Cytotoxic lymphocytes from normal donors. A functional marker of human non-T lymphocytes.

A phenomenon we have termed spontaneous lymphocyte-mediated cytotoxicity (SLMC) by non-thymus-derived lymphocytes from normal donors has been described. The phenomenon can be demonstrated using human and xenogeneic (mouse) cell lines as the target cell in a microplate 51Cr release assay which is simple and reproducible. In this paper, SLMC against xenogeneic targets has been evaluated as a potential marker of lumphocytotoxic function with respect to: (a) the nature of the target cell; (b) the variability of the cytotoxic function of lymphocytes from different donors, and from the same donor tested on different days; (c) the nature of the effector cell. Using buoyant density centrifugation of iron plus magnetpurified lymphocytes forming rosettes with sheep red blood cells (SRBC) (T-cells) or SRBC-rabbit 19S anti-SRBC-mouse complement (complement receptor lymphocyte), it has been demonstrated that the cytotoxic activity lies in the non-T-lymphocyte fraction, and is probably caused by the complement receptor-bearing lymphocyte. The potential usefulness of this phenomenon as a functional marker of non-T-LYMPHOCYTE CYTOTOXIC Ability, and for the assessment of serological factors ehich may affect this cytotoxicity, has been discussed.     

Mycoplasma-dependent activation of normal mouse lymphocytes: requirement for functional T lymphocytes in the cytotoxicity reaction mediated by Mycoplasma arthritidis.

Syngeneic and allogeneic target cells were killed in the presence of CBA mouse lymphocytes and viable Mycoplasma arthritidis. Medium supplementation had no effect on the response. Nonviable M. arthritidis was also capable of stimulating lymphocytotoxicity, although to a much lesser extent. Cytotoxicity was shown to be largely dependent upon the lymphocytes, since lymphocytes preincubated with mycoplasmas and treated to remove remaining organisms were highly toxic to target cells, whereas supernatants prepared from lymphocyte/mycoplasma mixtures exhibited minimal effects. A 6-h exposure of lymphocytes to mycoplasmas at a ratio of 100:1 was sufficient for commitment to target cell killing. Functional lymphocytes were required for the reaction, since gamma-irradiated lymphocytes did not develop cytotoxic potential despite the fact that the mycoplasmas replicated equally well in the presence of these and untreated lymphocytes. Furthermore, lymphocytes already activated with mycoplasmas lost cytotoxic potential after disruption. The kinetics and degree of lymphocytotoxicity induced by M. arthritidis and phytohemagglutinin toward 51Cr-labeled syngeneic fibroblasts were similar. Removal of most B cells and other adherent cells by column separation did not abrogate the cytotoxic effect. Lymphocyte suspensions treated with anti-Thy 1 antiserum and complement exhibited a marked decrease in their cytotoxic potential when added to labeled target cells in the presence of M. arthritidis. We conclude that the cytotoxic reaction is dependent upon the T-lymphocyte subpopulation.     

Formation of auto-rosettes by peripheral blood lymphocytes.

A mean of 3-4% (0-5-19-5%) of the peripheral blood lymphocytes of normal adults were shown to bind three or more autologous erythrocytes in vitro to form auto-rosettes. Marked individual fluctuations were observed. Increased percentages were observed in patients with cancer, but not in other selected groups, including a group who had undergone thymectomy for myasthenia gravis. Auto-rosette formation was shown to be a property of T cells by the demonstration of (a) simultaneous binding of autologous and sheep erythrocytes, (b) non-inhibition of auto-rosette formation by anti-immunoglobulin, and (c) formation of auto-rosettes by mitogen-stimulated T blasts. Auto rosette formation is a property of high percentages of human thymocytes, and of lymphocytes treated with neuraminidase or stimulated to blast-cell trans-formation by phytomitogens. It is suggested that auto-rosette formation by these cells is related to their relatively low content of cell-coat sialic acid, as compared with untreated T lymphocytes. The possible influence of the cell coat on lymphocyte function is discussed briefly.     

Spontaneous release of Fc gamma receptors from normal human peripheral blood lymphocytes in vitro.

