Activation of CD 4-, CD 8- thymocytes with IL 4 vs IL 1 + IL 2

Thymocytes from C57BL/6 mice were highly purified to obtain the CD 4-, CD 8- subpopulation which constitutes only 5% of all thymocytes. Substantial proliferation was induced in vitro with either IL-1 + IL-2 or with IL-4 in the presence of PMA. IL-1 and IL-2 synergized in inducing proliferation of these purified CD 4-, CD 8- thymocytes whereas neither synergized with IL-4. In order to determine whether stimulation with IL-1 + IL-2 acted via IL-4 or vice versa, cultures were treated reciprocally with affinity-purified anti-IL-2 or anti-IL-4 antibodies. Cultures with IL-4 were inhibited by anti-IL-4 but were unaffected by anti-IL-2. The CD 4-, CD 8- thymocytes cultured with IL-1 + IL-2 + anti-IL-2 were inhibited to baseline IL-1 stimulation. At low concentrations of IL-1 (1 U/ml) and IL-2 (100 U/ml), anti-IL-4 had no effect, whereas at higher levels of IL-1 (2 U/ml IL-1), and 100 or 200 U/ml IL-2, anti-IL-4 significantly reduced DNA synthesis. This result suggests that at higher concentrations the combination of IL-1 + IL-2 can induce cells to produce IL-4 which then contributes to overall proliferation. When CD 4-, CD 8- thymocytes were cultured with the low doses of IL-1 + IL-2 for 72 h, 62% expressed cell surface T3 complex (vs 11% at initiation) and 27% were F23.1+ (vs 5% at initiation). In contrast, culture with IL-4 led to no increase in numbers of T3+ cells and none were F23.1+; however, there was coexpression of Thy1 and 6B2 on 20% of cells at the end of culture (vs 4% at initiation). Thus, IL-1 + IL-2 causes expansion of a CD 4-, CD 8- thymocyte population expressing the alpha, beta-T cell receptor, whereas IL-4 induces cells to express a phenotype present in small numbers in the periphery of normal mice and in larger numbers in mice bearing the lpr gene.     

IL-2 and IL-4 stimulate different subpopulations of double-negative thymocytes

Double-negative (CD4-/CD8-) thymocytes from young adult mice can be separated into two distinct subpopulations on the basis of the binding of mAb 7D4 directed against the receptor for IL-2. The 7D4+ cells have predominantly nonrearranged TCR beta-chain genes and express incomplete 1.0-kb beta-messages, whereas the 7D4- cells have rearranged beta-genes and express complete 1.3-kb as well as incomplete 1.0-kb beta-messages. These two populations of double-negative thymocytes also differ in their responses to IL-2 and IL-4. The 7D4+ cells are nonresponsive to IL-2 alone or IL-2 plus PMA but they are stimulated to proliferate by the combination of IL-4 and PMA. In contrast, the 7D4- cells vigorously proliferate in response to IL-2 alone or IL-2 plus PMA but they respond poorly to IL-4 alone or IL-4 plus PMA. These results suggest that IL-2 and IL-4 may be involved in the stimulation of immature thymocytes at distinct steps of their differentiation. IL-4 together with PMA stimulate immature thymocytes which seem to express the IL-2R but do not respond to IL-2.     

Developmentally controlled expression of IL 2 receptors and of sensitivity to IL 2 in a subset of embryonic thymocytes

At various times of gestation murine fetal thymocytes were tested for IL 2 receptor (IL 2-R) and T cell differentiation antigen expression. The majority of 14 to 15 day fetal thymocytes were IL 2-R and Thy-1 antigen positive, yet negative for the Lyt and L3T4 marker. A subset of IL 2-R-positive fetal thymocytes could be induced by recombinant IL 2 to proliferate over at least 10 days. Growth of these proliferating cells could not be enhanced by syngeneic feeder cells nor suppressed by monoclonal anti-I-A or anti-I-E antibodies. No antigen-specific effector functions could be induced in the proliferating Thy-1, IL 2-R-positive cells. As a whole, the results suggest a developmentally controlled rather than antigen-induced expression of IL 2-R during embryogenesis of thymocytes.     

