Effector Cell Subpopulations Research Articles

Passive adoptive immunotherapy of low-immunogenic BALB/c lymphoma by syngeneic alloimmune T lymphocytes.

The virus-induced BALB/c lymphoma YC8 is known to be lysed in vitro by syngeneic lymphoid cells immune to non-H-2 antigens of B10.D2 and DBA/2 backgrounds. This tumor is weakly immunogenic in vivo and kills 100% of syngeneic mice with 1 X 10(3) cells given either intravenously (i.v.) or intraperitoneally (i.p.). We show here that i.v.-injected YC8 cells grow preferentially in the liver, where colonies become microscopically visible after 7-10 days, and, less frequently, in the kidneys and spleen but not in the lung. Passive adoptive immunotherapy of this tumor was carried out with alloimmune BALB/c anti-B10.A, anti-pool (donors were immunized with lymphocytes from 5 different strains), anti-A and anti-DBA/2 splenic and lymph node cells. When administered i.p. 1, 3 or 5 days after tumor cells had been given i.p. and with a schedule of 5 subsequent daily inocula, anti-DBA/2 lymphocytes cured 100%, 80% and 60% of animals respectively. A weaker effect was obtained with anti-pool immune cells whereas anti-B10.A and anti-A lymphoid cells had not therapeutic effects. When given i.v., the anti-DBA/2 immune lymphocytes were able to cure both i.v. and i.p. tumor-injected mice. A significant effect was observed also when the onset of immunotherapy was delayed until 7 or 10 days after tumor injection. By depleting the BALB/c anti-DBA/2 immune cells with appropriate monoclonal antibodies and complement, it was found that Lyt 1+ 2-cells played the major role in eradicating the neoplasm. in vitro phenotypic and functional analysis showed that the immune cell population included 70% of Thy 1+, 38% of Lyt 1+ and 18-20% of Lyt 2+ cells. Immune lymphocytes were not cytotoxic in vitro to YC8 or DBA/2 targets whereas they proliferated after restimulation with DBA/2 but only weakly with YC8 cells. This shows that it is possible to cure mice bearing a disseminated lymphoma which expresses non-immunogenic antigens recognized by BALB/c anti-DBA/2 immune T lymphocytes. These immune lymphocytes had no cytotoxic activity in vitro and their major effector cell subpopulation displayed the Thy 1+, Lyt 1+ phenotype.

Allosuppression: evidence for the involvement of both "noncytotoxic" and cytotoxic T cells.

Allogeneic suppressor cells were generated by priming to a minor histocompatibility antigen. C57BL/10 female mice were primed to the male-specific minor transplantation antigen, H-Y. After boosting, anti-male-primed cells could potently suppress the secondary antibody response of male spleen cells in vitro. Anti-male suppressor cells were H-2-restricted, radiation-resistant T cells which could act on either T or B cells in the responding population. Mapping the restriction elements for anti-male suppressor cells revealed two distinct subpopulations of effector cells. The majority subpopulation was restricted to H-Y in the context of the H-2Db molecule. These cells were probably cytotoxic T cells as they were inhibited by culture of the suppressor cells in pyrilamide (a histamine receptor antagonist, which prevents the maturation of cytotoxic T cells from their precursors). The second subpopulation of suppressor cells was restricted to H-Y in the context of H-2K or I region-coded molecules. as neither H-2Kb nor H-2Ib molecules can act as restriction elements for anti-male-specific cytotoxic T cells, this subpopulations was almost certainly not composed of conventional cytotoxic cells. Furthermore, these cells were not affected by culture in pyrilamide. Together these two populations act to completely inhibit the anti-trinitrophenyl (TNP) plaque-forming cell response of male spleen cells in vitro.

Heterogeneity of human NK cells: comparison of effectors that lyse HSV-1-infected fibroblasts and K562 erythroleukemia targets.