Certain normal human peripheral blood lymphocyte (PBL) membrane receptors for the Fc region of IgG (i.e. Fc gamma receptors) are known to be released spontaneously when incubated at 37 degrees in serum-free medium into the culture supernatant. However, the mechanisms involved in spontaneous Fc gamma receptor release are not clear. In this study we have shown that Fc gamma receptors appear to be released in two stages. Stage 1 occurs within the first hour of incubation, and is probably mediated by limited proteolysis at the cell surface. Stage 2 occurs between 2 hr and 4 hr, and requires active synthesis of Fc gamma receptors. The kinetics of Fc gamma receptor release from activated PBL was found to be slightly different from normal, but no appreciable increase in Fc gamma receptor 'activity' was found in the culture supernatants when compared with supernatants from normal 'resting' cells. It is doubtful whether these mechanisms operate in vivo since spontaneous release of Fc gamma receptors was completely inhibited in the presence of serum. The serum factors that prevent receptor release were found to be confined to two distinct fractions corresponding to those that contain the major serum protease enzyme inhibitors-alpha 2-macroglobulin and alpha 1-trypsin inhibitor.     

Concanavalin A-activated suppressor cells in normal human peripheral blood lymphocytes.

A 24-hr preincubation of human peripheral blood lymphocytes in the presence of Con A renders them suppressive for the response of untreated cells to soluble antigens, allogeneic cells and Con A.     

In vitro IgE formation by peripheral blood lymphocytes from normal individuals and patients with allergic bronchopulmonary aspergillosis.

Peripheral blood lymphocytes from five normal donors and five individuals with allergic bronchopulmonary aspergillosis (ABPA) were used for measurement of in vitro IgE formation in tissue culture preparations. IgE was measured by a sensitive radioimmunoassay using concentrated tissue culture medium (TCM) and expressed as ng IgE/ml of TCM. The results demonstrated that IgE was formed by unstimulated lymphocytes from one of five normal donors (range 1.5-2.4 ng/ml) and in two of five ABPA donors (range 2-4 ng/ml). However, when different dilutions of pokeweed mitogen were added to the tissue culture, three of five normal donors and three of five ABPA donors in remission showed enhanced IgE formation. At the time of an ABPA exacerbation in one individual, there was a definite increse in IgE synthesis up to 40 ng/ml which was suppressed by pokeweed mitogen to 17.3 ng/ml. Clear differences between normals and ABPA patients in remission are not apparent.     

Kinetics of cellular cytotoxicity mediated by cloned cytotoxic T lymphocytes

Kinetic methods can provide significant information concerning the mechanism of cellular cytotoxicity reactions. Previous kinetic studies of cytotoxic T lymphocytes (Tc) have been hampered by the heterogeneity of the effector cell population tested. We therefore examined the kinetics of lysis mediated by cloned, IL 2 and antigen-dependent murine Tc cells with strong cytotoxic activity that is restricted to distinct tumor-associated antigens on P815 mastocytoma target cells. Initial velocity measurements for cytotoxicity mediated by these clones fit a simple Michaelian kinetic model. Specific activity values obtained from these initial rate measurements are compared to those obtained for polyclonal Tc preparations, NK cells, and activated killer cells. Whereas the initial rate of lytic programming mediated by these cloned cells was very rapid, the rate of cytolysis mediated by the cloned cells decreased significantly within one hour. Since this decrease was observed over a wide range of E:T ratios, it is unlikely to result from product inhibition or a significant decrease in the concentration of unlysed target cells, but may be due to a decrease in the rate of programming and/or effector cell recycling. These results indicate that a simple Michaelian kinetic model is not adequate for tumor cell cytolysis by Tc cells in vitro.     

Antibody-dependent cellular cytotoxicity mediated by CD16+ lymphocytes from HIV-seropositive homosexual men

We investigated the ability of peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus (HIV)-seropositive asymptomatic and mildly symptomatic homosexual men with known time of seroconversion to mediate antibody-dependent cellular cytotoxicity (ADCC) specific for HIV. PBMC from HIV-seronegative and -sero-positive subjects lysed T (CEM) cells persistently infected with HIV to a significantly greater degree than uninfected CEM cells in the presence of HIV antibody-positive serum in a 4-hr 51Cr release assay. The response was mediated by CD16+ cells. ADCC responses were lower in PBMC of 13 men tested 9 to 25 months (average, 16.1 months) after seroconversion to HIV as compared with seronegative subjects, and were further decreased in 11 men tested 26 to 38 months (average, 31.6 months) after seroconversion. Decreases in numbers of circulating CD16+ cells appeared to contribute to depression in ADCC activity. The suppressive effect of HIV infection on ADCC effector cell activity may be important in the immunopathogenesis of acquired immune deficiency syndrome.     

