Studies on cytotoxicity generated in human mixed lymphocyte cultures. III. Natural killer-like cytotoxicity mediated by human lymphocytes with receptors for IgM

Stimulation of human peripheral blood lymphocytes with allogeneic cells in mixed lymphocyte culture (MLC) results in increased NK-like cytotoxicity against K562 targets. The effector cells of this cytotoxicity were shown to include both Fcμ+ and Fcμ? cells, as shown by EAμ rosette separation and by combined rosette formation and single-cell analysis. Peak cytotoxic activity of Fcμ+ cells was found after 3 days of MLC stimulation. The Cytotoxicity against KS62 targets mediated by Fcμ+ cells could not be inhibited at all with alloantigen-bearing cells and could only be partially inhibited with another NK-sensitive target (MOLT- 4). This cytotoxicity could be generated from either Fcγ+ or FCγ? cells. These results indicate considerable heterogeneity of NK-like effectors and their precursors.     

In vitro generation of human activated lymphocyte killer cells. II. N-acetyl-D-galactosamine inhibits a distinct subpopulation of human activated lymphocyte killer cells generated in mixed lymphocyte culture

A range of monosaccharides was tested for its ability to inhibit the generation of cytotoxic cells during mixed lymphocyte culture. The most discriminatory effect was produced by N-acetyl-D-galactosamine (NADG). The presence of this sugar at the initiation of the coculture significantly inhibited in a dose-dependent manner the induction of a subset of nonspecific activated lymphocyte (ALK) cells preferentially able to lyse the K562 target cell (natural killer, NK-like cells) but had no effect on the generation of either specific cytotoxic T lymphocytes or another separate subset of ALK cells mediating lysis of an NK-insensitive melanoma cell line. The addition of conditioned medium containing interleukin 2 and interferon (IFN) at the start of culture reversed the inhibitory effect of the sugar. Under conditions of limiting dilution, the frequency of NK-like precursors ranged from 1/50 to 1/1200 with different mononuclear cells (MNC) and in all cases the presence of NADG from Day 0 of culture selectively decreased the frequency of these precursors. At the concentrations used NADG had no effect on NK-like cell cytolysis once generated. The addition of recombinant gamma-IFN did not abrogate the inhibitory effect of NADG and in MLC of some individuals decreased the frequencies of ALK cell precursors. These data provide further evidence for the heterogeneity of ALK cells and indicate that what is usually referred to as NK-like cell activity in in vitro culture is mediated by a subpopulation of MNC which are activated and induced to differentiate along a pathway independent of that of other ALK subsets.     

Functional characteristic of TTK-lymphocytes in vitro, cytotoxicity and mixed lymphocyte culture (MLC)

A simple method of cryopreservation (TTK) of lymphocytes is presented. The functional properties of TTK-lymphocytes are examined by the lymphocyte toxicity micro test and in the mixed lymphocyte culture (MLC). 51Cr-release technique is performed as a measure for reversible and irreversible cell damages in the course of the Cryopreservation process.     

Cytotoxicity of human peripheral lymphocytes in cell-mediated lympholysis; antibody-dependent cell-mediated lympholysis and natural cytotoxicity assays after mixed lymphocyte culture.

Lymphocytes that have been purified by Ficoll-Hypaque centrifugation lose antibody-dependent and natural cytotoxic activities upon culture in tissue culture medium supplemented with human plasma. However, stimulation of peripheral lymphocytes in the mixed leukocyte culture (MLC) appears to enhance killer (K) and natural killer (NK) activities in addition to generating cytotoxic T ymphocytes. Enhancement of NK and antibody dependent activities appears to correlate with cell division as measured by 3H-thymidine uptake. However, elimination of dividing cells in the MLC by addition of 5-bromodeoxyuridine has no effect on NK and K cells activities. Since this treatment abolishes cell-mediated lympholysis mediated by cytotoxic T lymphocytes, it is a useful probe for determining the relative activities of NK, K, and cytotoxic T lymphocyte effector cells after lymphocyte stimulation.     

Activation of suppressor T cells in human autologous mixed lymphocyte culture.

Co-culture of autologous T and mitomycin-C treated B cells results in increased DNA synthesis in the responding T cells. T cells thus activated in AMLC exerted suppressive effects on both the proliferative and cytotoxic responses of fresh unstimulated T cells to allogeneic cells in MLC. The suppressor cells generated are sensitive to treatment with mitomycin-C. AMLC-activated cells treated with mitomycin-C failed to suppress both cytotoxicity and proliferation in fresh primary MLC. It appears that the AMLC reaction reflects an immunologic homeostatic mechanism. Since this reaction is defective in patients with CLL and SLE and the homologous mouse syngeneic MLC is defective in NZB mice, the failure of this T-B interaction may be related to the pathogenesis of certain lymphoproliferative and autoimmune disorders.     

