T-cell growth factor production by adult, but not childhood. Acute lymphoblastic leukaemic cells

The production of T-cell growth factor (TCGF) by acute lymphoblastic leukaemia (ALL) cells was determined in seven children and in three adults. A significant production of TCGF by adult, but not childhood, ALL cells was observed. The adult ALL cells were classified as "non-T-non-B" by surface marker analysis. It is suggested that TCGF production may not be confined to the cells of T-lineage.     

Regulation of T-cell functions by L-lactate

Lactate is a product of glycolytically active macrophages. After stimulation with concanavalin A accessory cell-depleted splenic T-cell populations were found to produce only minute amounts of T-cell growth factor (TCGF); but substantial amounts of TCGF were produced if the cultures were supplemented either with splenic adherent cells or with lactate but not with interleukin-1 (IL-1). IL-1 was capable, however, of supporting TCGF production by the thymoma subline EL4-6.1. TCGF production in cultures of accessory cell-depleted splenic T-cell populations was demonstrable with 10(-3) M L-lactate, and optimal responses (plateau level) were obtained with 4-6 X 10(-2) M L-lactate. Cultures of macrophages were found to accumulate up to 5 X 10(-2) M lactate. Our experiments indicate, therefore, that lactate serves as a regulatory signal by which macrophage-like accessory cells enhance helper-T-cell functions. Lactate is apparently not the only mediator of accessory cell function since plateau levels of TCGF production were markedly lower with lactate than with splenic accessory cells; but L-lactate was found also to determine the magnitude of T-cell-mediated immune responses in vivo and in cultures of unfractionated lymphocyte populations. The production of interferon in accessory cell-depleted and concanavalin A-treated T-cell cultures, however, was not significantly affected by lactate. Concanavalin A-stimulated splenic T-cell populations were found to consume glucose rapidly and to release lactate into the supernatant. This indicates that the cells contain more lactate and pyruvate than they can utilize by their respiratory metabolism. The administration of external lactate or pyruvate was found to inhibit the utilization of glucose by the mitogenically stimulated T cells.     

Differentiation antigens in human T-cell activation: Evidence that anti-VH antibodies react with a membrane structure on human T Lymphocytes distinct from the antigens detected by monoclonal antibodies of the OKT and Leu series

The effect of a panel of monoclonal antibodies and heteroantibodies on T-cell proliferation in various assay systems has been examined. The antibodies tested were directed against T-cell differentiation antigens, HLA-DR antigens, and structures defined by an anti-human V_H antiserum. As the test cell system highly purified subpopulations of T-cell growth factor (TCGF)-dependent T-cell lines activated either by mitogen or antigen were used. A survey of the data indicates the following: (1) Mitogenic and antigenic triggering of T lymphocytes are mediated through partly different membrane structures. (2) Antigenic stimulation by purified protein derivative (PPD) as well as polyclonal activation induced by OKT3/anti-Leu 4 monoclonal antibodies can be inhibited by heteroantibodies raised against human immunoglobulin V_H fragments thus pointing to a possible connection between the antigens detected by these antisera. (3) There does not seem to be differences between the two major subpopulations of T lymphocytes (i.e., helper/inducer and suppressor/cytotoxic cells) as to how they respond to antigens or mitogens in the investigated assay systems. (4) A clear distinction was found between T blasts specific for PPD and allogeneic cells as compared to cytotoxic T cells (CTL), as the T4 and T8 antigens seem to be functionally important for antigen recognition among CTL but not for the blasts proliferating in response to PPD and allogeneic cells. (5) An inhibitory effect of OKT3/anti-Leu 4, OKIal, and anti-HLA-DR on TCGF-dependent growth was detected, possibly indicating a steric relationship between these antigens and TCGF receptors on mitogen-induced T blasts. (6) Soluble factors obtained after incubating adherent cells with OKIal and anti-HLA-DR antibodies seemed to have an inhibitory effect on overall T-cell proliferation stressing the importance of studying the T-cell activation process at different levels in these kinds of experiments. (7) The results further suggest a complexity in the build up of antigen receptors on the various T-effector cells, perhaps also involving receptors for growth factors, HLA-DR antigens, and receptors for the latter.     