Cytotoxic T lymphocytes against peptides derived from cyclin D1 isolated from peripheral blood of normal individuals.

We have studied the state of CTL tolerance to the self proteins cyclin D1, mdm2 and p53. These proteins were selected because they are often overexpressed in human tumours and may consequently represent targets for tumour-reactive CTL. In a murine model we found that peptides derived from these widely expressed self proteins can stimulate CTL responses in normal mice. The murine experiments revealed that the CTL repertoire is not completely tolerant to widely expressed self proteins that are frequently overexpressed in tumours.

Recently, we have extended these studies to the human system and analysed the state of CTL tolerance to cyclin D1. The cyclin D1 sequence was screened for HLA-A2.1 binding motifs and 5 motif containing peptides were synthesised. In vitro binding assays with T2 cells were used to identify A2.1-binding peptides. These peptides were used to coat dendritic cells isolated from the peripheral blood of HLA-A2.1 positive, healthy individuals, and the peptide coated DC were then co-cultured with autologous lymphocytes. Two peptides stimulated CTL that lysed the peptide loading mutant cell line T2 only in the presence of the relevant peptides. In contrast, the CTL showed some lysis of C1R-A2 cells without added peptides. Further experiments will show whether the CTL recognised cyclin D1 peptides are produced by natural antigen processing in C1R-A2 cells.     

Production of osteoclast-activating factor by normal human peripheral blood rosetting and nonrosetting lymphocytes

This study has tested the ability of human mononuclear cells and the subpopulations of lymphocytes that rosette (E-RFC) or do not rosette (non-E-RFC) with sheep red blood cells to synthesize DNA and to produce a mediator with bone resorbing activity (osteoclast-activating factor, OAF) in response to stimulation with phytohemagglutinin (PHA). Mononuclear cells were stimulated by PHA both to synthesize DNA and to produce a factor that caused bone resorption. E-RFC enriched for T lymphocytes and depleted of macrophages synthesized considerable DNA in response to stimulation with PHA, but were unable to produce significant bone resorbing activity in tissue culture unless macrophages were re-added to the E-RFC. In contrast, mononuclear cells depleted of E-RFC (non-E-RFC enriched for B cells) did not synthesize DNA in response to stimulation with PHA, but did produce a mediator that caused significant bone resorption in tissue culture. In most of the experiments, unstimulated non-E-RFC produced as much OAF as the PHA-stimulated non-E-RFC. These results suggest that both activated T lymphocytes (E-RFC) and B lymphocytes (non-E- RFC) can produce OAF.     

Use of cryopreserved normal peripheral blood lymphocytes for isolation of human immunodeficiency virus from seropositive men.

The possibility was investigated of using frozen stocks of phytohemagglutinin (PHA)-stimulated normal human peripheral blood lymphocytes (PBL) in cocultivation with human immunodeficiency virus (HIV)-infected lymphocytes for the isolation of HIV. Fresh and cryopreserved PBL from eight healthy volunteers were compared for their susceptibility to HIV infection in vitro. Fresh lymphocytes, as well as lymphocytes that were stimulated with PHA before or after cryopreservation, displayed comparable susceptibilities to HIV infection in vitro. In addition, HIV was recovered in all cases when lymphocytes stimulated with PHA before or after cryopreservation were cocultured in parallel with PBL from 15 patients with acquired immune deficiency syndrome. However, the cryopreserved PBL were less efficient in isolating HIV from asymptomatic men.     

Antibody-dependent direct cytotoxicity of human lymphocytes. II. Studies on peripheral blood lymphocytes, synovial fluid cells and sera from patients with rheumatoid arthritis.

Antibody-dependent direct cytotoxicity (ADDC) by human lymphocytes was evaluated in patients with rheumatoid arthritis and normal controls. Purified peripheral blood and synovial fluid lymphocytes mediated normal ADDC when compared to control subjects. No correlation could be obtained between the percentages of T, B and null cells in effector cell populations and the degree of 51Cr released from target cells. Sera from 50% of patients with rheumatoid arthritis inhibited ADDC by normal lymphocytes; the degree of inhibition did not correlate with the titre of IgM rheumatoid factor. The pathogenic implications of these findings are discussed.     

Inhibition of the pokeweed mitogen-induced response of normal peripheral blood lymphocytes by humoral components of colostrum.