Zinc inhibits the mixed lymphocyte culture

The mixed lymphocyte culture (MLC) is an established clinical method for bone marrow transplantation, as it serves as an in vitro model for allogenic reaction and transplantation. We previously showed that cytokine release into the supernatant is a more specific and sensitive parameter for cross-reactivity in the MLC than the common measurement of cell proliferation. Therefore we tried to find an inhibitor of the MLC in vitro with the least side effects in vivo, measuring interferon (IFN)-γ as one of the most important cytokines in posttransplant medicine. Earlier studies showed that zinc is an important trace element for immune function with both stimulatory and inhibitory effects on immune cells. We found that slightly elevated zinc concentrations (three to four times the physiological level), which do not decrease T-cell proliferation in vitro nor produce immunosuppressive effects in vivo, suppress alloreactivity in the mixed lymphocyte culture. In this report we analyzed the mechanism whereby zinc influences the MLC to possibly find a nontoxic way of immunosuppression.     

Inhibition of proliferation without affecting the generation of cytotoxicity in the human mixed lymphocyte reaction

Phosphoramide mustard (PM) is considered to be the major tumoricidal metabolite of cyclophosphamide in vivo. The effects of this metabolite in vitro on several immune functions of human lymphocytes have been investigated. Very low concentrations (10(-7) to 10(-9) M) of PM added to lymphocyte cultures inhibited proliferation of the lymphocytes in response to mitogens and alloantigens. At these concentrations, inhibition of proliferation appeared to be due to a direct action of PM on the proliferative cells. Thus, concanavalin A-stimulated lymphocytes still acquired IL-2 receptors (Tac antigen) normally in the presence of PM (10(-6) to 10(-9) M). Only exceedingly high concentrations of PM (10(-5) M or greater) prevented the acquisition of Tac antigen. Similarly, the inhibition of proliferation was probably not related to endogenous IL-2 levels: addition of exogenous IL-2 to PM-containing cultures did not result in any restoration of proliferation. Further evidence that PM directly affected proliferative cells was that low concentrations of PM inhibited the proliferation of T cells continuously growing in IL-2. The exposure time to PM necessary for inhibition was essentially identical to those for lymphoproliferative responses to mitogens and alloantigens. Paradoxically, however, the generation of cytotoxic lymphocytes in mixed lymphocyte reactions (MLRs) and mixed lymphocyte tumor cell cultures (MLTCs) was very resistant to PM. In parallel MLRs and MLTCs the cytotoxic responses were resistant to approximately 1000-fold more PM than were the proliferative responses. Only at 10(-5) M PM were these inhibited. These data suggest that clonal expansion of cytotoxic lymphocytes or their precursors by proliferation is not an absolute requirement for the generation of cytolytic activity.     

Cytotoxic T cells generated in the autologous mixed lymphocyte reaction. I. Primary autologous mixed lymphocyte reaction

In the course of the culture of an autologous mixed lymphocyte reaction (AMLR), T cells proliferated in response to autologous non-T cells, and differentiated to cytotoxic T cells (AMLR killers). DNA synthesis was necessary to generate AMLR killers, as the elimination of autoreactive proliferating cells with BUdR and UV light completely abrogated AMLR killer cytolysis. Amlr killers lysed various lymphoid cell lines, including autologous B cell lines, autologous or allogeneic mitogen blasts stimulated by Con A, PHA, or pokeweed mitogen, variious nonlymphoid cell lines derived from human, mouse, or rat, and weakly normal autologous or allogeneic non-T cells. KMT-17, methylcholanthrene-induced rat fibrosarcoma, was the only resistant cell line to have been tested. AMLR killers had characteristics similar to NK cells, Major histocompatibility antigens were not the target antigens for AMLR killers. AMLR killers distinguished the blasts stimulated by alloantigens as self from the blasts stimulated by mitogens as non-self.     