Initial characterization of sheep T-cell growth factor and its species-restricted activity on human, rat, and mouse cells

Sheep T-cell growth factor (TCGF) was prepared from concanavalin A-activated sheep peripheral blood cells and subsequently characterized by ammonium sulfate precipitation, gel exclusion chromatography, and isoelectric focussing. The TCGF was found in the 60-80% ammonium sulfate fraction and was shown to have an apparent molecular weight of 32,500 and an isoelectric point in the range pI 5.2-5.5. The ability of the sheep TCGF to promote proliferation of activated human, sheep, mouse, and rat cells was compared with that of human TCGF prepared by phytohemagglutinin stimulation of lymphocytes from multiple donors and TCGF prepared from concanavalin A-stimulated rat and mouse spleen cells. Human TCGF was found to act across all species barriers, rat TCGF supported the growth of cells of all species except human, and mouse only promoted the growth of activated mouse and rat cells. Sheep TCGF was unique in being unable to support the growth of any cells except autologous cells.     

The production of T-cell growth factor (TCGF) in vivo in sheep

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000–20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.     

Class I MHC antigens in the generation and expression of promiscuous cytotoxic cell function

In recent years investigators from a number of laboratories have described antigen nonspecific, lymphocyte-mediated cytotoxicity generated by TCGF alone, in the absence of antigen or mitogen. The exact origin of the cells mediating this cytotoxicity, in either mouse or humans, is unknown. We found that when mouse spleen cells are incubated with higher than normal concentrations of TCGF, good levels of cytotoxicity toward allogeneic, NK, and untransformed self target cells are generated by day 5 or 6 in culture. We were unable to block the lysis of any of these target cells with antibodies to target cell class I antigens. However, generation of this cytotoxicity from naive spleen cells was very strongly blocked by anti-class I MHC antibodies. When T cells from spleen were extensively purified, they did not respond to TCGF at any concentration unless adherent cells were added back. Generation of cytotoxicity under these conditions was also blocked by class I antibodies. Generation of promiscuous killing activity by PMA and ionomycin, on the other hand, was class I independent. Our data suggest that pre-CTL, under the influence of TCGF, can be activated to CTL and that under the continued influence of TCGF can be driven into a so-called "promiscuous" state of cytotoxicity. Possible roles for class I antigens in this process are discussed.     

Radiosensitivity of murine T-lymphocyte cytotoxicity

A biphasic curve was observed when surviving allogeneic lytic activity was plotted as a function of irradiation delivered before sensitization. Flow cytometry analysis demonstrated that the number of cells was reduced in response to increasing dose and that subset precursors Lyt 1+2+ were proportionally more radiosensitive than the other subsets. Paradoxically, the presence of exogenous T-cell growth factor (TCGF) in limiting dilution analysis changed the shape of the survival curve, and the mere addition of TCGF or Lyt 2- TCGF-producing cells abrogated the irradiation effect even though they were not shown to be the target of low dose irradiation in flow cytometry analysis. It is proposed that TCGF acted by enhancing the proliferation of surviving cells. This effect may be responsible for the relative radioresistance at higher doses due to enhanced availability of TCGF for the remaining cells.     

Mitogenic and mitogenically defective phytohemagglutinin isolectins stimulate T-cell growth factor (interleukin 2) production and response in fresh and cultured human T lymphocytes

