Spontaneous Lymphocyte-mediated Cytotoxicity Research Articles

The elevated natural killer sensitivity of targets carrying surface-attached C3 fragments require the availability of the iC3b receptor (CR3) on the effectors.

Human serum-treated Raji and Daudi cells were shown to bind C3 fragments on their surface as a consequence of their capacity to activate C via the alternative pathway. C3 molecules were detectable on the cell surfaces up to 24 h after serum exposure. The C3 fragment-coated cells showed increased sensitivity to spontaneous lymphocyte-mediated cytotoxicity. The effector lymphocytes involved in the enhanced cytotoxicity were NK cells with low buoyant density, carrying both CR3 and FcR. Blocking of the FcR and CR3 with F(ab)2 fragments from Leu-11c or Leu-15 mAb, respectively, did not influence the lysis of targets that did not carry C3 fragments. In contrast, the accessibility of CR3 on the effector lymphocytes was essential for the C3 fragment-mediated enhancement of cytotoxicity. In addition to the Leu-15 antibody, N-acetyl-D-Glucosamine, a compound known to block iC3b binding to CR3, also abrogated the C3 fragment-imposed effect. Our previous experiments showed that the C3 fragments bind to acceptor sites on target cells. The present experiments show that the C3 fragments fixed onto the target bind to CR3 on effector cells. These data substantiate the hypothesis that the bivalent C3 fragments, which are fixed on the targets, promote their interaction with lytic lymphocytes by bridging the two cells.

Human spontaneous lymphocyte-mediated cytotoxicity (SLMC) against malignant and normal tissue-derived target cell lines tested in autologous and allogeneic combinations by the microcytotoxicity assay.

Effector cell types and effector mechanisms of human spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied in a 44-h microcytotoxicity titration assay. Peripheral blood lymphocytes from cancer patients and controls were used as effector cells either unfractionated or after fractionation by rosetting techniques or affinity chromatography. The possible immunoglobulin dependency of the reactions was studied by incorporation of specific Fab fragments of rabbit anti-human IgG antibodies in the incubation mixtures. Twelve different target cell lines of either high or low sensitivity to SLMC and with or without easily detectable HLA antigens were used. Most of the target cells were cell lines derived from transitional cell carcinoma of the urinary bladder (TCC). Both allogeneic and autologous lymphocyte target cell combinations were tested. Although high- and low-sensitivity target cells differed significantly in susceptibility to lysis, the predominating SLMC was displayed by Fc-receptor-positive lymphocytes in both allogeneic and autologous combinations. Addition of the Fab anti-immunoglobulin reagent to the incubation mixtures resulted in strong inhibition of cytotoxicity regardless of the type of target cells used and in allogeneic as well as in autologous lymphocyte target cell mixtures. However, in some combinations no inhibition was seen and inhibition was usually not complete, suggesting that both immunoglobulin-dependent (i.e., ADCC-like) and immunoglobulin-independent mechanisms were involved in the cytotoxicity reactions. The results of the microcytotoxicity assay were compared with those obtained with aliquots of the same lymphocytes and target cells in an 18-h 51Cr-release assay. While similar results were obtained with high-sensitivity target cells, with low-sensitivity targets and in some autologous combinations the two assay systems registered lymphocyte/target cell interactions which differed with regard to specificity, effector cell type, and immunoglobulin dependency.

Lymphocyte-dose response relationship in spontaneous lymphocyte mediated cytotoxicity (SLMC) measured by microcytotoxicity assay.