In this study, we compared the natural killer (NK) cells that lyse HSV-1-infected NK(HSV-1) or uninfected [NK-(FS)] fibroblasts to those that lyse K562 erythroleukemia cells [NK(K562)]. Activity against all three targets was found in Percoll gradient fractions enriched for large granular lymphocytes, which suggests that these effector cells have a common morphology. In competition studies between 51Cr-labeled targets and unlabeled targets, both the infected and the uninfected fibroblasts competed for lysis of NK(HSV-1) and NK(FS) activity, whereas K562 cells competed poorly. In contrast, when 51Cr-labeled K562 cells were used, the unlabeled K562 cells competed well, but HSV-1-infected and uninfected fibroblasts competed poorly. Panning studies and complement elimination experiments using monoclonal antibodies were performed to describe cell surface markers on the NK cell populations. Treatment with an antibody to an la framework antigen reduced NK(HSV-1) but not NK(K562) activity. In contrast, the majority of NK(K562) effectors were recognized by antibodies to the E-rosette receptor (Lyt-3 and OKT11A), whereas NK(HSV-1) activity was much less sensitive to this antibody. OKM1, OKT10, and Leu-7 (HNK-1) markers were found on a portion, but not all, of the cells that lysed both the HSV-FS and K562 targets, while treatment with HLA plus complement totally abrogated both NK activities. Taken together, these data are consistent with the concept that human NK cells are heterogeneous and that we are dealing with at least three subpopulations of effector cells--one that kills the infected or uninfected fibroblasts; one that kills K562 cells; and a third population that may be able to kill all three targets. Patient studies provide additional evidence for heterogeneity within the NK cells that lyse the fibroblasts and K562 cells. We have studied a number of individuals who have normal NK activity with one target (HSV-FS or K562) but have low or no activity against the other. These patients provide strong evidence not only that NK cells are heterogeneous but also that these NK subpopulations can be regulated independently of each other in vivo.

Quantification of natural cytotoxicity by human lymphocyte subpopulations isolated by density: Heterogeneity of the effector cells

Natural cell mediated cytotoxicity has been expressed as percent cytotoxicity, as the slope of the titration curve obtained by testing different effector: target cell ratios, and as lytic units. Objections can be raised to each method as used. The present report involves the study of cytotoxicity by subpopulations of lymphocytes obtained by Percoll density gradient centrifugation. The subpopulations vary greatly in cytotoxic activity, making accurate comparisons by traditional means difficult. A method was therefore developed for making objective comparisons between activities of subpopulations of lymphocytes. Cytotoxicity titration curves, which were sigmoidal on linear-linear plots, were found to best fit the sigmoidal curves described by the Von Krogh equation. A best fitting scale family of curves having the identical maximum cytotoxicity and shape parameter was fitted simultaneously to cytotoxicity measurements obtained by titrating all subpopulations of effector cells obtained from each patient. Values expressing relative cytotoxicities were obtained from the ratios of the scale parameters of the different curves or by obtaining lytic units from the fitted curves. In addition to the enriched natural cytotoxic activity found among the lighter cells obtained by Percoll density gradient centrifugation, activity was also increased among cells sedimenting at the very bottom of the gradient. This was found in subpopulations of cells obtained from 16 21 gradients in tests against K562 and in subpopulations obtained from both of 2 gradients in tests against Daudi cells. These findings are consistent with the existence of at least 2 subpopulations of lymphocytes which can mediate natural cytotoxicity, and which are separable by density.

Natural cytotoxic cells against solid tumors in mice. IV. Natural cytotoxic (NC) cells are not activated natural killer (NK) cells.

Natural cell-mediated cytotoxicity (NCMC), measured against a variety of tumors, is mediated by at least two sub-populations of effector cells: natural cytotoxic (NC) and natural killer (NK). The studies described in this report show that target lysis by NC cells requires a prolonged (18- to 24 h) assay period, whereas NC-susceptible targets can be lysed in short-term (4h) [3H]-proline assays using allo-sensitized CTL or mitogen-activated cytotoxic populations. NC cells are not "activated" during the long-term assay as indicated by: (1) demonstrating that lysis of NC-susceptible targets in long-term (20 h) 51Cr assays is still the function of a Qa-5- effector cell (NC) and (2) the fact that preincubation of the NC effector cell with susceptible targets for 18 h did not result in the activation of an NK-like population (kinetics of target lysis were comparable to those noted with fresh NC cell preparations). We show that NC cell activity is preserved in both the beige mutant and the PL/J mouse strains, both of which exhibit low NK cell activity, even in long-term assays. These combined studies support the view that NC and NK cell activities are the function of distinct cell types and not the property of a single cell class under different states of activation.

In vitro effect of cimetidine on human cell-mediated cytotoxicity : I. Inhibition of natural killer cell activity

The effect of cimetidine on human natural killer cell activity against K562 myeloid cells was studied. A bimodal inhibitory dose-related effect was found, as the result of the interaction of the drug with both target and effector cells. Cimetidine inhibits natural killer effector cells at very low molar concentrations and both effector and glass-adherent cells exerting suppressor function of natural killer cell activity at intermediate concentrations. High doses of the drug, besides affecting the effector cell subpopulations, seem to trigger an active protective mechanism in the target cells, thus resulting in the total abrogation of natural killer cell activity.

