Lymphokine regulation of activated (G1) lymphocytes. II. Glucocorticoid and anti-Tac-induced inhibition of human T lymphocyte proliferation

The regulation of the first cell cycle of human, activated (G1) PBL was analyzed by flow cytometry and [3H]thymidine incorporation. Endogenous IL 2 production was blocked in situ by pharmacologic concentration of DEX (100 to 1000 nM), resulting in an 80 to 90% reduction of thymidine uptake. Although T lymphocyte activation (G0-G1a transition) by PHA was unaltered, cells remained in the G1a phase of the cell cycle due to insufficient RNA synthesis for proliferation. The addition of IL 2-containing supernatants reversed this inhibitory effect of DEX by allowing the cells to synthesize more RNA (G1a-G1b transition). Such cells could enter the S phase and proliferate. Similar studies were performed on cells treated with a monoclonal antibody (anti-Tac) against the IL 2 receptor. In these studies, IL 2-induced RNA synthesis, and subsequent proliferation of DEX-treated and PHA-stimulated cells was inhibited by anti-Tac. Anti-Tac did not, however, inhibit the effect of endogenous IL 2 (PHA-stimulated PBL without DEX treatment), although it did bind equally well to such cells. Thus, IL 2 directly or indirectly regulates human T cell proliferation at the level of RNA synthesis. Furthermore, anti-Tac can inhibit the mitogenic signal given by endogenous IL 2, but not by in situ produced IL 2, an observation of importance to further investigations of the mechanisms by which IL 2 interacts with specific receptors to elicit proliferation.     

The regulation of G0-S transition in mouse T lymphocytes by polyamines

While the role of polyamines in DNA synthesis during the S phase of the cell cycle has been repeatedly postulated, recent studies point also to polyamine involvement in the early phase of the G0-S transition. In order to determine polyamine-dependent steps in the cell cycle we have studied the effects of inhibitors of polyamine biosynthesis and exogenous polyamines on the proliferation of T lymphocytes as well as on the expression of some growth-regulated genes. The ability of Con A-stimulated mouse T lymphocytes to enter DNA synthesis was markedly inhibited by methylglyoxal bis(guanylhydrazone) in a dose-dependent manner. This inhibitory effect was stronger in the presence of fetal calf serum containing a high level of activities of polyamine oxidases than in the presence of horse serum. Putrescine and spermine added to T splenocyte culture instead of mitogen-Con A stimulated [3H]thymidine incorporation with kinetics similar to that observed with Con A. The growth-stimulating effects of polyamines were concentration-dependent. Polyamines at optimal growth-stimulating concentrations (10 microM spermine and 80 microM putrescine) induced the expression of genes encoding the cytoskeletal proteins beta-actin, vimentin, and alpha-tubulin to an extent and with kinetics similar to those of Con A. The results presented herein suggest that polyamines are capable of stimulating the transition of G0 cells to the S phase and that this effect may be mediated by their influence on the gene expression.     

Antigen presentation by human dermal fibroblasts: activation of resting T lymphocytes

We have shown that human dermal fibroblasts, exposed to interferon-gamma (IFN-gamma) to induce surface class II major histocompatibility complex (MHC) antigens, were capable of presenting tetanus toxoid (TT) antigen to human TT-specific T cell clones. Antigen presentation by fibroblasts was antigen dependent, required HLA-DR expression by fibroblasts, and was MHC restricted. In contrast, we now report that IFN-gamma-treated fibroblasts are unable to present TT antigen to purified resting T cells obtained from the peripheral blood of TT-immune donors. In addition, although IFN-gamma-treated fibroblasts were able to stimulate alloreactive T cell clones, they were unable by themselves to stimulate primary allogeneic responses in resting T cells. The failure of fibroblasts to stimulate resting T cells was not due to suppressor effects by fibroblasts, because induction of TT and alloantigen responses in resting T cells by monocytes was not inhibited by the presence of fibroblasts. On the contrary, IFN-treated fibroblasts were synergistic with small numbers of monocytes in activating resting T cells. In addition, the failure of antigen presentation by fibroblasts to resting T cells was reversed by the addition of recombinant human interleukin 2 (rIL 2) to cultures, but not of purified human interleukin 1 (IL 1). These results emphasize that the requirements for activation of resting T cells differ from those of T cell clones. Although fibroblasts can efficiently present antigen to T cell clones, antigen presentation by fibroblasts to resting T cells requires the addition of exogenous IL 2. It is postulated that fibroblasts differ from classical antigen-presenting cells in that fibroblasts are incapable of stimulating the production of IL 2 in resting T cells.     