L4-PHA (L4) and E4-PHA (E4) lectins isolated from Phaseolus vulgaris have different mitogenic properties. The mechanisms of the differences in mitogenic behavior were sought in the interaction of lectin, lymphocyte subsets, and T-cell growt factor (TCGF) also known as interleukin 2 (IL-2). TCGF activity in culture supernatants ( L4S ; E4S ) from L4- and E4-stimulated, freshly isolated lymphocytes was assayed as stimulation of DNA synthesis in TCGF-dependent continuous T-cell cultures (CTC). E4S contained less TCGF than did L4S . Addition of partially purified TCGF does not increase the stimulation of fresh lymphocytes by L4 or E4. L4 and E4 equally stimulate both helper (OKT4+) and suppressor (OKT8+) cells. The ability of L4 to further stimulate CTC is slowly lost (15 greater than 30 greater than 45 days). It is concluded that production of TCGF is not rate limiting in E4 and L4 stimulation of lymphocytes. The growth of CTC, which requires the presence of TCGF, remains sensitive to, but not dependent on, L4 for at least 30 days.     

Human T-cell cultures with selective autotumor reactivity

Summary T-cell cultures derived from the blood of 14 patients with solid tumors were propagated with T-cell growth factor (TCGF). The cultures were initiated from lymphocytes exposed to autologous tumor-biopsy cells. TCGF was added either immediately or 3–10 days later. In the former culture type the cell yield on day 7 was considerably higher. The cytotoxic potential of the cultured cells was assayed on two occasions, between days 7 and 10 and between weeks 5 and 8. Cells of all but two cultures had the potential to lyse autologous tumor-biopsy cells.On the population level, cytotoxicity was specific for autologous tumor in those cultures that were driven to growth with TCGF after the 3rd day. These lymphocytes did not lyse allogeneic tumor-biopsy cells. In contrast, all five cultures initiated in the presence of TCGF exhibited a broader cytotoxic potential, i.e., in addition to the stimulator autologous-tumor cells, they also lysed other targets. Another difference between the two culture types was their behavior toward K562. Tested on the 7th day they all lysed K562; however, this function declined in strength or disappeared later in the cultures exposed to TCGF after the 3rd day.Reexposure of the lymphocytes to autologous tumor-biopsy cells after 2 weeks of culture period, but not on the 7th day, induced DNA synthesis. This secondary response was specific inasmuch as allogeneic tumor cells had little or no effect.One of the autotumor restimulated cultures was tested for cytotoxic potential. It increased against the autologous but not against other tumors or K562 cells.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Properties of T-cell growth factor obtained from diucifon-stimulated human lymphocytes

The properties of T-cell growth factor (TCGF), obtained by diucifon (Dc) stimulation of human mononuclear cells (MNC) (TCGF-Dc) have been studied. Taking into account the fact that Dc alone does not, like other TCGF inductors, cause proliferation, differences between TCGF-Dc and TCGF were suggested. Partial purification of supernatant from cells, activated by Dc was performed on Sephadex G-100 column. TCGF-Dc biological activity in these fractions was determined in the system of mitogen activated human MNC and mice thymocytes, as well as in the system of concanavalin A transformed cells. 2 peaks of TCGF-Dc activity have been revealed that are indicative of TCGF-Dc molecular mass heterogeneity. In contrast to TCGF, low molecular mass TCGF-Dc (8000-12000) and TCGF-Dc from the whole supernatant were capable of absorbing on intact human MNC. TCGF-Dc may be constantly present on MNC membrane, but TCGF-Dc fixation is not sufficient for proliferation induction, the receptor activation is necessary as well. Receptors to TCGF-Dc were suggested to consist of fixing and triggering sites.     

Inability of immunocompetent thymocytes to produce T-cell growth factor under adenosine deaminase deficiency conditions

Five density-defined subpopulations of rat thymocytes were separated by isopycnic centrifugation on a discontinuous density Ficoll gradient and compared with respect to their response to Con A stimulation under normal and adenosine deaminase (ADA) deficiency conditions. This study shows that (a) immunocompetent (low-density) thymocytes and splenic T lymphocytes produce T-cell growth factor (TCGF) in response to mitogenic stimulation in normal culture conditions, but are unable to synthesize effective TCGF in the presence of an adenosine deaminase inhibitor and excess substrate (ADA deficiency conditions), and (b) high-density immunoincompetent thymocytes proliferate and differentiate into mature T cells in response to Con A if effective exogenous TCGF is added to the culture medium but are unable to do so under ADA deficiency conditions.     

