Differential production of interferon and lymphotoxin by human tonsil lymphocytes.

Lymphotoxin (LT) production, interferon (IF) production, and DNA synthesis were investigated after mitogen stimulation and in the mixed lymphocyte culture (MLC) reaction using human tonsil lymphocytes. Both LT and IF were assayed in parallel and from the same lymphocyte supernatants. An analysis of the PHA, PWM, one-way and two-way MLC reactions showed that the amounts of LT and IF produced could not be correlated. Polyriboinosinic: polyribocytidylic acid (poly(I: C)) failed to induce either LT production or [³H]TdR incorporation but did induce IF production. Removal of glass-adherent cells (GAC) had no effect on mitogen induced LT production but their removal reduced LT production in MLC reactions. GAC were necessary for IF production and optimal [³H]TdR incorporation in both mitogen stimulated cultures and in MLC reactions. IF and LT activities were shown to be the result of different molecules by using a Sephadex G-75 column. These results indicate that mitogen stimulation differs from MLC reactions in the cell type or control mechanisms involved for LT production, and that in mitogen stimulated cultures all three of these in vitro phenomena are probably the results of either different cell types or of different cell to cell interactions.     

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.     

Undermethylation of interferon-gamma gene in human T cell lines and normal T lymphocytes.

The relative levels of DNA methylation at CCGG sequences within and around the interferon-gamma (IFN-gamma) gene in normal human tissues and cell lines were examined by Southern blot analysis using isoschizomeric restriction enzymes, HpaII and MspI. On the test of normal tissues, the IFN-gamma gene was undermethylated only in a small population of T lymphocyte, whereas the gene was fully methylated in T cell-depleted lymphocytes and uterus cells. In TCL-Fuj cell line which is a T cell line producing a high level of IFN-gamma spontaneously, the IFN-gamma gene was undermethylated. Moreover, the extent of DNA methylation was inversely correlated to the level of expression of the IFN-gamma gene in several T cell lines including sublines derived from TCL-Fuj cells. However, partial or complete unmethylation at the CCGG sites of IFN-gamma gene was observed in a promyelocytic leukemia cell line and two epithelial cell lines that fail to produce IFN-gamma irrespective of induction. These results suggest that undermethylation of IFN-gamma gene is necessary but not sufficient for its efficient expression.     

Generation and characterization of continuous lines of CD8+ suppressor T lymphocytes

The ability to grow normal T lymphocytes in long term culture has advanced our understanding of T cell biology. The growth of CD4+ cell lines allowed a further evaluation and appreciation of functional subtypes within this group. Cytotoxic CD8+ T cells have been characterized as well. The routine and continuous culture of Ag-nonspecific CD8+ Ts cells has been difficult to achieve. We have found that CD8+ T cells that suppress T cell proliferation and lack cytotoxic activity against T cells can be routinely obtained from PWM or PHA-stimulated PBMC. Continuous culture of T cell blasts from PWM or PHA-stimulated PBMC resulted in the growth of CD4+ and CD8+ T cells. These lines developed suppressor cell activity within 7 days after stimulation with PWM and 3 to 4 wk after stimulation with PHA. Concomitant with the development of suppressor activity was the loss of CD4+ T cells resulting in homogeneous lines of CD8+ suppressor cells. These cell lines have been maintained in continuous culture for greater than 6 mo by addition of rIL-2 twice weekly and restimulation with feeder cells and PHA every 2 wk. Activity of these cell lines was relatively resistant to irradiation or treatment with mitomycin C. Both cell lines suppressed proliferation of autologous or heterologous CD4+ T cells stimulated with PWM, OKT3, or tetanus toxoid but failed to suppress proliferation of CD4+ T cells in a mixed lymphocyte reaction. CD4+ T cells stimulated with PWM produced equivalent amounts of IL-2 in the presence or absence of Ts cells but failed to express the IL-2R (TAC) on their surface in the presence of Ts cells. By contrast, CD4+ T cell lines or cytotoxic CD8+ T cell lines failed to suppress proliferation of CD4+ T cells. With these results we describe methods for the generation and continuous culture of Ag-nonspecific CD8+ Ts cells and define some of their properties. These cells lines should be helpful in further elucidating the functional and phenotypic repertoire of CD8+ Ts cells.     