Induction of nonspecific, proliferative dependent suppressor T lymphocytes in human mixed lymphocyte cultures

The stimulation of highly purified human T and B cells by soluble and insoluble protein A was studied. Insoluble protein A, such as protein A conjugated to Sepharose beads (S-pro A), or Staphylococcus aureus Cowan I strain bacteria (SpA CoI), markedly stimulated B cells, but did not affect T cells. SpA CoI stimulated B cells independently of the presence of T cells. While soluble protein A failed to stimulate either T or B cells alone, it greatly stimulated the mixture of T and B cells. Mitomycin treatment revealed that the response to soluble protein A was ascribed mainly to the T-cell response with the B-cell helper effect, though partially to the B-cell response with the T-cell helper effect as well. The response of T cells to protein A was enhanced by both the adherent population and the nonadherent B-cell population. This T-B cooperation was mediated by direct cell-to-cell interaction rather than soluble mediators. The binding experiments also demonstrated that the amount of protein A bound to T cells was far less than that to B cells. These results point out the significance of B-cell participation in T-cell activation. The mechanism by which protein A activates T and B cells was also discussed.     

In vitro regulation of human lymphocyte proliferation by selenium

A chemoprotective role for dietary selenium in malignancy has been well documented in numerous epidemiological and experimental studies. The precise mechanisms of this relationship are not understood, but may be related to observations that selenium can inhibit the proliferation of various normal and neoplastic cells, both in vivo and in vitro. In this study, we present evidence that selenium at physiologic concentrations can effectively inhibit the overall proliferation of human lymphocyte populations in response to various immune stimuli in vitro, including mixed lymphocyte response and response to soluble antigen (tetanus toxoid). This inhibition was reversible, indicating that selenium was not toxic to the lymphocytes at these concentrations. Preliminary data from our laboratory indicate that the antiproliferative effects of selenium may be specific for certain lymphocyte subsets. Similar modulation of immune responses in vivo could enhance various humoral and cellular immune mechanisms. Together with published evidence that selenium can inhibit tumor cell proliferation, these data may help to explain the decreased incidence of cancer associated with elevated selenium intake.     

Promotion of human T lymphocyte proliferation by IL-4

The capacity of human rIL-4 to support the proliferation of mitogen-stimulated T cells directly as well as by increasing IL-2 production or enhancing IL-2 responsiveness was investigated. IL-4 augmented proliferation of T cells stimulated with PHA, Con A, immobilized mAb to the CD3 molecular complex (OKT3), or PMA. IL-4 increased the number of mitogen-stimulated cells entering the cell cycle as well as enhancing ongoing proliferation of mitogen-activated lymphoblasts. Facilitation of initial activation by IL-4 was not inhibited by mAb to the p55 component of the IL-2R, anti-Tac, and, therefore, was not dependent on endogenous IL-2 activity. However, IL-4-mediated enhancement of ongoing T cell proliferation stimulated by PHA or OKT3 was partially but not completely blocked by anti-Tac. Analysis of the supernatants from PHA-stimulated T cell cultures indicated that IL-4 increased the production of IL-2 by mitogen-activated cells. Moreover, IL-4 increased the amount of IL-2 mRNA that accumulated in mitogen-stimulated T cells. In addition, IL-4 markedly augmented IL-2R expression by PHA-stimulated T cells. Although IL-4 promoted ongoing DNA synthesis of mitogen-stimulated T cells in an IL-2-dependent manner, it was also able to sustain their proliferation directly. Thus, IL-4 supported proliferation of PMA-activated T cells in a manner that was not inhibited by anti-Tac. Furthermore, IL-4 could augment proliferation and IL-2R expression of T cells stimulated with PHA in the presence of cyclosporin A, which blocks endogenous cytokine production or anti-Tac. Finally, IL-4 was noted to enhance proliferation of both CD4+ and CD8+ T cell subsets. The results indicate that IL-4 enhances proliferation of mitogen-activated human T cells by a number of mechanisms, including the direct promotion of cell cycle entry and subsequent DNA synthesis, enhanced production of IL-2, and increased responsiveness to IL-2 in part by up-regulation of IL-2R expression.     

