour cell lines. However, compared with the lymphocyte fractions, small and large monocytes displayed greater antibody-dependent cellular cytotoxicity (ADCC) activity against human B erythrocytes. These results indicate that NK found to have activity against both tumour cells lines were larger lymphocytes, not small monocytes. Thus, countercurrent centrifugal elutriation (CCE) can provide a sensitive method to obtain enriched fractions of large lymphocytes contained tumoricidal activity against human sarcoma and neuroblastoma cell lines.     

Antibody-dependent cell-mediated cytotoxicity: heterogeneity of effector cells in human peripheral blood.

We have compared antibody-dependent cell-mediated cytotoxicity (ADCMC) of human peripheral blood leukocytes (PBL) in three model systems. target cells were 51Cr-labeled mouse mastocytoma cells, chicken erythrocytes (CRBC), and human erythrocytes (HRBC) coated with appropriate heterologous or isologous antisera. Effector cells were characterized on the basis of their adherence, phagocytosis, radiosensitivity, and sedimentation velocity(s) at 1 g. In predominantly mononuclear (Ficoll-Isopaque-purified) PBL preparations (MPBL) HRBC were lysed by an adherent, phagocytic population of cells that was markedly radio-resistant. Sedimentation velocity analysis further established that these effector cells were restricted to rapidly sedimenting fractions (s greater than 4.5 mm/hr). On the other hand, mastocytoma cells were lysed by a population of MPBL that was nonadherent, nonphagocytic, and relatively radiosensitive. These cells mainly restricted to slowly sedimenting fractions (s greater than 4.5 mm/hr) following 1 g velocity sedimentation. CRBC appeared to be susceptible to lysis by both types of mononuclear effector cell. In some experiments, enriched populations of polymorphonuclear leukocytes (PMN) were isolated. These cells were found to lyse both HRBC and CRBC very efficiently, whereas mastocytoma cells were lysed very little if at all by the same effector populations. Taken together, these results suggest that antibody-coated mastocytoma cells are lysed uniquely by effector cells in human peripheral blood with the physical properties of lymphocytes, whereas antibody-coated HRBC are lysed by both monocytes and PMN, but not by lymphocytes. Antibody-coated CRBC would appear to be lysed by all of the three effector cell types tested.     

Intercellular communication in cell-mediated cytotoxicity. Fluorescein transfer between H-2 d target cells and H-2 b lymphocytes in vitro

Fluorescein permeable junctions occur infrequently between unsensitized C57BL/6 lymphocytes (H-2^b) and DBA/2 mastocytoma cells (H-2^d), but the incidence of such junctions increases significantly with lymphocytes from animals sensitized against the mastocytes. The mastocytoma cells were labeled intracellularly with fluorescein using permeant (non-fluorescent) fluorescein dipropionate which is hydrolyzed to free fluorescein within the cells; they were additionally stained with neutral red. As an in vitro model of cell-mediated cytotoxicity the Brunner system was used in which close contact is a prerequisite of cell lysis. With contacts of aggressor cells to targets fluorescein permeable junctions were found in 17 out of 23 experiments, or 168 times, when sensitized spleen cells from female C57BL mice took part in the reaction. This incidence is significantly greater than in control experiments with normal spleen cells, when fluorescein permeable junctions were observed only 28 times and in only 9 out of 23 experiments.     

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.     

High nonspecific reactivity of normal lymphocytes against mycoplasma-infected target cells in cytotoxicity assays.

Several rat tumor cell cultures were deliberately infected with three species of mycoplasma commonly found as contaminants of cell lines grown in vitro, and the effect of mycoplasma infection on the results of cytotoxicity assays was examined. Lymph node cells and spleen cells from normal animals showed an apparently high spontaneous cytotoxic activity against tumor cells infected with either M. arginini or M. hyorhinis, but the reactivity against cells infected with M. orale was not significantly higher than that against uninfected cells. The high reactivity towards tumor cells infected with M. arginini and M. hyorhinis bore a close resemblence to natural cell-mediated immunity in that spleen cells were much more reactive than lymph node cells, spleen cells from nude mice were as effective as spleen cells from normal mice, and the reaction crossed both strain and species barriers. However, closer examination revealed that the cytotoxic effects were directly caused by depletion of arginine or other essential nutrients from the medium. These findings imply that a cautious approach should be taken when interpreting certain aspects of spontaneous cell-mediated cytotoxicity, and that the greatest care be taken to ensure that the cells used as targets in any cytotoxicity test are mycoplasma-free.     

A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.     

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.     

Cytokines secreted by IL-2-activated lymphocytes induce endogenous nitric oxide synthesis and apoptosis in macrophages

IL-2-activated killer (LAK) cells secrete inflammatory cytokines such as IFN-gamma and TNF-alpha, which can induce NO synthesis (NOS). In this study, we investigated IL-2-activated lymphocyte-mediated macrophage apoptosis via NOS. LAK cells and their culture supernatants induced NOS in murine macrophages. NOS was markedly inhibited by blocking antibodies to IFN-gamma and TNF-alpha, suggesting the key role of these lymphocyte cytokines in mediating NOS. Endogenous NO production inhibited macrophage proliferation and induced apoptosis in concordance with p53 accumulation and caspase-3 activation, processes that were inhibited by N(G)-monomethyl-l-arginine (a NOS inhibitor) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (a NO scavenger). Our study demonstrated a novel, noncontact-dependent mechanism of macrophage suppression by IL-2-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages as a result of endogenous NOS induced by cytokines secreted from IL-2-activated lymphocytes.     

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.     

The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity.

A mouse monoclonal antibody, anti-HM1.24 (IgG2a/kappa), binds to a surface antigen preferentially overexpressed on multiple myeloma (MM) cells, and exhibits potent antitumor cell activity against MM cells by antibody-dependent cell-mediated cytotoxicity (ADCC). To develop an antibody-based immunotherapy against MM, a humanized anti-HM1.24 antibody, in which all FRs correspond to naturally processed human FRs, has been successfully constructed with the aid of both the hybrid variable region and two-step design methods. This humanized anti-HM1.24 antibody (IgG1/kappa) is able to effectively induce ADCC against human myeloma KPMM2 and ARH77 cells in the presence of human PBMCs as effectively as a chimeric anti-HM1.24 antibody. The humanized anti-HM1.24 antibody, therefore, could be expected as a potent immunotherapeutic agent for MM patients.