Arachidonic acid and glucose uptake by freshly isolated human adipocytes

Fatty acid (FA) and glucose transport into insulin-dependent cells are impaired in insulin resistance (IR; type 2 diabetes mellitus). Studies done on the effects of FAs on glucose uptake, and the influence of insulin on FA uptake by adipocytes, have yielded contradictory results. In this study, isolated human adipocytes were exposed to arachidonic acid (AA) and to insulin, and FA uptake as well as glucose uptake was measured. AA uptake into adipocyte membranes and nuclei was also investigated. Glucose uptake was inhibited by 57 +/- 8% after 30 min of exposure to arachidonate. AA was significantly taken up into adipocyte membranes (49.6 +/- 29% and 123 +/- 74%) at 20 and 30 min of exposure, respectively, and into nuclei (147.6 +/- 19.2%) after 30 min. Insulin stimulated AA uptake (24.1 +/- 14.1%) at 30 min by adipocytes from a non-obese subject, while inhibiting it (16.6 +/- 12%) in adipocytes from an obese subject. These results suggest that: (1) AA inhibits glucose uptake by adipocytes exposed over a short period, probably by a membrane-associated mechanism, (2) insulin-dependent AA uptake is dependent on the body mass index (BMI) of the donor and the insulin sensitivity of their adipocytes.     

Radiation-induced DNA single-strand breaks in freshly isolated human leukocytes.

Single-strand breaks are a major form of DNA damage caused by ionizing radiation, and measurement of strand breaks has long been used as an index of overall cellular DNA damage. Most assays for DNA single-strand breaks in cells rely on measuring fractionated DNA samples following alkali denaturation. Quantification is usually achieved by prelabeling cells with radioactive DNA precursors; however, this is not possible in the situation of nondividing cells or freshly isolated tissue. It has previously been demonstrated that the alkali unwinding assay of DNA strand breaks can be quantified by blotting the recovered DNA on nylon membranes and hybridizing with radiolabeled sequence-specific probes. We report here improvements to the technique, which include hot alkali denaturation of DNA samples prior to blotting and the use of carrier DNA that is non-complementary to the radiolabeled probe. Our method allows both single- and double-stranded DNA to be quantified with the same efficiency, thereby improving the sensitivity and reproducibility of the assay, and allows calibration for determination of absolute levels of DNA strand breaks in cells. We also used this method to assay radiation-induced DNA strand breaks in freshly isolated human leukocytes and found them to have a strand break induction rate of 1815 strand breaks/cell/Gy.     

Production of lymphotoxin, IFN-gamma and IFN-alpha, beta by murine T cell lines and clones

The PCl-6 T cell line, derived from mice sensitized by skin painting with picryl chloride (PCl), shows antigen dependence for DNA synthesis and for lymphotoxin (LT) production. These cells produce LT, but not interferon (IFN), when exposed to 2,4,6-trinitrobenzene sulfonic acid- (TNBS) coupled syngeneic spleen cells. Concanavalin A (Con A) induces IFN production by PCl-6 cells, and IFN levels, but not LT titers, are increased by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). These results support the noncoordinate regulation of these two lymphokines. Line 32, a T cell growth factor- (TCGF) dependent T cell line and its Ly-1.2+, 2.2- derivative clone, 32H1, produce both antiviral and antiproliferative activity after exposure to several different mitogens. Tests for acid lability, sensitivity to anti-mouse IFN-alpha, beta antisera, and antiproliferative activity on non-mouse target cells indicates that an Ly-1+ clone, in the absence of both TCGF and accessory cells, can produce at least three separate lymphokine activities after Con A exposure: IFN-gamma (Type II), IFN-alpha, beta (Type I), and LT.     

Mechanisms of lymphocyte-mediated cytotoxicity. I. The effects of anti-human lymphotoxin antisera on the cytolysis of allogeneic B cell lines by MLC-sensitized human lymphocytes in vitro