Titrations of spontaneous lymphocyte-mediated cytotoxicity (SLMC) in a 44-hour microcytotoxicity assay (MA) by serial dilutions of effector lymphocytes were performed to characterize the lymphocyte-dose response relationship, and to investigate the methodological basis for comparative cytotoxicity studies. The following lymphocyte fractions were used: unseparated, E rosetting, non-E rosetting, EA rosetting, and non-EA rosetting. Assays were performed against target cell lines from normal and malignant urothelium with high or low sensitivity to SLMC in allogeneic and autologous combinations, and against an osteosarcoma cell line. Analysis of SLMC titration curves showed that two different mathematical models could be used to describe the relationship between lymphocyte concentration and SLMC, I: linear relationship (log lymphocytes, CI) with exclusion of both high and low CI values, and II: sigmoid relationship (log lymphocytes, CI) which was transformed to linear relationship (log lymphocytes, log CI/100 - CI), including also high and low values. It is pointed out that perfect quantitative comparisons of SLMC in different combinations were complicated by statistically significant differences between the curve slopes. However, estimations based on experimental results in a range close to 50% cytotoxicity index (CI) may be acceptable, as the differences between slopes were small.

Increase of T Lymphocytes Bearing Immunoglobulin G Receptors During Circulation in Human Renal Cell Carcinoma

We studied the membrane markers and functions of lymphocytes from venous and arterial blood in kidneys from patients with renal cell carcinoma. The proportion of T lymphocytes that bore the receptors for the Fc portion of immunoglobulin G increased significantly in venous blood compared to arterial blood in kidneys with tumors. The assay of antibody-dependent cellular cytotoxicity demonstrated that lysis of target chicken erythrocytes was significantly less when caused by lymphocytes from the arterial blood (38 plus or minus 18 per cent, standard error) than those from the venous blood (54 plus or minus 21 per cent) in kidneys with tumors. There was no significant difference in the spontaneous lymphocyte-mediated cytotoxicity assay between the venous and arterial blood. Increment of the antibody-dependent cellular cytotoxicity activity seemed to correlate with the increased proportion and absolute number of T lymphocytes bearing immunoglobulin G.

Immunological characterization of cell lines established from malignant and normal human urothelium

Cell lines from normal or malignant human urothelium, described elsewhere by morphological, cell kinetic and genotypical criteria, have been characterized further by their response to antibody or lymphocyte mediated cytotoxicity in vitro. Four groups of cell lines were included: (1) two fast proliferating cell lines from normal urothelium, (2) three fast proliferating cell lines from transitional cell carcinoma (TCC) together with two transformed sublines of normal derived cell lines, (3) six slowly proliferating TCC lines, and (4) a line from squamous cell carcinoma. HLA antigen expression was demonstrated in the cell lines of groups 1 and 3, but not in lines from group 2 or 4. The sensitivity to spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells in group 1 and 2 exceeded that of cells from group 3 by a factor of 8. The TCC line, HU 456, was more susceptible to SLMC than T 24. Specifically increased cytotoxicity of lymphocytes from patients suffering from interstitial cystitis (IC) against HU 609 from normal urothelium indicated that this line expresses tissue specific antigens, and at the same time that an immune reaction may be part of the pathogenesis of IC. By a sensitive test for antibody-dependent, cell-mediated cytotoxicity (ADCC) antibody activity against HU 456 was found in only one of nine TCC patients and none of five clinical controls. A pronounced non-disease-related blocking serum activity, frequently found, may account for some of the negative findings. The SLMC against HU 456 could be inhibited but not abolished by Fab fragments of rabbit anti-human immunoglobulin, indicating an ADCC mechanism as part of the SLMC.

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells: VII. The effect of immunodeficiency disease

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseases—immunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients' lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients' or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.

Spontaneous and antibody-dependent cell-mediated cytotoxicity by human T cell subpopulations.

Human peripheral blood non-T cells, T cells and their subpopulations (Tmu, Tgamma, Tphi, Tgamma-depleted cells, and Tmu-depleted cells) were assayed for their capacity to mediated spontaneous lymphocyte-mediated cytotoxicity (SLMC) or natural killer activity against K562 tumor cell line and antibody-dependent cellular cytotoxicity (ADCC) against chicken erythrocytes coated with antibody. Non-T cells, unseparated T cells, Tgamma cells, and Tmu-depleted (Tgamma-enriched) cells were found to have both SLMC (NK activity) and ADCC. Tmu, Tphi, and Tgamma-depleted cells had minimal or no SLMC and ADCC activity. This study demonstrates that SLMC and ADCC activity in T cells is mediated by Tgamma cell subpopulations. These two cytotoxic reactions were either mediated by two distinct subsets of Tgamma cells or by a single effector cell using two different mechanisms.

Establishment and characterization of a squamous cell carcinoma of the vulva in tissue culture and immunologic evaluation of the host

A continual epithelial cell culture has been established from a squamous cell carcinoma of the vulva metastatic to the right inguinal lymph node. Initial characterization of the cell line (designated LT2), including morphology, growth properties, karyotyping, and secretion of carcinoembryonic antigen (CEA) and the beta subunit of human chorionic gonadotropin (HCG-β) is reported. The LT2 cell line satisfies the following criteria of malignancy: (1) establishment in culture for more than one and one-half years exceeding more than 70 generations; (2) heteroploid karyotype; (3) abnormal cytology; (4) growth in soft agar suspension; and (5) tumorigenicity in nude mice. This cell line has also been found to be free from contamination by fibroblasts, mycoplasma, bacteria, and fungi. With the LT2 cell line, the specific tumor-host immunorelationship was assayed by autologous (host lymphocyte versus LT2 cells) cytotoxicity and lymphoblast transformation in the presence of tumor extracts or mitomycin C-treated LT2 cells. In vivo patient immunocompetence was evaluated specifically by skin testing with autologous tumor extracts and nonspecifically with bacterial recall skin test antigens and dinitrochlorobenzene. In vitro nonspecific immunocompetence was evaluated by total peripheral white blood cell and lymphocyte counts, T cell, active T cell and B cell determinations, lymphocyte response to the mitogen phytohemagglutinin, and spontaneous lymphocyte-mediated cytotoxicity versus cultured bronchogenic carcinoma cells. Correlation of immunocompetence and the status of the tumor-host relationship with the patient's clinical course, and the potential significance of LT2 CEA and HCG-β production are discussed. The patient's response to bacillus Calmette-Guerin immunotherapy is evaluated.

Heterogeneity among human lymphocyte effector cells mediating spontaneous lymphocyte‐mediated cytotoxicity

The spontaneous lymphocyte-mediated cytotoxicity (SLMC) of lymphocytes from normal individuals was studied using the lymphocyte titration assay. Various techniques were used to study selectively several lymphocyte subpopulations. The activity of these subpopulations was tested in both the antibody-dependent cellular cytotoxicity (ADCC) and lymphocyte titration assays. Surface marker studies were performed on each lymphocyte subpopulation. The results from these studies indicate that effector cells mediating SLMC are a heterogeneous lymphocyte population composed of Fc-receptor-positive lymphocytes both with and without C3 receptors that consistently segregate with cells that do not form rosettes with sheep red blood cells. Our data suggest that, in many respects, the effector cells in SLMC and ADCC are identical. However, the mechanism appears to be different.

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells: IV. The suppressive effect of normal pregnancy

The effects of pregnancy and other donor characteristics on spontaneous lymphocyte-mediated cytotoxicity (SLMC) were studied. Sex, age, and stage of the menstrual cycle had no significant effect on the SLMC of the control donors, the SLMC was significantly lower in pregnant donors in the second or third trimester. However, this lowered level of activity was still within the 95 per cent confidence limits of the normal population and parity had no cumulative effect. At delivery, fetal cord blood lymphocytes possessed substantial cytotoxic activity, further evidence that the fetus is immunologically competent at birth. The relevance of these data to the immunologic coexistence of the mother and fetus is discussed.