Detection of autologous mixed lymphocyte reaction responding cells and their precursor frequency in NZB mice

The autologous mixed lymphocyte reaction (AMLR) can be detected in older NZB mice after treatment of the responding cell population with monoclonal anti-I-A^d and complement and supplementation of the culture medium with T-cell growth factor (TCGF) from young animals. The addition of TCGF to cultures containing responding cells alone that had not been pretreated with anti-I-A plus complement resulted in high levels of background proliferation. This is indicative of a high number of preexisting I-A-positive, activated, TCGF-responsive T cells in these mice. These activated cells could also be removed by treatment with anti-I-A antibody and panning on anti-mouse Ig plates, or by BUdR and light killing of those cells proliferating in the presence of TCGF or purified IL-2. Prior treatment of the responding cells with anti-Lyt 2 and complement did not effect the AMLR. An NZB AMLR responding cell line was established using these methods. This line retained haplotype specificity in a proliferation assay. Limiting dilution analysis of the precursor frequency of AMLR responding cells in the nonautoimmune C58 and BALB/C strains in culture medium with TCGF gave a frequency of between 1 in 35,000 and 1 in 88,000. In young, AMLR-positive, NZB mice, supplementation with TCGF yielded precursor frequencies within the normal range. In older NZB mice, the addition of TCGF resulted in increased background proliferation of preactivated, IA⁺ T cells. After removal of these cells with anti-I-A plus complement, AMLR responding cells were found at normal frequency levels when stimulated in the presence of TCGF. In the oldest animals tested (greater than 18–20 weeks), normal precursor frequencies could not be demonstrated even after this treatment, representing a true decline in the AMLR responding cell number. AMLR deficiency in NZB mice appears therefore to be the result of the combined effects of decreased lymphokine production, excessive T-cell activation, and finally decreased numbers of AMLR responding cells.     

Possible involvement of the T4 molecule in T cell recognition of class II HLA antigens: evidence from studies of proliferative responses to SB antigens

The T4 molecule has been identified as a marker of human T cell differentiation, but the function of this molecule remains to be defined. We have investigated its possible functional involvement in T cell proliferative responses to class II HLA antigens encoded by the recently described SB locus. The responses of SB-primed cells (specific for each of four different SB antigens) were studied with the use of two proliferation-inducing stimuli, SB antigen or TCGF. The proliferative responses to both stimuli were found to be mediated by T4+, T8- cells. Monoclonal antibodies against some epitopes on the T4 molecule (OKT4A and OKT4B) substantially blocked antigen-stimulated proliferative responses; antibodies against other epitopes of the T4 molecule (OKT4, T4C, T4D) blocked less well. Inhibition of SB-specific proliferation by antibodies to the T4 molecule was maximal only when the antibodies were incubated with the responder cells before the addition of stimulator cells. Proliferative responses of SB-primed cells stimulated with TCGF alone were not inhibited by any of the OKT4-related antibodies, but were completely inhibited by the anti-Tac monoclonal antibody, which reacts with the TCGF receptor. These results lend further support for the hypothesis that the T4 molecule is involved in T cell recognition of and/or activation by class II HLA antigens. We suggest that 1) the T4 molecule binds a nonpolymorphic epitope on class II HLA molecules, and 2) this interaction may facilitate, but not be an obligate requirement for, T cell activation by class II antigens.     