Goat and rabbit anti-human lymphotoxin sera, IgG and F(ab')2 reagents were investigated for their capacity to effect a specific alloimmune lymphocyte-mediated cytotoxic reaction. The cytotoxic reaction employed human peripheral blood or adenoid lymphocytes sensitized in MLC to allogeneic B lymphocyte cell lines and lysis was measured in a short-term 51Cr-release assay. A polyspecific anti-LT sera (anti-WS), made against unfractionated whole supernatants from lectin-activated lymphocytes and its IgG and F(ab')2 fragments, was found to be a potent inhibitor of this reaction when the anti-WS reagent was present throughout the assay period. Absorption studies indicated the anti-WS was inhibiting cytolysis at the level of effector cell or its products. Two broadly defined antibody specificities were involved in the cytolytic-inhibitory activity of the polyspecific anti-LT; i) antigens present on the normal lymphocyte cell surface; and ii) lymphocyte surface antigens associated with activated cells. These results correlate with the previously defined antigenic structure of the LT Cx and alpha H classes. Anti-LT sera reactive with the smaller m.w. alpha and beta classes and subclasses were not inhibitory, although the anti-beta sera showed a moderate enhancing activity. The results indicated that several anti-LT antibody specificities may be required to inhibit alloimmune cytolysis. The results suggest LT molecules may mediate lymphocyte-induced alloimmune cytolysis as a multi-component toxin system, rather than as an individual toxin.     

Stimulation of human T lymphocytes by Leishmania lipophosphoglycan-associated proteins.

Lipophosphoglycan (LPG) is a glycoconjugate present on the surface of Leishmania promastigotes that has been reported to promote intracellular survival of these parasites, to protect mice against leishmaniasis, and to elicit T cell responses in infected mice and humans. We investigated whether LPG and its components could elicit proliferative responses and cytokine secretion from leishmaniasis patient PBMC. LPG prepared by standard methods (LPG-1) stimulated patients T cells to proliferate and secrete IFN-gamma. LPG was fractionated into several components. An LPG-1-specific T cell line was shown to respond to the core region but not to the repeating saccharide units. LPG-1 was fractionated to yield an LPG-free- associated protein complex and an LPG-2 fraction that was more than 95% depleted of associated protein. The ability of LPG-2 to stimulate T cells was significantly decreased over that of LPG-1. In contrast, LPG-AP stimulated T cell proliferation and IFN-gamma production. Therefore, proteins associated with LPG were effective in eliciting patient T cell responses, whereas the glycolipid enriched moiety was weakly effective or ineffective at stimulating these responses.     

Alpha-fetoprotein-mediated uptake of fatty acids by human T lymphocytes.

The binding to resting and activated T lymphocytes of two radiolabelled fatty acids (oleic and arachidonic) was studied in the presence or in the absence of alpha-fetoprotein (AFP) as carrier protein. Fatty acid binding by resting and activated T lymphocytes was determined at 4 degrees C as a function of the concentration of fatty acid and AFP. Under the conditions employed, the following observations were made: (1) in the presence of AFP, fatty acids (oleic and arachidonic acid) are bound to cells by a two-component pathway; one is a saturable process, evidenced when the fatty acid to AFP (FA/AFP) molar ratio was fixed at 1 and the concentration of the fatty acid and the protein varied from 0.1 to 3.2 microM, and the second is a nonsaturable function of FA/AFP molar ratio and was linearly related to the unbound fatty acid concentration in the medium over the entire range studied; (2) in the absence of AFP, the nonsaturable process appears to be the only component of fatty acid binding; 3) at all tested concentrations of free (unbound) fatty acid in the medium, net fatty acid binding by either resting or activated T cells was considerably greater in the presence than in the absence of AFP; (4) in the presence of AFP, fatty acid binding was much higher in activated T cells than in resting T cells, whereas in the absence of AFP, nonsignificant differences were observed between activated and resting T cells; and (5) the time course of fatty acid and AFP binding at 4 degrees C revealed that, at equilibrium, the number of fatty acid molecules bound to the cell was much greater than that of AFP suggesting an accelerated dissociation of the fatty acid upon interaction of the AFP-fatty acid complex with putative cell receptors. It is concluded to the existence of an AFP/AFP-receptor pathway that facilitates the binding of fatty acids to T lymphocytes, particularly upon their blast transformation. This pathway may fulfill the increased requirement for fatty acids characteristic of proliferating cells and may serve to regulate the endocytosis of fatty acids with modulatory effects on lymphocyte function and to protect cells from their cytotoxic potential when internalized in excess.     