Relation between sister chromatid exchange,cell proliferation and proportion of B and T cells in human lymphocyte cultures

Summary Human B and T lymphocytes differ in the rate of cell proliferation and frequency of sister chromatid exchange (SCE) when cultured separately in short-term cultures. This difference could theoretically be responsible for part of the variation in the SCE-frequency previously observed among healthy subjects since there is individual variation in the proportion of B and T cells in the peripheral blood. We have therefore studied cell proliferation and SCE-frequency in conventional short-term cultures of lymphocytes from 28 healthy subjects with different proportions of B and T cells. The percentage, of B or T lymphocytes did not correlate with the SCE-frequency, nor with the rate of cell proliferation in culture. However, a significantly higher SCE-frequency was found in slowly proliferating cultures than in cultures with a high rate of turn over. Thus, the rate of cell proliferation appears to be an important determinant of the SCE-frequency in conventional lymphocyte cultures. Although the data do not exclude attribution of the difference in SCE-frequency between rapidly and slowly growing cultures to differences in subpopulations of lymphocytes, it appears less likely that B and T cells constitute these tentative subpopulations.     

Protein phosphokinase activities of resting and proliferating human lymphocytes : Changes upon phytohemagglutinin stimulation and in acute lymphoblastic leukemic cells

Transformation of normal human peripheral lymphocytes by phytohemagglutinin (PHA) to blast-like cells is accompanied by an enhancement of the protein phosphokinase activity. This activity becomes maximal approx. 70 h after exposure to the mitogen and amounts to a 3- to 4-fold stimulation in the 10 000 g supernatant and 8- to 10-fold in the nuclear fraction. This augmented activity is due to the increased level of some of the multiple forms of lymphocyte protein kinase, specially the one which is active on exogenous casein and elutes from DEAE-cellulose at 125 mM phosphate (casein-kinase S-C₃ and N-C₂). Acute lymphoblastic leukemic cells have a protein kinase pattern upon chromatography on DEAE-cellulose which is similar, but not identical, to that of PHA-stimulated lymphocytes. The most remarkable difference is the presence in the 10 000 g supernatant of leukemic cells of a protein kinase form which was either absent or barely detectable in resting or PHA-treated normal lymphocytes. This protein kinase is active on casein and is not stimulated by cAMP. The results obtained are discussed in connection both with the known enhanced protein phosphorylation in PHA-stimulated cells and with the protein kinase changes observed in other cellular systems.     

Simple sugars inhibit proliferation of human T lymphocytes in autologous and allogeneic mixed lymphocyte reactions

Several oligo- and monosaccharides were studied for their capacity to modulate lymphocyte proliferation in human allogeneic and autologous mixed lymphocyte reactions (MLR). A defined subset of sugars showed a marked inhibitory effect on lymphocyte proliferative response in the majority of the allogeneic MLR combinations studied. The inhibitory effect disappeared when sugars were added to allogeneic MLR 96 hr after the beginning of culture. These sugars also showed a significant inhibitory power on autologous MLR, performed by using T- and non-T-enriched lymphocytes from the same donor. The reported data suggest that carbohydrate determinants are involved in the proliferative response of human lymphocytes in both autologous and allogeneic MLR.     

Liquid-holding experiments with human lymphocytes: III. Experiments with G0 and G1 cells

Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the GO (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sisterchromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in GO cells and that G1 cells can repair both DEB and MNU induced lesions.     

Lymphokine regulation of activated (G1) lymphocytes. I. Prostaglandin E2-induced inhibition of interleukin 2 production

PGE2-induced inhibition of the proliferatory response of PHA-stimulated human PBL and Con A-stimulated murine thymocytes was analyzed by flow cytometry. It was found that the activation process (G0-G1a transition) was not influenced by PGE2 over a wide range of concentrations (10(-10) to 10(-6) M), nor was the formation of IL 2 receptors inhibited. Similarly, the viability of human lymphocytes was practically unaltered. In contrast, the IL 2-dependent cell cycle event (G1a-G1b transition), which is required for proliferation, was inhibited in a dose-dependent fashion. The addition of IL 2-containing supernatants to such cultures prevented the PGE2-mediated block in the G1a phase and reconstituted a normal lymphocyte proliferation. Furthermore, lower IL 2 titers were measured in supernatants from PHA-stimulated human PBL treated with PGE2. These findings strongly suggest that PGE2 primarily exerts its inhibitory effect on lymphocyte proliferation through an inhibition of IL 2 production.     