The murine IL 2 receptor. III. Cellular requirements for the induction of IL 2 receptor expression on T cell subpopulations

The accessory cell requirements for the induction of the IL 2 receptor by the lectin Con A on murine T cell subsets were directly assayed with anti-IL 2 receptor monoclonal antibodies. Substantial levels of IL 2 receptor expression were induced on T lymphocytes of the MHC class I-restricted, suppressor/cytotoxic phenotype (L3T4-, Ly-2+) in the presence and absence of accessory cells. In contrast, high levels of IL 2 receptor expression could only be induced on T cells of the MHC class II-restricted, helper/inducer phenotype (L3T4+, LY-2-) in the presence, but not in the absence, of accessory cells. Ia- cells such as the P388D1 macrophage line or cultured fibroblasts (DAP X 3) were as efficient as the Ia+ B cell hybridoma LB in providing accessory cell function for the L3T4+, Ly-2- subset. PMA, but not purified human IL 1, could substitute for accessory cells for both IL 2 receptor expression and IL 2 secretion by the L3T4+, Ly-2- subset. These data suggest that IL 2 receptor induction on the L3T4+, Ly-2- subset is complex, possibly requiring a T cell-accessory cell interaction, whereas the lectin may directly trigger IL 2 receptor expression on L3T4-, Ly-2+ T cells.     

Cytochalasans and PMA induce IL-2 receptors on CD8+ lymphocytes.

The cytochalasans, fungal metabolites that interact with actin, can affect lymphocyte proliferation; high concentrations inhibit lectin-induced proliferation and low concentrations augment it. The phorbol ester tumor promoter, PMA, alone is not mitogenic for primary lymphocytes but enhances the activity of mitogenic lectins. Because the cytochalasans have been reported to increase intracellular Ca2+ and because PMA activates protein kinase C, lymphocytes were treated with PMA and cytochalasin B (CyB) to determine if this combination would induce DNA synthesis. While this treatment by itself did not cause proliferation, lymphocytes cultured with PMA and CyB overnight, washed, and recultured with IL-2 proliferated to the same degree as lymphocytes stimulated with Con A. Three different cytochalasans, cytochalasin B, cytochalasin D, and chaetoglobosin C, all of which bind to cellular actin with different affinities and only one of which affects glucose transport, induced IL-2 receptors in combination with PMA. Flow cytometric analysis with an antibody to the IL-2 receptor alpha subunit confirmed the induction of receptors on CD8+ cells. However, no IL-2 was produced after the exposure of lymphocytes to the combination of cytochalasans and PMA. Therefore, there was sufficient signal to induce IL-2 receptor expression but not to induce IL-2.     

Induction of IL 2 receptor expression and cytotoxicity of thymocytes by stimulation with TCF1

We investigated the role of T cell cytotoxicity inducing factor 1 (TCF1) in the induction of a cytotoxic T cell response. We found that help-deficient thymocyte cultures supplied with saturating amounts of purified IL 2 did not develop CTL in a 5-day culture. The expression of cytotoxicity was dependent on the addition of TCF1 derived from the T cell hybridoma K15. TCF1 also induced proliferation of thymocytes in the presence of IL 2. Only the PNA- thymocyte subpopulation responded to TCF1 with proliferation and cytotoxicity in the presence of IL 2. The monokine IL 1 also induced proliferation in this subpopulation but failed to induce cytotoxicity. IL 1 was further distinguished from TCF1 by inhibition of IL 1-induced but not TCF1-induced proliferation by anti-IL 1 antibodies. In addition, using anti-IL 2 receptor antibodies (AMT 13), we showed that TCF1 in the presence of IL 2 substantially increased IL 2 receptor expression in thymocytes. IL 1 had the same effect on induction of IL 2 receptor expression as TCF1. Because some effects of IL 1 and TCF1 are distinct and some overlap, we discuss whether IL 1 and TCF1 induce different subsets of PNA- thymocytes.     

Mitogenic stimulation of thymocytes results in the calcium-dependent phosphorylation of c-ets-1 proteins.

Human, murine and chicken c-ets-1 proteins migrate in SDS-polyacrylamide gels as multiple species. We show here that most if not all of this heterogeneity is due to phosphorylation events occurring predominantly on serine and to a lesser extent on threonine residues. These phosphorylations can be specifically and rapidly stimulated by treatment with the calcium ionophore A23187 or abolished by lowering the extracellular calcium concentration to less than 0.1 microM. The products encoded by c-ets-2 are also phosphorylated in a Ca2+-dependent manner, indicating that these modifications have been conserved in the products encoded by different members of the same gene family. In thymocytes, where the expression of c-ets-1 is elevated as compared with other cell types, c-ets-1 protein phosphorylation occurs after stimulation with mitogenic doses of concanavalin A, is short lived and is strictly dependent upon extracellular Ca2+ sources. This suggests that the c-ets-1 gene product may play a role in the Ca2+-mediated early events linked to T-cell activation.     

