Changes in inducibility of IL-2 receptor alpha-chain and T cell-receptor expression during thymocyte differentiation in the mouse

Within the thymus, developing T cells must acquire the competence to respond to appropriate signals by inducing the expression of genes required for immunologic function; one such gene encodes the 55-kDa-chain of the IL-2R (IL-2R alpha). Previously, we showed that most cortical-type thymocytes lack the competence to make this particular response, while most medullary-type cells respond like mature T lymphocytes. The noninducibility of cortical-type cells was striking, because most of their presumed precursors were inducible. To test the relationship between this apparent loss of competence and the positive and negative selection processes that may occur in the thymic cortex, we have assayed the inducibility of thymocyte populations, staged carefully with respect to their expression of TCR. Using size fractionation to enrich for dividing cells, we concentrated and thereby revealed defined developmental intermediates. We report that, although CD4+CD8- thymocytes behave as mature T cells, a significant fraction of CD4-CD8+ cells are noninducible. These noninducible thymocytes are dividing cells, which appear to be in a major developmental continuum between CD4-CD8- blasts and CD4+CD8+ blasts. Furthermore, the noninducible blasts as yet lack surface TCR expression. We also demonstrate the functional similarity of these CD4-CD8+ cells to a major subset of dividing CD4-CD8- precursor cells, which appear to have lost IL-2R alpha expression. These results suggest that precursors of cortical thymocytes lose competence to be induced to express IL-2R alpha several stages before their acquisition of cell-surface TCR complexes. The implications of this characterization are discussed in terms of the possible relationships between IL-2R alpha gene regulation and intrathymic fate determination.     

A requirement for IL-2/IL-2 receptor signaling in intrathymic negative selection

The nature of the signals that influence thymocyte selection and determine the fate of CD4(+)8(+) (double positive) thymocytes remains unclear. Cytokines produced locally in the thymus may modulate signals delivered by TCR-MHC/peptide interactions and thereby influence the fate of double-positive thymocytes. Because the IL-2/IL-2R signaling pathway has been implicated in thymocyte and peripheral T cell survival, we investigated the possibility that IL-2/IL-2R interactions contribute to the deletion of self-reactive, Ag-specific thymocytes. By using nontransgenic and transgenic IL-2-sufficient and -deficient animal model systems, we have shown that during TCR-mediated thymocyte apoptosis, IL-2 protein is expressed in situ in the thymus, and apoptotic thymocytes up-regulate expression of IL-2RS: IL-2R(+) double-positive and CD4 single-positive thymocytes undergoing activation-induced cell death bind and internalize IL-2. IL-2-deficient thymocytes are resistant to TCR/CD3-mediated apoptotic death, which is overcome by providing exogenous IL-2 to IL-2(-/-) mice. Furthermore, disruption or blockade of IL-2/IL-2R interactions in vivo during Ag-mediated selection rescues some MHC class II-restricted thymocytes from apoptosis. Collectively, these findings provide evidence for the direct involvement of the IL-2/IL-2R signaling pathway in the deletion of Ag-specific thymocyte populations and suggest that CD4 T cell hyperplasia and autoimmunity in IL-2(-/-) mice is a consequence of ineffective deletion of self-reactive T cells.     

Changes in homing receptor expression on murine lymphokine-activated killer cells during IL-2 exposure

The effects of IL-2 on the expression of homing receptors by lymphocytes of NK or lymphokine activated killer (LAK) cell derivation has not yet been evaluated. We developed a murine model to evaluate the potential of LAK cells to localize into peripheral lymph nodes since LAK cells are used to treat human cancers which have metastasized to these tissues. Using a frozen section binding assay, LAK cell adhesion to the lymph node microvasculature was easily demonstrable. Inhibition studies demonstrated that LAK cell binding to lymph nodes was mediated by mechanisms previously described in T cells. LAK cell surface expression of the 85- to 95-kDa homing receptor recognized by the antibody MEL-14 on LAK cells was assessed by indirect immunofluorescence. The percentage of cells which bound MEL-14 decreased slightly over 3 days of IL-2 exposure (from 73 to 60%), particularly in the large granular lymphocyte (cytotoxic effector) subpopulation (45% MEL-14+). The expression of another homing-related molecule, leukocyte function-associated Ag-1, markedly increased during activation of LAK cells. Despite the expression of these homing receptors, we observed almost no LAK cell localization into lymph nodes in vivo. Furthermore, IL-2 pretreatment of recipient animals did not increase the adhesion of LAK cells to lymph node microvasculature or enhance their extravasation. IL-2 activation of non-T, non-B lymphocytes results in significant changes in both the expression and function of cell surface homing receptors. Our results indicate that in vitro analysis does not always predict in vivo localization potential.     