ヒト腎腺癌由来細胞株 (KU-2) に対する正常ヒトリンパ球の細胞障害能

In order to solve the problems related to the resistance of human being against hypernephroma, in vitro cytotoxicity of the peripheral blood lymphocytes (PBL) from healthy persons was examined using an established cell line (KU-2), which was derived from human renal cell carcinoma, as the target cells.The original of the KU-2 was a metastatic hypernephroma in the lung, which was capable of producing ervthropoietin, and the KU-2 has been established in our Department as a monolayer cell-line from the tumor successively transplanted in nude mice.As the effector cells in the cytotoxicity assay, PBL were isolated by Ficoll-Conary method from the blood drawn from 3 healthy male and one healthy female adults. T cells and B cells separated from the PBL after removing macrophages by the carbonyl iron-powder treatment were also used as the effector cells. T cells were obtained as sheep erythrocyte-rosette (ER) forming cells, and B cells as non-ER forming cells. PBL, T cells, and B cells thus obtained were used as the effector cells either as such or after stimulation with phytohemagglutinin (PHA). In vitro cytotoxicity of the effector cells was assayed by % cytotoxicity in the microcytotoxicity assay and also by morphological observation of time lapse cinematographs. Identification of the effector cells responsible for the spontaneous lymphocyte-mediated cytotoxicity (SLMC) was made by scanning electron microscopic observation and by immunofluorescence technique for detection of surface Ig and Fc receptors on the cells.Cytotoxicity against KU-2 of PBL, T and B cells as such and after stimulaton with PHA showed (1) the highest Cytotoxicity by PHA stimulated PBL and PHA stimulated T cells (PHA induced cytotoxicity), (2) high cytotoxicity by individually isolated non PAH-stimulated B cells (SLMC), absence of cytotoxicity by individually isolated non PHA-stimulated T cells, and (3) discrepancy between the cytotoxic effects demonstrated under time lapse cinematography and % cytotoxicity determined by microcytotoxicity assay with regard to cytotoxicity by individually isolated PHA stimulated B cells. Furthermore, (4) presence of long microvilli on the surface of B cells connected closely with KU-2 cells as target cells was demonstrated under scanning electronmicroscopic observation, and (5) presence of surface Ig and Fc receptor as surface markers on B cells playing major role in SLMC was observed by immunofluorescence technique.Above mentioned findings indicated strong possibility that cytotoxicity generated by both PHA stimultaed T cells and non PHA-stimulated B cells were responsible for high cytotoxicity generated by PHA-stimulated PBL.

Lymphocyte Mediated Natural Cytotoxicity in Neoplasia

Peripheral blood lymphoid cells from normal donors, patients with non-malignant diseases, and patients with malignant diseases were evaluated for cytotoxicity against a cultured carcinoma cell line, using a chromium release assay. The natural spontaneous lymphocyte-mediated cytotoxicity was significantly higher in tumor-free groups. The observed decreased response in patients with malignancies correlated with depressed responsiveness to phytohemagglutinin stimulation. Removal of cell subpopulations bearing Fc receptors significantly decreased the cytotoxicity, while depletion of phagocytic mononuclear cells did not. We suggest that natural cytotoxicity is a measure of cell membrane integrity of lymphocytes and should be included as a routine test for evaluation of general immune competence.

Surface markers on human b and t lymphocytes. VI. Cytotoxicity against cell lines as a functional marker for lymphocyte subpopulations.

The spontaneous lymphocyte mediated cytotoxicity (SLMC) of cells from normal donors against 19 different established cell lines was analysed. All normal lymphocytes were cytotoxic in all combinations tested in a17h 51-Cr release assay. ALMC was found to be mediated by a minor subpopulation of lymphocytes with Fc/C3 receptors. Non-cytotoxic SRBC binding T-lymphocytes could be induced to become cytotoxic by the addition of Con A to the incubation medium. SLMC and lectin-induced cytotoxicity were briefly characterized. It is argued that SLMC is a non-specific non-immunological reaction which must be taken into consideration when lymphocytes from cancer patients are tested against tumour-cell lines in vitro. Futhermore, SLMC and letin-induced cytotoxicity are proposed as functional markers for Fc/C3-binding lymphocytes and SRBC binding lymphocytes respectively.