L3T4+, B2A2+ thymocytes from infant mice produce IL 2 after interaction with accessory cells expressing self class II antigens

The presence of a thymocyte population in infant (3 to 10 day old), but not adult mice, that produces IL 2 after self class II MHC antigen stimulation is described. The responding thymocyte expresses the antigenic phenotype: Thy-1+, Ly-1+, Ly-2-, L3T4+, B2A2+. Cell-cell mixing experiments and limiting dilution analysis were consistent with a loss of the self-Ia-reactive IL 2-producing thymocyte in the adult, with no evidence for active suppression found. Although thymocytes from adult animals failed to generate IL 2 after self class II stimulation, a similar frequency of IL 2-producing cells was demonstrated in both infant and adult after allo stimulation. These findings, taken with the recent demonstration of IL 2 receptors on neonatal thymocyte subpopulations, demonstrate a mechanism for IL 2 production in the thymus that may play a role in intrathymic differentiation of T cells.     

Mitogenic activation and proliferation of mouse thymocytes : Comparison between isotope incorporation and flow-microfluorometry

Staining with acridine orange and flow microfluorometry was used to measure the distribution over the cell cycle of Concanavalin A (ConA)-stimulated mouse thymocytes. These data were compared with data on isotope incorporation. Mitogen-induced [¹⁴C]thymidine incorporation into DNA increased 10–20-fold upon addition of 2-mercaptoethanol or spleen-derived glass adherent cells. However, the proportion of proliferating cells as measured by flow microfluorometry increased only by a factor of two. Removal of nylonwool adherent cells nearly abolished ConA-induced thymidine incorporation; this was restored by the addition of glass-adherent cells. The fraction of mitogen-activated cells as measured by flow microfluorometry was hardly affected by nylonwool filtration and addition of mercaptoethanol or glass adherent cells enhanced the proportion of activated cells that can enter DNA synthesis. From these and similar experiments it is concluded that DNA synthesis measured as thymidine incorporation is only proportional to the number of mitogen-activated cells under restricted conditions. Differences in thymidine incorporation in different lymphocyte populations or measured under different conditions are difficult to interpret in terms of number of proliferating cells. The cytofluorographic method is a simple and rapid way to solve these problems quantitatively and rapidly.     

Comparative effects of Saccharomyces cerevisiae cultivation under copper stress on the activity and kinetic parameters of plasma-membrane-bound H+-ATPases PMA1 and PMA2

The major yeast plasma membrane H⁺-ATPase is encoded by the essential PMA ₁ gene. The PMA ₂ gene encodes an H⁺-ATPase that is functionally interchangeable with the one encoded by PMA ₁ , but it is expressed at a much lower level than the PMA ₁ gene and it is not essential. Using genetically manipulated strains of Saccharomyces cerevisiae that exclusively synthesize PMA₁ ATPase or PMA₂ ATPase under control of the PMA₁ promoter, we found that yeast cultivation under mild copper stress leads to a similar activation of PMA₂ and PMA₁ isoforms. At high inhibitory copper concentrations (close to the maximum that allowed growth), ATPase activity was reduced from maximal levels; this decrease in activity was less important for PMA₂ ATPase than for PMA₁ ATPase. The higher tolerance to high copper stress of the artificial strain synthesizing PMA₂ ATPase exclusively, as compared to that synthesizing solely PMA₁ ATPase, correlated both with the lower sensitivity of PMA₂ ATPase to the deleterious effects of copper in vivo and with its higher apparent affinity for MgATP, and suggests that plasma membrane H⁺-ATPase activity plays a role in yeast tolerance to copper. Received: 19 October 1998 / Accepted: 6 January 1999     

Leukemia L 1210 cells induce depletion of Lyt 2+ thymocytes

The thymocytes were analyzed on the 7th day after i.p. inoculation of 10(6) leukemia L 1210 cells to syngeneic DBA/2 Wf mice. A three-fold decrease of the total number of thymocytes was found as well as 1.7-fold decrease of the per cent of thymocytes with Lyt 2+ phenotype, while the per cent of cells with phenotypes Thy 1.2+ and Lyt 1+ was unchanged.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.     