Phenotypic and functional characterization of c-kit expression during intrathymic T cell development.

We have studied the expression and function of c-kit on subsets of mouse thymocytes. c-kit was primarily expressed on subpopulations of CD4-CD8-CD3- triple negative (TN) cells. The strongest c-kit expression was associated with subsets that represent the least mature TN cells, including CD44+CD25- TN, and a subpopulation of CD25+ TN. These cells were also Thy-1lo, H-2Khi TSA-1hi, HSAlo, B220-, Mac-1-, and Gr-1-. Additionally, the recently described pre-TN thymocyte population (CD4loCD3-CD8-) was also c-kit+. CD25+ TN thymocytes proliferated in the presence of IL-7 and stem cell factor (the ligand for c-kit), and this proliferation was completely inhibited in the presence of anti-c-kit. Furthermore, the addition of anti-c-kit to 2-deoxyguanosine-treated fetal thymic lobes undergoing reconstitution with fetal liver-derived precursor cells inhibited their T cell differentiation potential. These observations indicate an important role for c-kit/stem cell factor interactions during early thymocyte development.     

P2X7 receptor-dependent cell death is modulated during murine T cell maturation and mediated by dual signaling pathways

Extracellular ATP causes apoptosis and/or necrosis of the hemopoietic lineage through the activation of P2X7 receptors. In this study, we investigated P2X7 receptor-mediated cell death during murine T cell maturation. The expression level and activity of P2X7 receptors, as measured by induction of cell death and pore formation, were higher in splenocytes than thymocytes. Flow cytometric analysis revealed that cell shrinkage was induced by activation of the P2X7 receptor in murine lymphocytes and the responding cells were T cells. Splenic T cells were more responsive than their thymic counterpart. These observations indicate that the system of P2X7 receptor-mediated cell death in T cells could be modulated during T cell maturation. Furthermore, decreased extracellular Cl- suppressed ATP-induced cell shrinkage in splenocytes without inhibiting ERK1/2 phosphorylation, which is reported to mediate necrotic cell death. Treatment with U0126 (a MEK inhibitor) suppressed ATP-induced ERK1/2 phosphorylation without inhibiting cell shrinkage. Moreover, decreased extracellular Cl- and treatment with U0126 suppressed ATP-induced cell death. These observations indicate that the activation of P2X7 receptor leads to T cell death by two independent pathways, one of which is cell shrinkage dependent and the other of which involves the phosphorylation of ERK1/2. In conclusion, we demonstrate increasing P2X7 receptor activity during T cell maturation and the existence of two essential pathways in P2X7 receptor-mediated T cell death. Our findings suggest that ATP-induced cell death of peripheral T lymphocytes is important in P2X7 receptor-regulated immune responses.     

IL-4 inhibits IL-2 receptor expression and IL-2-dependent proliferation of human T cells

Recent studies have shown that IL-4 can affect lymphocyte responses to IL-2. To evaluate the effects of IL-4 on T cell responses to physiologically relevant stimuli, we studied normal human T cells cultured with a low concentration of anti-CD3 mAb and IL-2 in the presence and absence of added IL-4. The addition of IL-4 to cultures of T cells stimulated with anti-CD3 mAb and IL-2 reduced the proliferative response by 49 to 59%. The inhibitory effect was observed in 3-, 5-, and 7-day cultures. Inhibition was dose-dependent with maximal inhibition at concentrations greater than or equal to 5 to 10 U/ml IL-4. IL-4-mediated inhibition occurred early during the T cell response, inasmuch as addition of IL-4 after stimulation for 24 h did not result in significant inhibition. Phenotypic analyses of cells cultured in the presence of anti-CD3 mAb, IL-2, and IL-4 suggested that the mechanism of regulation by IL-4 involves the inhibition of IL-2R expression. The proportion of both CD4+ and CD8+ cells that expressed IL-2R in response to IL-2 was diminished in the presence of IL-4, although HLA-DR levels were unaffected. Soluble IL-2R was also reduced in supernatants of cultures stimulated with anti-CD3 mAb, IL-2, and IL-4 as compared to cultures stimulated with anti-CD3 mAb and IL-2. These findings indicate that when normal human T cells are stimulated in vitro in a manner that approximates a physiologic interaction with Ag in vivo, rIL-4 provides a potent inhibitory signal to IL-2 responsive cells that is likely mediated by IL-4-induced inhibition of IL-2R expression.     

Impaired T cell functions during amphibian metamorphosis: IL-2 receptor expression and endogenous ligand production.

T cell functions are impaired during defined developmental stages of amphibian metamorphosis (Marx et al., 1987). Here we show, using a fluorescent anti-human IL-2 receptor antibody and flow cytometry, that during these stages, the splenocytes of Xenopus laevis, the South African clawed toad, have a progressively diminished capacity to express IL-2 receptors (IL-2R), after in vitro lectin stimulation. Preincubation with human rIL-2 specifically blocks binding of the anti-IL-2R antibody. Separation of an endogenous ligand bound to the IL-2R leads to a substantial increase in available epitope recognized by the anti-IL-2R antibody when pre- and postmetamorphic splenocytes are employed, but not when splenocytes of the prometamorphic stages are treated similarly. Thus, the cells from the prometamorphic stages are not producing significant quantities of the ligand. Finally, we demonstrate that human rIL-2 is not by itself mitogenic in the toad, but it can act as a co-stimulator of antigen-induced mitogenesis. Thus, an absence of an endogenous ligand (autologous IL-2?), coupled with a reduced capacity to express IL-2 receptors may be responsible for impaired T cell clonal expansion in metamorphosing Xenopus. Inhibition of T cell functions during this period is vital, since adult cells forming within the larval body bear surface proteins not found on larval cells (Flajnik et al., 1986).     

IFN-alpha and IL-12 induce IL-18 receptor gene expression in human NK and T cells

IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-alpha or IL-12 to induce IFN-gamma. Here we show that in human NK and T cells IFN-alpha and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-alpha enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-alpha or IL-12 enhanced IL-18-induced NF-kappaB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-alpha and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.     

Steroid sensitivity of thymocyte subpopulations during intrathymic differentiation. Effects of 17 beta-estradiol and dexamethasone on subsets expressing T cell antigen receptor or IL-2 receptor

We have studied the effects of the steroid hormones, 17 beta-estradiol and dexamethasone, on the relative proportion of thymocyte expression of CD4 (L3T4), CD8 (Ly-2), TCR and IL-2R, used to identify different stages of thymocyte differentiation. After short-term in vivo steroid treatment, a significant decrease in the number and proportion of the CD4+/CD8+, double positive subpopulation was observed in parallel with a proportional increase in the percentage of the CD4+/CD8- single positive, of the CD4-/CD8-, double negative and, to a lesser extent, of the CD8+/CD4- subsets. Either steroid treatment increased the proportion of cells expressing either the epsilon-chain of the CD3 complex and/or the beta-chain of the TCR (beta-TCR) (TCR+/CD3+) and the 55 kDa protein of the IL-2R (IL-2R+), related to the increase of CD4+ SP thymocytes and of DN cells, respectively. Furthermore, the increased proportion of CD3+ cells could also be partially related to the increase of both the CD4+ and DN subsets. A decrease of the effect on either DN/IL-2R+ cells or CD4+ SP cells was selectively observed after long-term treatments with 17 beta-estradiol or DEX, respectively. It is concluded that after short-term administration, different steroid hormones mediate a similar selective depletion of DP, TCR-/CD3-, IL2R- cells presumably in an intermediate stage of differentiation. However, either steroid effects evolve differently in long-term treatment schedules, resulting in different effects on early (DN/IL2R+) and late (SP/IL2R-) steps of thymocyte differentiation.     

Broad programming by IL-2 receptor signaling for extended growth to multiple cytokines and functional maturation of antigen-activated T cells

Coincident production of IL-2 and induction of high-affinity IL-2R upon TCR engagement has precluded a clear distinction for the biological outcome of signaling through TCR/costimulatory molecules vs the IL-2R. Using a novel transgenic mouse on the IL-2Rbeta(-/-) genetic background, this study has separated the relative outcome of signaling through the TCR and IL-2R. We show that stimulation through the TCR and CD28 or CD40 ligand directly leads to T cell activation and several rounds of proliferation in an IL-2-independent fashion. However, this stimulation is insufficient for extended T cell growth to multiple cytokines or differentiation into CTL or IFN-gamma-secreting effector T cells. IL-2 is required for these functions in part by regulation of cyclin D3 and granzyme B. Somewhat less efficiently, IL-4 stimulation of these transgenic T cells redundantly rescued many of these activities. These data demonstrate a fundamental requirement for IL-2 and perhaps other common gamma-chain-dependent cytokines to promote selective gene expression by Ag-activated T cells for their subsequent growth and differentiation into effector T lymphocytes.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

IL-2 receptor signaling through the Shb adapter protein in T and NK cells

We have investigated the effect of hypoxia on the excitatory synaptic transmission in the substantia gelatinosa neurons using perforated-patch-clamp configuration. Brief periods of hypoxia induced a depression in the evoked excitatory postsynaptic current (eEPSC) amplitude. The hypoxia-induced depression of eEPSC was not observed in the presence of theophylline, a nonselective adenosine receptor antagonist, and DPCPX, a selective adenosine receptor A1 antagonist. Application of adenosine (100 microM) also depressed eEPSC in a similar way as with hypoxia. This adenosine-induced depression of eEPSC was inhibited by DPCPX. Hypoxia and exogenous adenosine decreased the frequency of the spontaneous excitatory postsynaptic current (sEPSC) but not the amplitude of sEPSC and increased the paired-pulse ratio. From these results, it is suggested that acute hypoxia depresses the excitatory synaptic transmission by activating the presynaptic adenosine A1 receptor.     

Changes in gene expression induced by a phorbol diester: expression of IL 2 receptor, T3, and T cell antigen receptor

Phorbol esters cause an apparent differentiation of human T leukemic cell lines. It was shown previously that TPA induces the expression of the interleukin 2 (IL 2) receptor and the T3 complex on some T cell lines, including CCRF-CEM. We demonstrate that expression of the IL 2 receptor correlated with an induction of the 3.5 and 1.5 kb IL 2 receptor mRNA. In addition, the TPA-induced expression of the T3 polypeptides was found to be accompanied by induction of a putative T cell antigen receptor heterodimer on CEM cells. This was demonstrated by the co-precipitation of the T cell receptor with T3 from digitonin-solubilized cells. The cells expressed high levels of T3 delta- and T cell receptor beta-chain mRNA in the absence of TPA. The effect of TPA was to cause a rapid accumulation of T cell receptor alpha-chain mRNA. This suggested that the alpha-chain gene was rearranged before TPA induction and that expression of the T cell receptor/T3 complex on the cell surface was regulated by the level of alpha-chain expression. It was also shown that cloned sublines of CEM cells which expressed different T cell antigen phenotypes differed in their response to TPA.     

Specific signaling pathways triggered by IL-2 in human V gamma 9V delta 2 T cells: an amalgamation of NK and alpha beta T cell signaling

The global immune response can be simplified into two components: the innate and the acquired systems. The innate immune response comprises primarily macrophages and NK cells, while B and T cells orchestrate the acquired response. Human Vgamma9Vdelta2 T cells represent a minor T cell subpopulation in blood (1-5%) that is activated via the TCR by small nonpeptidic molecules. Their percentage dramatically increases during the early phase of infection by intracellular pathogens, and they display many characteristics of NK cells, which places them at a unique position within the immune system. Our aim was to explore the behavior of these cells when they are activated by a receptor that is common to NK and alphabeta T cells, and to determine signaling pathways and biological responses induced in these cells through this receptor. Thus, we investigated whether Vgamma9Vdelta2 T cells behave as NK cells or as alphabeta T cells. We demonstrated that IL-2 activates not only STAT3, STAT5, the phosphatidylinositol 3-kinase pathway, and extracellular signal-regulated kinase-2 pathway, but also STAT4 as in NK cells, and the p38 mitogen-activated protein kinase pathway as in alphabeta T cells. Moreover, IL-2 induces the production of IFN-gamma in Vgamma9Vdelta2 T cells as observed in NK cells. Due to their double profiles, Vgamma9Vdelta2 T cells are at the interface of the innate and the acquired immune response and may therefore not only modulate the activity of innate cells, but also influence Th1/Th2 differentiation.     

An essential role for the IL-7 receptor during intrathymic expansion of the positively selected neonatal T cell repertoire

Intrathymic T cell development is a multistage process involving discrete phases of proliferation as well as differentiation. From studies on IL-7 or IL-7Ralpha-deficient mice, it is clear that the IL-7 receptor (IL-7R) plays a critical role during the initial stages of intrathymic CD4-8- precursor development. In contrast, the role of IL-7R in later stages of thymocyte development are unclear. Here, we have used various approaches to investigate directly the role of the IL-7R in thymocyte positive selection and the recently described phase of postselection proliferation. First, we show that positive selection involves selective up-regulation of IL-7Ralpha- and IL-7Rgamma-chains, with the majority of CD4+ and CD8+ cells being IL-7R+. Second, MHC class II+ thymic epithelium-which drives postselection proliferation-expresses IL-7 mRNA. Finally, analysis of positive selection and postselection proliferation in thymocytes from IL-7Ralpha-/- neonates shows that positive selection occurs normally, whereas postselection expansion is drastically reduced. Thus, our data provide the first evidence that, as well as playing a role during early phases of thymic development, IL-7R mediates intrathymic expansion of positively selected thymocytes, which may aid in establishment of the neonatal peripheral T cell pool.     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.