Immortalization of human T lymphocytes by oncogenes

Immortalized human T cell lines were established by cotransfecting c-Ha-ras and c-myc oncogenes to lymph node lymphocytes. The cell lines kept growing for 3 months after establishment without a decrease in growth rate. The cells did not require interleukin-2(IL-2) for their growth, but addition of IL-2 stimulated the growth of these cells. Flow cytometric analysis revealed that these cells were T cells expressing CD4 or CD8 antigens. A CD4 positive (CD4⁺) cell line produced IL-6, indicating that the cell line belongs to helper T cells. The CD8 positive (CD8⁺) cell line possessed cytotoxicity to tumor cells, indicating that the cell line were killer T cells. Both cell lines were able to proliferate in serum-free medium indefinitely.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.     

Alloreactive cloned T cell lines. VI. Multiple lymphokine activities secreted by helper and cytolytic cloned T lymphocytes

Culture supernatants generated by alloantigenic or lectin stimulation of a cloned helper T lymphocyte, designated L2, contain interleukin 2 (IL 2), granulocyte/macrophage colony-stimulating factor (CSF), B cell stimulating factor (BCSF), macrophage (Ia+)-recruiting factor (MIRF), (Ia+)-inducing activity, gamma-interferon, Fc receptor-enhancing activity, macrophage migration inhibitory factor (MIF), macrophage activation factor (MAF), interleukin 3 (IL 3), and a factor responsible for prolonging the synthesis and secretion of the fourth and second components of complement by guinea pig peritoneal macrophages. Erythropoietin was not detected. A spontaneously arising variant of L2, designated L2V, produces much lower quantities of macrophage-stimulating activities, IL 2, and interferon. However, when compared to L2, L2V produces much higher levels of BCSF, equivalent amounts of IL 3, and slightly smaller amounts of CSF. Unlike L2V, a cytolytic clone, designated L3, secretes lymphokines that primarily affect macrophage function. The time course of lymphokine production by L2 cells indicates that for the six lymphokine activities studied there are three different times at which maximal or near maximal levels are reached, as follows: 1) IL 2, 12 to 24 hr; 2) IL 3 and CSF, 24 to 48 hr; and 3) (Ia+)-inducing activity, MAF, and interferon, 48 hr or later. Only IL 2 activity disappears during the 8-day culture cycle. The time course data and the differential production of activities by the three types of lymphocyte clones suggest that at least four terminal effector lymphokine molecules account for the ten biologic activities tested.     

Cloned lines of human cytotoxic T lymphocytes with specificity for influenza virus

The generation of human cytotoxic T cell clones with specificity for influenza virus and some of their characteristics are described. The clones were generated by limiting dilution of peripheral blood lymphocytes after two in vitro stimulations with autologous influenza A/USSR virus-infected cells and were grown in T cell growth factor. The majority of the virus-specific clones showed cross-reactivity for different influenza A virus subtypes but did not recognize influenza B virus-infected cells. The HLA specificity of two clones was further analyzed. One clone, LL33, was specific for HLA-Bw60, the other, clone WH5, for HLA-A1. Clone WH5 also seemed to recognize the serologically related HLA-A26 as restriction element for the recognition of the viral antigen. Whereas the virus-specific CTL clones had the OKT3+,4-,8+ phenotype, another clone, WH 49, exhibiting natural killer-like activity, was found to have the OKT3+,4+,8- phenotype.     

Activation of human T lymphocytes by bryostatin

The immunologic effects of bryostatin (Bryo), a PKC activator with antineoplastic activity, were assessed and compared to PMA. Bryo induced IL-2R expression on CD4+ and CD8+ human T lymphocytes with a dose response comparable to PMA. However, Bryo induced only a marginal proliferative response as compared with the vigorous response induced by PMA. Bryo mediated functional receptor expression because the proliferative response was enhanced by addition of rIL-2. Furthermore, the proliferative response was inhibited by the relatively specific Ca+, phospholipid-dependent protein kinase (PKC) inhibitor, H-7, indicating a role of PKC in Bryo-induced activation. Addition of the calcium ionophore, ionomycin, to Bryo-stimulated lymphocytes resulted in the production and secretion of IL-2 with a concomitant proliferative response. This effect of the calcium ionophore could be inhibited by cyclosporine with identical results obtained in PMA-stimulated cultures. A most intriguing finding was that Bryo could effectively antagonize PMA-induced T cell proliferation. Although this mechanism of inhibition is unclear, a discussion with respect to differential effects on potential intracellular PKC isoforms is provided. These studies indicated that Bryo has potent immunopotentiating properties that share some similar effects of the phorbol ester, PMA, but offers the additional property of modulating other phorbol ester effects on proliferation.