Differential effect of anti-beta 2-microglobulin on IL 2 production and IL 2 receptor expression in the primary mixed lymphocyte culture reaction

The mechanism of inhibition of the proliferative response in primary mixed lymphocyte culture (1 degree MLC) by antibodies to beta 2-microglobulin (beta 2m) was investigated. It is demonstrated that anti-beta 2m antibodies inhibit the production of interleukin 2 (IL 2). In contrast, the expression of IL 2 receptor is not affected by anti-beta 2m. The addition of purified exogenous IL 2 to the antibody-treated 1 degree MLC can completely restore the proliferative response, indicating that anti-beta 2m does not interfere with IL 2 binding to its receptor. Similarly, anti-beta 2m does not interfere with the capacity of IL 2-dependent T cell lines or T cell clones to respond to exogenous IL 2. The inhibition of cell proliferation and IL 2 production by anti-beta 2m is maximal when the antibody is added at the beginning of 1 degree MLC culture, and no effect of anti-beta 2m is seen when added after 3 days of culture. Anti-beta 2m has no effect on mitogen-induced cell proliferation and IL 2 production. Anti-beta 2m acts on the responder cell population, as demonstrated in experiments in which responder cells or stimulator cells are treated separately with the antibody. The expression of HLA-class II antigens (i.e., HLA-DR and DQ (DC) on the T cells activated on 1 degree MLC is not affected by anti-beta 2m. These studies indicate that the HLA-beta 2m class I antigen complex plays a role in T lymphocyte activation via release of IL 2, and suggest the existence of different mechanisms for activation of IL 2 producers and IL 2 responders in 1 degree MLC.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Effect of magnesium ions on Ca2+, Mg2+-ATPase on plasma membranes of thymocytes]

Ca2+, Mg(2+)-ATP-ase of plasmatic membranes of thymocites were studied as affected by Mg ions. A complex character of the reaction rate dependence on Mg2+ concentration in the range of 0.002-25. OmM was revealed. The data obtained indicate the existence of Mg(2+)-binding centre of ATP-ase which regulates the enzyme catalytic activity.     

PARP-2 deficiency affects the survival of CD4+CD8+ double-positive thymocytes

Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADP-ribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2-/-) cells, similar to Parp-1-/- cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4+CD8+ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2-/- DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2-/- have a reduced expression of T-cell receptor (TCR)alpha and a skewed repertoire of TCRalpha toward the 5' Jalpha segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRalpha recombination is compromised despite normal amounts of Bcl-xL. These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRalpha rearrangements preceding a positively selected TCR.     

TGF beta 1 inhibits Ca2+-calcineurin-mediated activation in thymocytes

TGFbeta1 is a polypeptide growth modulatory and differentiation factor involved in many biological processes including immune homeostasis and self-tolerance. Tgfb1 knockout mice die around weaning age due to severe inflammation in most major organ systems, but the mechanism underlying this disease is not understood. In this study we demonstrate that Tgfb1(-/-) CD4(+)CD8(+) and CD4(+)CD8(-) thymocytes are hyperresponsive to receptor-mediated and receptor-independent mitogenic stimulation. A suboptimal concentration of ionomycin in the presence of PMA fully activates Tgfb1(-/-) thymocytes, whereas the inhibitors of Ca(2+) influx and calcineurin, EGTA and FK506, eliminate the hyperresponsiveness. Hence, the hypersensitivity of Tgfb1(-/-) thymocytes is due to a lowered threshold for Ca(2+)-dependent activation. Further, we demonstrate that the hypersensitivity of thymocytes results from the absence of TGFbeta1 and not from the inflammatory environment because the thymocytes are hyperresponsive in preinflammatory-stage Tgfb1(-/-) mice. Our results suggest for the first time that TGFbeta1 functions to inhibit aberrant T cell expansion by maintaining intracellular calcium concentration levels low enough to prevent a mitogenic response by Ca(2+)-independent stimulatory pathways alone. Consequently, TGFbeta1 prevents autoimmune disease through a Ca(2+) regulatory pathway that maintains the activation threshold above that inducible by self-MHC-TCR interactions.