Colostral whey at dilutions up to 1 : 100 inhibited both the uptake of tritium-labelled thymidine and the differentiation of pokeweed mitogen-stimulated peripheral blood lymphocytes from normal adults into immunoglobulin-containing plasma cells. Upon gel filtration (Sephadex G-200), inhibitory activity was associated with high molecular weight fractions. Secretory component, but not monomeric, polymeric or colostral IgA, IgM, IgG, lactoferrin, casein or alpha-lactalbumin, inhibited the response to mitogen. Supernatants from cultures of colostral cells did not induce the differentiation of adult peripheral or cord blood lymphocytes into IgA-containing cells and did not stimulate the uptake of tritium-labelled thymidine.     

Antibody dependent cell mediated cytotoxicity and phagocytosis of senescent erythrocytes by autologous peripheral blood mononuclear cells

Human red blood cells (RBCs) have a life span of 120 days in circulation, after which they are removed primarily by resident macrophages. Autoimmune antibodies are commonly found on effete RBCs and appear to contribute to their removal from the circulation. In this article, we focused on senescent erythrocytes and studied their removal, in comparison to young RBCs, in two RBC-depletion in vitro assays: antibody dependent cell mediated cytotoxicity (ADCC) and erythrophagocytosis. The results were determined prior to and following the addition of anti-D antibodies to the systems. Old (O-RBC) and young (Y-RBC) erythrocytes were separated by differential centrifugation. When incubated with autologous peripheral blood mononuclear cells as attacking cells, both anti-D treated O-RBCs and anti-D treated Y-RBCs were phagocytized and underwent contact lysis. However, O-RBCs had a significantly higher tendency to be phagocytized (p = 0.05) and a higher predisposition to undergo lysis (p = 0.043) than did Y-RBCs. When incubated with attacking cells without anti-D antibodies, O-RBCs were phagocytized while Y-RBCs were not phagocytized at all (p = 0.046). No contact lysis of either source of target cells occurred when incubated with attacking cells alone without anti-D sera. These in vitro results suggest that ADCC may serve as an additional pathway of elimination of senescent erythrocyte in addition to the classical phagocytosis pathway.     

Antibody-dependent direct cytotoxicity of human lymphocytes. I. Studies on peripheral blood lymphocytes and sera of patients with systemic lupus erythematosus.

Antibody-dependent direct cytotoxicity (ADDC) is generally believed to be unrelated to T-cell function in experimental animals. The role of ADDC in humans and its clinical usefulesss was evaluated in patients with systemic lupus erythematosus (SLE) and normal controls. Peripheral blood lymphocytes from patients with active SLE were unable to lyse antibody-coated target cells in vitro to the same degree as lymphocytes from patients with inactive SLE and controls. Sera from patients with active SLE suppressed ADDC by lymphocytes derived from normal controls and this abnormality was not corrected by overnight incubation or by extensive washing of lymphocyte preparations. Although there was poor correlation between ADDC and the proportions of B cells and null cells in effector lymphocyte populations from SLE patients and controls, it is concluded that this assay provides another means of determining immune competence in man.     

Isolation of lymphocytes from peripheral blood of dogs

Previous methods for the isolation of canine peripheral lymphocytes have not consistently produced cells of adequate purity. By means of centrifugation over a layer of Ficoll-sodium diatizoate of appropriate density, followed by passage through a simple column using nylon fibers, relatively pure lymphocyte fractions were isolated from canine peripheral blood. This simple and rapid technique requires no special equipment. Sixty percent of the total lymphocyte population was recovered. The fractions were composed of 96% normal small lymphocytes with a viability of 98%. The lymphocytes thus obtained were suitable for a lymphocytotoxicity assay.     

Stimulation of human peripheral blood lymphocytes by Tamm-Horsfall urinary glycoprotein.

Tamm-Horsfall urinary glycoprotein (THP), prepared by salt precipitation of pooled urine from normal individuals, stimulated purified human peripheral blood lymphocytes (PBL) to undergo blastoid transformation. the response was measured by tritiated thymidine uptake into DNA after 6 days in culture. Several batches of THP stimulated, in varying degrees, all samples of PBL tested and the response approached that seen with the mitogens phytohaemagglutinin (PHA), Concanavalin A (Con A) and pokeweed mitogen (PWM) after 4 days in culture. The response usually exceeded that seen after 6 days with tuberculin purified protein derivative (PPD) in Mantoux positive lymphocyte donors.