Tumour-reactive lymphocytes stimulated in mixed lymphocyte and tumour culture

Summary Lymphocytes from cancer patients were stimulated in mixed culture with autologous tumour (MLTC) or pooled allogeneic lymphocytes (MLC). Both protocols induced increased uptake of ³H-thymidine at 5 days and the appearance of lymphoblasts. Blasts were isolated on discontinuous Percoll gradients and either expanded as bulk cultures or cloned directly under limiting dilution conditions in the presence of conditioned medium containing IL-2. Results with MLTC-blast-CTC have been reported elsewhere. MLC-activated cultures lysed autologous tumour but not autologous lymphoblasts. Lysis of some allogeneic tumours, lymphoblasts from members of the inducing pool, and K562 was also apparent. MLC activated cultures did not undergo restimulation in response to autologous tumour or lymphocytes but were restimulated by leukocytes from pool members.MLTC clones showed autologous tumour-specific cytotoxic activity or cross-reactive proliferative responses with tumours of the same site and histology. The majority of MLC clones cytotoxic for autologous tumour were also specific and did not lyse allogeneic tumour, K562, or lymphoblasts from the inducing pool. Two clones lysed autologous tumour and pool members. None of the clones tested proliferated in response to autologous tumour following MLC activation but some were responsive to pool members and one clone was restimulated by autologous monocytes. No association was found between clone phenotype and function. The implication of these data is that the effector cells with activity against autologous tumour induced in MLC arose largely by transstimulation of in vivo-activated tumour reactive lymphocytes by IL-2 release rather than expansion of NK-like effectors or sharing of antigenic specificities between tumour and allogeneic lymphocytes. Since MLC activation of cancer patients lymphocytes does not induce proliferative responses to autologous tumour it is unlikely to be a useful procedure in preparing cells for immunotherapy protocols. Abbreviations used in this paper: PBL, peripheral blood lymphocytes; TIL, tumour infiltrating lymphocytes; MLTC, mixed lymphocyte tumour culture; IL-2, interleukin-2; MLC, mixed lymphocyte culture; LSM, lymphocyte separation medium; BSS, balanced salt solution; HuSe, human serum; PBS, phosphate-buffered saline; CTC, cultured T cells; PHA, phytohaemagglutinin; CM, cultured medium; NK, natural killer; FcR, receptor for the Fc portion of IgG     

Ouabain inhibition of human lymphocyte cytotoxicity.

Ouabain, a specific inhibitor of the membrane Na⁺-K⁺-ATPase, is known to inhibit the proliferation of human lymphocytes in culture induced by any of a variety of agents. Here we show that the cytotoxic activity of human lymphocytes precultured with ouabain for 1 day against antibody-coated target cells (ADCC) was inhibited in a concentration-dependent manner; a 2-day exposure resulted in a drastic and irreversible loss. Ouabain acted synergistically with the respiratory chain inhibitor antimicin A to block killing, but did not act synergistically with the competitive inhibitor of glucose utilization 2-deoxyglucose. T-cell cytotoxicity (CML) was more sensitive to ouabain than was ADCC; it was inhibited when the drug was only present during the assay. We conclude that prolonged exposure to ouabain has a selective effect not only on the proliferation of immunocompetent cells, but also on the different function of cytotoxicity, whether in ADCC or in CML, and that one of the victims of the treatment may be energy production.     

Formaldehyde-induced cytotoxicity and sister-chromatid exchanges in human lymphocyte cultures

The role of the error-prone misrepair pathway in mutagenesis was examined for a series of mutagens in umuC⁺ and umuC36 strains of Escherichia coli. Mutagenesis by ENU, MNU, MNNG and EMS was independent of the umuC⁺ gene function, while mutagenesis by MMS, 4NQO, γ-rays and UV was largely umuC⁺-dependent. Residual mutagenesis following UV-treatment of a umuC^? strain showed the same mutational specificity seen in the umuC⁺ strain. In contrast, the umuC mutation altered specificity substantially in an excision-repair-defective strain that showed a UV-spectrum strikingly different from that seen in an excision-repair-proficient strain. Only one of nine trpE frameshift mutations examined was reverted by UV-light and its reversion was umuC-dependent. In comparison, the dependence of frameshift mutagenesis following ICR 191 treatment was site-specific, suggesting at least two mechanisms of frameshift mutagenesis, one dependent upon misrepair, the other not.The results, together with those of previous reports (Kato and Nakano, 1981; Shinoura et al., 1983), suggest that the umuC⁺ gene exerts it's mutator activity via misrepair of DNA lesions provoking the induction of all types of mutational events, though following UV-irradiation mainly transition events are recovered.     

Progressive changes in cytotoxic potential during mixed lymphocyte culture.

In a previous study it was shown that at least one round of DNA synthesis is required for initial expression of cytotoxic function in mouse lymphocytes responding to alloantigen in vitro. In the experiments reported here we ask whether subsequent rounds of cell division are required simply for clonal expansion of this initial level of cytotoxic function within the population, or whether the amount of cytotoxicity per cytotoxic cell is altered during subsequent rounds of cell division. The amount of cytotoxicity per unit number of cells at various stages of culture was compared with the frequency of cytotoxic cells as estimated principally by effector-target cell conjugates. Our results strongly suggest that the amount of cytotoxicity per cell (cytotoxic potential) is not a static property of cytotoxic cells, but can be modulated up or down during the course of a reaction.     

Effect of BUdR on proliferation and development of cytotoxicity in mixed leukocyte culture

The effect of the thymidine analog, 5-bromo-2′-deoxyuridine (BUdR) on the proliferation of, and development of cytotoxicity in, mouse lymphocytes in mixed leukocyte culture was measured. It was found that BUdR inhibited the development of cytotoxicity only to the extent that it inhibited mitosis. This is in contrast to the effect of BUdR in differentiating cell systems, where differentiation can be inhibited by concentrations of BUdR that do not inhibit cell proliferation. Possible implications of these results are discussed.