Inhibition of lymphocyte proliferation by monoclonal antibody directed against the T3 antigen on human T cells

Peripheral blood mononuclear cells from 40% of normal donors are mitogenically unresponsive to UCHT1, a monoclonal antibody reactive to the T3 surface molecule on human T lymphocytes. Cell preparations from non-UCHT1 responders were used to examine whether and how interaction of UCHT1 with the T3 molecule affects T-cell functionality. It was found that UCHT1 profoundly (greater than 85%) suppressed lymphocyte proliferation induced by plant mitogens (phytohemagglutinin (PHA) and concanavalin A (Con A], recall antigen (candidin), and allogeneic non-T cells. The antibody abrogated both the production of interleukin 2 (IL-2) by and the expression of IL-2-specific receptors on T lymphocytes stimulated by PHA or allogeneic non-T cells. UCHT1 was maximally suppressive when added to cells within 2 hr (PHA stimulation) or 1 day (allogeneic non-T cell activation) after the initiation of the culture period. The inhibiting activity of UCHT1 could be related to its ability to modulate T3 molecules from the T-cell surface: both actions displayed the same antibody concentration dependence and had a comparable time dependence. Moreover, after modulation, unresponsive lymphocytes regained responsiveness to PHA in parallel with reexpression of surface T3 molecules. These findings are consistent with the idea that the human T3 molecule functions as an essential signal transducer during the early phases of T-cell activation.     

DNA synthesis and proliferation of human lymphocytes in vitro: III. Fate of cycling cells in aging cultures of phytohemagglutinin stimulated human lymphocytes

There are few data available on cell cycle events that occur when proliferation of normal cells in culture is curtailed due to “natural aging” of the culture conditions. Stathmokinetic and cytofluorometry studies were performed on PHA-stimulated human lymphocyte cultures for eight consecutive days. Cell proliferation peaked on day 5 and then gradually decreased. Percent labeled mitosis curves performed each day demonstrated that, for those cells which progressed to mitosis, the cell cycle time remained constant at 18 ± 1 hour throughout the entire period of culture. However when the fate of all cells pulse-labeled with ³H-thymidine (S phase cells) was followed daily, only 64 ± 5% of labeled cells reached mitosis on day 3 and <20% on day 6. When the growth fraction was estimated by standard methods (with the labeling index) and used to predict future cell counts in the culture, proliferation was greatly overestimated; but after correcting the growth fraction for labeled cells not reaching mitosis, proliferation was accurately predicted by a newly derived “dividing fraction.” Flow cytofluorometry confirmed accumulation of cells in S and G₂ + M phases, and mitotic indices ruled out accumulation in M phase. Assessment of non-viable cells with cytofluorometry demonstrated that death occurred in all phases of the cell cycle. We conclude that with increasing age of culture, an increased fraction of cycling PHA-stimulated lymphocytes fail to progress all the way to mitosis and are arrested in S or G₂ phases. These observations provide evidence against the existence of a specific “restriction point” in G₁ or at the G₁/S interface in aging proliferating human lymphocyte cultures, but it remains to be determined whether cells arrested in S or G₂ phases retain the capacity to complete the cell cycle in more favorable culture environments.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

Abstract. The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry.
Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lumphocytes on the basis of their nucleolar antigen content.     

Different nucleolar antigen expression in resting and proliferating human lymphocytes as studied by fluorescence microscopy and flow cytometry

The aim of this study was to investigate the use of a nucleolar antigen to discriminate between proliferating and resting cells. Antinucleolar antibodies (Si87) were obtained from a scleroderma patient. The specificity of immunostaining was verified and morphological changes in nucleoli were monitored using a fluorescence microscope. Fluorescence of propidium iodide-stained DNA and nucleolar immunofluorescence were measured by flow cytometry. Following phytohaemagglutinin stimulation the number of nucleoli of normal human peripheral blood lymphocytes increased about 3-fold, accompanied by enlargement of nucleolar size. Simultaneously a mean increase in total immunofluorescence per cell by a factor of three was detected. The method developed and applied here allows a discrimination between resting and proliferating human lymphocytes on the basis of their nucleolar antigen content.     