Detection of antibody-dependent cell-mediated cytotoxicity by automated flow cytometry. Comparison to a chromium release assay and characterization of the effector cell subpopulation

Antibody-dependent cell-mediated cytotoxicity (ADCC) against chick red blood cells (CRBC) can be detected by flow cytometric (FCM) analysis of cellular DNA content. When compared to a standard chromium release assay FCM analysis shows several advantages: (1) equivalent cytotoxicity can be detected after 1 h compared to 4 h for 51Cr; (2) equivalent cytotoxicity can be seen at a 5-fold lower effector-to-target ratio; and (3) no radiolabeling is needed. When mouse spleen cells were fractionated based on adherence to the plastic, adherent cells showed the highest ADCC by both FCM and 51Cr release.

Differential effects of ouabain on human cell-mediated cytotoxicity: I. Inhibition of mitogen-induced cellular cytotoxicity and antibody-dependent cellular cytotoxicity against chicken red cell targets

The effects of ouabain, a known inhibitor of lymphoproliferation, were studied in relation to the cytotoxic effector function of human peripheral blood mononuclear leukocytes (MNL) against chicken red blood cell (CRC) targets. MNL effectors lysed 51Cr-labeled CRC targets in the presence of PHA (mitogen-induced cellular cytotoxicity—MICC) or rabbit anti-CRC antibody (antibody-dependent cellular cytotoxicity—ADCC) in the absence of ouabain. The addition of ouabain to the cytotoxic reaction caused profound diminution of MICC with greater than 90% suppression of killing at ouabain concentrations of 5 × 10 −4 M; ADCC was much more resistant to the effects of ouabain with only 60 to 70% inhibition of killing at similar ouabain concentrations ( P < 0.01). Similar ouabain inhibition of MICC occurred whether the effector cell populations were unseparated MNL, depleted of monocytes, enriched for T cells, or depleted of T cells, suggesting a generalized activity by ouabain against all effector cells active in MICC. Ouabain inhibition of MICC could be overcome by increasing PHA concentrations, indicating that ouabain inhibition was not due to irreversible toxic effects on effector cells. Increasing the concentration of anti-CRC antibody resulted in increased killing in this ADCC system and, paradoxically, ADCC cultures with the highest antibody concentrations were more completely inhibited by ouabain. This enhanced inhibitory effect of ouabain on ADCC cultures with the highest antibody concentrations was not observed when the effector cell population was first depleted of phagocytic cells, suggesting a preferential inhibitory action by ouabain against monocyte effectors in ADCC. Thus, the differential inhibitory effects of ouabain on MICC and ADCC against CRC targets may be in part explained by the differing ouabain sensitivities of the various effector cell subpopulations involved in these cell-mediated cytotoxic events.

IgM Antibody-Dependent Cell-Mediated Cytotoxicity in the Moloney Sarcoma Virus System: the Involvement of T and B Lymphocytes as Effector Cells

Antibody-dependent cell-mediated cytotoxicity in the Moloney sarcoma virus (MSV) system was analyzed with respect to the subpopulations of effector cells involved in tumor target cell destruction when IgM was used as the sensitizing antibody. With unfractionated sera from animals that had undergone regression primary MSV tumors it was found that macrophages did not contribute to the cytotoxicity induced by normal spleen cells that were syngeneic to the target cells. The IgM fraction of MSV regressor sera was found to induce cytotoxicity against the target cells by immunoadsorbent column-fractionated normal spleen cells, which were either depleted of T cells or B cells, according to the specificity of the columns. Immune IgM was also found to potentiate the activity of MSV regressor spleen cells that had been similarly fractionated. Furthermore, IgM antibody was found to induced cytotoxicity by normal spleen cells which had been depleted of either T or B cells by the appropriate antiserum (anti-T or anti-Ig) in the presence of complement and subsequent recovery of the viable cells by trysinization, filtration, and washing. However, spleen cells treated with both anti-T and anti-Ig sera simultaneously in the presence of complement and subsequet recovery of viable cells, were not induced to be cytotoxic against the IgM-coated tumor target cells. Further support oy T cells was provided by an experiment showing the induction with IgM of cytotoxicity against the target cells by normal thymocytes.