The human receptor for T-cell growth factor. Evidence for variable post-translational processing, phosphorylation, sulfation, and the ability of precursor forms of the receptor to bind T-cell growth factor

The T-cell growth factor (TCGF) receptor on phytohemagglutinin-activated normal peripheral blood T-cells is characterized as a glycoprotein with an apparent Mr = 55,000 that contains N-linked and O-linked carbohydrate with only approximately 33,000 daltons of peptide structure (p33) as evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There are two N-linked glycosylated intermediate precursor forms (apparent Mr = 35,000 (p35) and 37,000 (p37]. This receptor differs from the TCGF receptor on HUT-102B2 cells (apparent Mr = 50,000) because of differences in post-translational processing. Experiments with the carboxylic ionophore monensin demonstrate blockade of the transition of the p35 and p37 receptor precursor forms to the mature receptor, presumably secondary to inhibition of Golgi-associated receptor processing. We identify the primary translation product of TCGF receptor mRNA as intermediate in size between the p33 and the p35/p37 forms. We further demonstrate that the p33, p35, and p37 precursor forms, but not the primary translation product, are all capable of binding TCGF. Thus, the removal of the signal peptide and/or conformational changes of the primary translation product are necessary for ligand binding; however, the extensive post-translational modifications are not. Lastly, we demonstrate that at least some TCGF receptors are phosphorylated and sulfated, and that TCGF receptors on phytohemagglutinin-activated normal T-cells are more heavily sulfated than those on HUT-102B2 cells.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

Rat lymphocyte subsets: Cellular requirements for the generation of T-cell growth factor

In this study, the cells producing T-cell growth factor (TCGF) in the rat MLR were characterised with respect to the antigens defined by $\text{W}\text{3}\text{13}\text{,}\text{W}\text{3}\text{25}$, and OX8 monoclonal antibodies. Unfractionated lymphocytes and cells depleted of OX8 positive cells were found to be fully capable of producing TCGF, whereas lymphocytes depleted of $\text{W}\text{3}\text{13}\text{and}\text{W}\text{3}\text{25}$ positive cells had lost this ability. Parallel experiments demonstrated that cells selected by the fluorescence-activated cell sorter for the expression of $\text{W}\text{3}\text{13}\text{and}\text{W}\text{3}\text{25}$ defined antigens were potent producers of TCGF. Further studies suggested a functional role for the antigen defined by $\text{W}\text{3}\text{25}$ antibody because the addition of this antibody to a MLR abrogates TCGF production. These findings suggest that the important immunomodulatory functions of $\text{W}\text{3}\text{25}$ positive lymphocytes could be exercised via the synthesis of essential lymphokines.     

The induction of the human T-cell growth factor receptor precedes the production of RNA and occurs in the presence of inhibitors of RNA synthesis

The receptor for T-cell growth factor (TCGF) is an activation antigen that is present in low amounts on a small fraction of resting T lymphocytes. The TCGF receptor on human T cells can be detected with the anti-Tac monoclonal antibody within 7-12 h of stimulating the cells with phytohemagglutinin (PHA). In the current studies, we examined human lymphocytes cultured alone, with PHA, or with PHA plus sufficient actinomycin-D to inhibit RNA synthesis. After varying intervals, aliquots of the lymphocytes were stained with acridine orange (AO) or pyronin-Y(PY) to measure RNA and/or with anti-Tac plus FITC goat anti-mouse Ig. Tac expression began to increase after 6-8 h incubation with PHA, whereas increases in PY or AO staining were not detected until 12 h or later. Furthermore, the initial increase in Tac expression was not affected by sufficient actinomycin-D to block all detectable nucleic acid synthesis. Therefore, it appears that the initial expression of TCGF receptors detected after lymphocyte activation does not require de novo production of RNA.     

Cytolytically active murine T-lymphocyte/polyoma virus-transformed fibroblast hybrids

It would be of great interest to obtain permanent T-cell lines retaining specific activity without either allogeneic or xenogeneic stimulation. Functionally active hybrids between cytolytic T cells and thymoma were previously reported, but they had to be selected in a TCGF-containing medium. This study contains new results and reports the preparation of a hybrid cell from a cytolytic T cell and a polyoma virus-infected fibroblast, in which the T-cell characteristics dominate over the polyoma-transformed characteristics. A differentiated T-cell function (i.e., cytolysis) persists and the differentiated line does not require TCGF. The loss of cytolytic activity during in vitro evolution may be due to a selection favouring transformed cells, as suggested by concomitant enhancement of the transformed phenotype and chromosome loss.