Volume regulation of human peripheral blood lymphocytes and stimulated proliferation of volume-adapted cells

Human peripheral blood lymphocytes exposed to hypotonic media (Ca/Mg-free, room temp.) first swell and then shrink. This shrinking response is characterized by a simple exponential with a half-time of 1.44±0.60 min (n=11) and its extent but not the half-time for a given hypotonicity is influenced by [K⁺]₀. Using K-selective electrodes, we observe a change in [K⁺]₀ when cells are diluted into hypotonic media. A half-time of 1.55±0.06 min (n=4) was obtained. A similar half-time was obtained by assay of total cell K using atomic absorption spectroscopy. At all osmolarities [K⁺]_i was decreased from control values and was constant as [K⁺]₀ was increased. Short-term incubation with ouabain (10⁻⁴ M) had no effect. Decreasing osmolarities progressively inhibited phytohemagglutinin-stimulated DNA synthesis, yet cell number and viability remained unaltered. Our evidence indicates that the volume response is mediated by a change in the passive permeability of the plasma membrane to K and/or to the accompanying anions, and that the consequently volume-adapted cells are growth-inhibited.     

Protein synthesis in resting and growth-stimulated human peripheral lymphocytes : Evidence for regulation by a non-messenger RNA

Stimulation of lymphocyte growth is accompanied by an early increase in the rate of protein synthesis. This increase is dependent upon the flow of inactive free ribosomes into polysomes, which is limited by a rate-controlling step at initiation [2]. Addition of actinomycin D (actD) to lymphocytes caused a gradual reduction in protein synthesis in resting cells, but rapidly inhibited both the elevation of protein synthesis and the activation of free ribosomes which normally follow exposure to mitogens. Since actD does not affect protein synthesis in enucleated lymphocytes [4], the effect in intact cells must be mediated by a nuclear event, which available data indicate is RNA synthesis. ActD prevented the accumulation of 80S initiation complexes which normally occurs in resting lymphocytes treated with pactamycin and cycloheximide, showing that its locus of action was at some point in initiation. The decline in rate of protein synthesis began without detectable lag when resting lymphocytes were treated with actD. However, after growth stimulation, a delay of ca 50 min occurred before the protein synthetic rate declined in response to actD. These observations agree with the hypothesis that the concentration of some moderately short-lived RNA is rate-limiting for protein synthesis in resting lymphocytes, and that an early event in growth stimulation is a rise in the amount of this component to levels which are no longer rate-limiting. This permits an increased flow of ribosomes into polysomes and a consequent rise in protein synthesis. Available evidence indicates that the regulatory RNA is neither mRNA nor rRNA, but may either be one of the small cytoplasmic RNAs whose function is unknown, or tRNA_i^met.     

rIL 2-induced proliferation of human circulating NK cells and T lymphocytes: synergistic effects of IL 1 and IL 2

Cells participating in the rIL 2-induced proliferation of resting PBMC were identified by using different methods of cell purification. NK cells recovered in the light density fraction of Percoll gradients responded, as already known, directly to rIL 2 by strong proliferation. In contrast, large T lymphocytes co-purifying with NK cells, and small T cells sedimenting in the high density area of the Percoll gradients, were virtually unresponsive when cultivated in the sole presence of rIL 2. However, the addition of either irradiated autologous monocytes or highly purified IL 1 allowed both kinds of T cells to undergo cell division. Stringent elimination of possibly contaminating NK cells (NKH-1+) and/or activated T cells (TNKTAR, Tac+, HLA-DR+) from the high density T cells by complement lysis did not impair rIL 2-induced cell proliferation, indicating entire responsiveness of these cells to the synergistic action of IL 1 plus IL 2. Both high density CD4+ and CD8+ participated in this phenomenon, with an apparent advantage for CD4+ cells. All Tac+ cells emerging in a 6-day culture of these cells expressed the WT31 antigen, which indicates that T cells involved in rIL 2-induced proliferation are conventional mature T cells. The relative precursor frequencies of NK cells, large T lymphocytes, and small T lymphocytes that proliferated in response to rIL 2 were analyzed by limiting dilution analysis. The frequencies of clonal growth of NK cells and low density T lymphocytes were approximately the same (1/103 vs 1/185), whereas that of high density T cells was four times lower (1/458). Thus, we clearly demonstrate that resting T cells, defined as such by morphological, density, and phenotypic criteria, are able to proliferate in response to IL 2 in the presence of IL 1 without antigenic or mitogenic triggering.