Production of lymphotoxin by human lymphocytes. II. Stimulation by purified tuberculin]

A total of 43 lymphocyte culture (LC) from 12 adults (aged 32--48 years) and 21 children 6--7 years of age were studied. In PPD-stimulated adults LC stimulation ratio ranged from 8 to 39%; their cytotoxic activity, i.e. lymphotoxin (LT) production, was determined by staining target L cells with crystal violet and measuring protein synthesis in surviving target cells by radioactive amino acid incorporation. In children blastogenic response occurred in 11 out of 21 PPD-stimulated LC studied, but only 8 supernatant culture fluids were toxic to L cells and 3 were not. These findings were confirmed by repeated tests 3 weeks later. Probable correlation of BTL and LT production with the functional state of specific immunity to tuberculosis is discussed.     

Elaboration of lymphotoxin by freshly isolated human T lymphocytes and continuous lymphoid-cell lines.

Freshly isolated human T lymphocytes were demonstrated to produce lymphotoxin (LT) after mitogenic stimulation with phytohemagglutinin (PHA). In contrast, freshly isolated B lymphocytes, stimulated with two B-cell mitogens [pokeweed mitogen (PWM) and Staphylococcus protein A (Staph A)] did not produce the lymphokine, although thymidine incorporation was increased in these cells. We also examined a series of nine continuous human lymphoid-cell lines with B-cell markers and observed the spontaneous release of either large or small amounts of cytotoxin, or none at all. Cytotoxin from one of the productive cell-lines (H4218) was compared in detail with that obtained from PHA-stimulated, freshly isolated human lymphocytes. The behavior of the two cytotoxins was found to be identical in respect to migration on polyacrylamide gel, neutralization with rabbit anti-human α-LT serum, ultracentrifugation on 5–30% sucrose gradients, and stability for 15 min at 75 °C. Observation of these identical parameters strongly suggests that the α-LT elaborated by PHA-stimulated, freshly isolated human lymphoid cells is the same as the cytotoxin obtained from the continuous human lymphoid-cell line H4218. Thus α-LT may also be produced in quantity from continuous lymphoid-cell lines by mass tissue-culture techniques, which are more readily applicable to large-scale production than is purification from freshly cultured human lymphoid tissues. Notably, in cultures of freshly isolated human lymphoid cells, only T cells, and not B cells, generated lymphotoxin. However, continuous human lymphoid-cell lines with B-cell markers can also secrete this lymphokine.     

IgA and IgA diphtheria antitoxin responses from human tonsil lymphocytes.

Human tonsil lymphocytes were stimulated with diphtheria toxoid and then cultured in a Marbrook culture system so that antibodies could be measured in the culture supernatant. Specific antibodies were measured with excess radiolabeled antigen and antisera specific for each immunoglobulin class. Good IgG and IgA diphtheria antitoxin responses have been obtained and responding culture supernatants were shown to neutralize toxin. The relationship between antitoxin response in vitro and immunization of donors with toxoid was investigated. It was found that at least two immunizations after the age of 6 months were necessary to prime the tonsils for an in vitro antibody response. The IgG and IgA in culture supernatants were demonstrated by immunodiffusion and were measured by radioimmunoassay. By sucrose density gradient ultracentrifugation, it was shown that 40% of the IgA produced in the cultures was greater than 7S. Evidence was obtained that neither the IgA nor the specific IgA antitoxin bears secretory piece. It appears that human lymphocytes from tonsils produce polymer IgA in vitro without secretory piece.     

Excretion of newly synthesized DNA by tonsil lymphocytes.

The main part of newly synthesized DNA is preferentially released in vitro both by non-stimulated or phytohemagglutinin-stimulated tonsil lymphocytes. Freshly isolated cells excrete DNA faster than phytohemagglutinin-stimulated ones. The acid-precipitable 3H-thymidine lost can be accounted for in the culture medium as labeled, double stranded DNA isolated on hydroxyapatite column.     

Lymphotoxin and interferon production by rossette-separated human peripheral blood leukocytes.

When normal (CBA × DBA)F₁ spleen cells are cultured for 4 days in polyacrylamide vessels, clones of cytotoxic lymphocytes (CLs) are generated. The specificity of these apparently spontaneous CL clones has been investigated by assaying cells from individual clones against pairs of different target cells. CL clones were found to discriminate between the two parental strain splenic blasts, between splenic blasts and syngeneic tumour cells, and between two F₁ splenic blasts induced with different mitogens (LPS and PHA). The CL clones generated spontaneously in culture also discriminate between semisyngeneic targets [DBA blasts and (CBA × DBA)F₁ blasts]. Significant cross-reactivity however, was detected when CL clones were assayed against normal P815 targets and TNP-modified P815 targets.     

Differential fluorochromasia of human lymphocytes as measured by flow cytometry.

Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.     

Enhancement of cytotoxicity of human peripheral blood lymphocytes by interferon

Human lung carcinoma cells persistently infected with mumps virus (Pc-10/MpV) were lysed with human peripheral blood mononuclear leukocytes (PBML) obtained from seropositive donors who had anti-mumps virus-neutralizing antibody in their sera. This cellular cytotoxicity was due not to the cytotoxic T lymphocytes but mainly to the non-T, non-B cells, possibly related to natural killer (NK) cells. Moreover, it was concerned not with antibody against mumps virus antigens but with alpha-interferon (IFN-alpha) produced in the mixture of human PBML and Pc-10/MpV cells, since this cellular cytotoxicity was suppressed by anti-human IFN-alpha rabbit serum. Exogeneous IFN-alpha augmented the cytotoxicity of non-T, non-B cells, not T cells, for the uninfected Pc-10 cells. IFN-gamma that had been induced by heat-killed Listeria monocytogenes in PBML had the same capacity to augment NK activity did IFN-alpha.     

Cytotoxic activity and interferon production by lymphocytes from patients with multiple sclerosis

Peripheral blood lymphocytes from MS patients and from healthy control donors were compared for their ability to mediate spontaneous and antibody-dependent cell-mediated cytotoxicity. They were also compared for their ability to respond to infection with various strains of measles and sSPE viruses with interferon production and enhanced NK activity. Neither SLMC nor ADCC against several different target cells was found to be impaired in the MS population. Furthermore, no defect was detected in the response of patients' lymphocytes to virus challenge in vitro in terms of both activation of NK cells and interferon production. Enhanced NK activity was also induced by an exogenous interferon preparation and by Poly I:C to the same extent in patients and controls.     

Relationship of lymphotoxin secretion and DNA synthesis in the human mixed lymphocytes reaction in vitro.

Influenza virus particles, inactivated with formalin, have been covalently bound to cyanogen bromide-activated Sepharose beads (Se-vi beads). Preservation of the hemagglutination properties of the viral particles enabled a strong binding of pigeon or human group O erythrocytes (PRBC or HoRBC) to these Se-vi beads. The conditions for preparation of PRBC- or HoRBC-Se-vi columns are described.Spleen cell suspensions from mice immunized with the above erythrocytes were considerably depleted of cells forming hemolytic plaques (PFC) against the corresponding erythrocytes after passage through these columns. In the case of cells from nonimmunized mice, the depletion is still greater and reaches up to 95–100%. However, the number of PFC reactive to unrelated erythrocytes is not affected in the filtered population. Specifically attached cells recovered from the Se-vi-RBC columns passed with normal spleen cells are considerably enriched in the number of PFC against homologous erythrocytes. Syngeneic irradiated hosts transferred with filtered cells are able to give a normal primary PFC response against heterologous, but not against homologous RBC up to the 12th day after immunization. These results are discussed in relation to the problem of precommitment of specific PFC precursor cells.     

Thymic hormonal activity on human peripheral blood lymphocytes in vitro. II. Enhancement of the production of immune interferon by activated cells

The thymic extract TP-1 significantly enhanced the production of immune interferon (IF) by human peripheral blood mononuclear cells in Con A- or Raji- but not PHA-induced cultures. TP-1 effect was more pronounced under conditions of low IF production. The effect was evident before cell proliferation took place and without necessary concurrent effect of TP-1 on the blastogenic response. TP-1 had no detectable effect on nonactivated cultures. The enhancement of IF production by TP-1 to the degree demonstrated here necessitates consideration of its possible effects in every assay of thymic hormones that involves lectin or allogeneic stimulation of lymphocytes.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.     

Mechanism of phorbol myristate acetate-induced lymphotoxin production by a human T cell hybridoma

Various hydroxyl radical scavengers markedly inhibited phorbol myristate acetate (PMA)-induced lymphotoxin (LT) production by a human T cell hybridoma, AC5-8. Among those we tested, tetramethylurea (TMU) was the most potent scavenger, and it was revealed that TMU must be added before 2 h have elapsed after PMA addition in order for LT production to be inhibited. In concordance with this fact, soluble NADPH dependent O2- forming enzyme(s) were activated several fold by PMA. PMA also induced DNA strand breaks, a process markedly inhibited by TMU. As expected, ADP-ribosyl transferase (ADPRT), which is well known to require DNA strand breaks for its enzymatic activity, was activated by PMA treatment. In addition, specific inhibitors for ADPRT, namely 3-amino-benzamide and nicotinamide, inhibited PMA-induced LT production. Taken together, these three successive events, activation of soluble NADPH dependent O2- forming enzyme(s), DNA strand breaks and activation of ADPRT, may be required for PMA-induced LT production by AC5-8.     

Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute