Thymocyte Cell Line Research Articles

Crosstalk pathway for inhibition of glucocorticoid-induced apoptosis by T cell receptor signaling.

Activation of the glucocorticoid receptor (GR) triggers apoptosis in T cells. However, activation of the T cell antigen receptor (TCR) blocks glucocorticoid-induced apoptosis, implying functional crosstalk between these two distinct signaling systems. By reconstructing or selectively blocking TCR-stimulated signaling pathways, we show here that TCR activation of the mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK) cascade via Ras is necessary and sufficient to inhibit GR-mediated death in immortalized T and thymocyte cell lines and in primary T cells. Moreover, we found that activation of various pathway components (TCR, Ras, MEK1) altered the transcriptional regulatory activity of GR. In contrast, phosphatidylinositol 3-kinase and Akt, which down-regulate other lymphocyte apoptosis pathways, did not inhibit glucocorticoid-induced apoptosis. Our findings, which link signaling from the TCR cell surface receptor to that from the GR intracellular receptor, demonstrate the importance of the integration of signal transduction pathways in defining regulatory circuits. Because the TCR/Ras/MEK pathway has been shown previously to be essential for positive selection of thymocytes, the TCR/Ras/MEK signaling to GR crosstalk described herein may affect T cell development and homeostasis.

Thymocyte development past the CD4+CD8+stage requires an active p38 mitogen-activated protein kinase

Activation of the p38 mitogen-activated protein kinase (MAPK) pathway is important for some T-cell functions, but its role in intrathymic development is unclear. To investigate the function of p38 MAPK during the late stages of thymocyte differentiation, pharmacologic and genetic manipulations were used to inhibit p38 MAPK activity in developing thymocytes. Ligation of the T-cell antigen receptor (TCR) on either thymocytes or a thymocyte cell line resulted in p38 MAPK activation. Selective pharmacologic inhibition of p38 MAPK activity with the pyridinyl imidazole drug SB203580 severely impaired the development of mature CD4(+) and CD8(+) single positive (SP) thymocytes from their CD4(+)CD8(+) double positive (DP) precursors in fetal thymic organ culture (FTOC). Further, pharmacologic or genetic suppression of p38 MAPK activity, the latter achieved by overexpressing a catalytically inactive p38 MAPK, resulted in a blockade of the DP-to-SP transition of a thymocyte cell line in a novel in vitro differentiation assay. Taken together, these data constitute the first demonstration that p38 MAPK plays a critical role in the DP-to-SP differentiation of thymocytes during late intrathymic development. (Blood. 2000;95:1356-1361)

Cutting Edge: A Role for p21ras/MAP Kinase in TCR-Mediated Activation of LFA-1

Abstract LFA-1 is a β2 integrin that plays well-characterized roles in adhesion of T lymphocytes to APC, T cell-mediated cytolysis, and leukocyte-endothelial cell interactions. Although it is clear that LFA-1 must undergo affinity or avidity changes to bind its cellular ligand ICAM-1, the intracellular signaling pathways involved are not well characterized. Here, we show that the Ras-mitogen-activated protein kinase (MAPK) signaling pathway is also involved in TCR-activated LFA-1 adhesion. Expression of a dominant negative form of p21ras in a thymocyte cell line inhibits, while constitutively active p21ras both enhances and sustains, subsequent TCR-triggered adhesion to isolated ICAM-1. However, the Ras/MAPK pathway alone is not sufficient for activating T cell LFA-1, as inhibition of both downstream MAPK/extracellular regulated kinase kinase (MEK) activity and phosphatidylinositol 3-kinase activity is required for complete inhibition of adhesion.

A role for Ras signaling in coreceptor regulation during differentiation of a double-positive thymocyte cell line.

Evidence suggests that the Ras/mitogen-activated protein kinase signaling pathway is required for positive selection of thymocytes. We have asked whether Ras activation is also sufficient to mediate changes in gene expression that are associated with positive selection. To accomplish this, we expressed a constitutively active form of Ras in the immature CD4+ 8+ DPK thymocyte cell line. DPK cells that express active Ras have reduced levels of CD8alpha and CD8beta at the level of cell surface protein and mRNA. These data provide evidence of a direct link between Ras signaling pathways and coreceptor regulation during positive selection. They also suggest that a sustained or potent Ras signal may play a critical role in directing thymocytes into the CD4 lineage. DPK cells that express active Ras, however, were not fully differentiated, indicating that Ras signaling provides only a partial signal for double-positive thymocyte differentiation.

Expression of bcl-x in T cells

The thymus is a major site of apoptosis where programmed cell death is involved in the deletion of self-reactive T cells. We have investigated the role of bcl-x in T cells by defining the expression of its two isoforms, bcl-xl and bcl-xs, in a series of human thymocyte cell lines and in human T lymphocytes using the ribonuclease protection assay. Bcl-xl was the predominant isoform expressed in T cell lines and in T lymphocytes, where expression was further enhanced by PMA/ionomycin. This broad expression supports a central role for bcl-x in thymocyte development perhaps through post-transcriptional regulation.

Development and Characterization of v-myc/v-raf-Transformed Murine Fetal Thymocyte Cell Lines

Transformed murine fetal thymocyte cell lines were derived by incubating fetal thymic organ cultures with a v-myc/v-raf-containing retroviral construct in order to model developmental stages within the early triple negative (CD3−CD4−CD8−) thymocyte population. The resulting 10 cell lines had a lymphoid morphology, were all CD44+, CD90+, and were triple negative by surface antigen analysis. The cell lines, however, were distinguishable by differences in the expression of T cell-associated and T cell-specific genes. The CD3 genes were observed to be discoordinately expressed, in that CD3γ chain gene expression was detected in 2 cell lines in the absence of CD3δ and ϵ expression. Expression of the CD3γ chain gene was observed in cell lines without the expression of other T cell-specific genes or T cell receptor rearrangement and may be one of the earliest T cell-specific genes to be expressed. The transcription factor Ikaros was expressed in all 10 cell lines, whereas the transcription factor TCF1α was expressed only in the 2 most differentiated lines. In 8 cell lines, expression of partial TCR β and/or TCRα transcripts was observed by Northern blot. In several lines, expression of rearranged TCRα transcripts in the absence of TCRβ transcripts was demonstrated; however, TCRβ DJ rearrangements were observed by Southern blot in all but 1 of these cell lines. Thus, these cell lines, ordered based on the general pattern of additive gene expression observed, may reflect various stages of triple-negative thymocyte differentiation and provide anin vitromechanism to elucidate some of the molecular events involved in early thymocyte development.

Phosphatidylinositol 4,5‐bisphosphate hydrolysis accompanies T cell receptor‐induced apoptosis of murine thymocytes within the thymus

Regulation of the development of thymocytes into mature T cells within the thymus is now known to involve antigen-induced deletion, by apoptosis, of potentially autoreactive thymocytes, and it can be mimicked either by stimulating the T cell receptor (TcR) complex by monoclonal antibody (mAb) or by ionophore-induced elevation of cytosolic [Ca2+]. To identify signaling pathways employed by the TcR complex of immature thymocytes, we examined the effects of anti-CD3 and anti-TcR beta constant (c) region mAb, staphylococcal enterotoxin B (SEB) and pharmacological agents on the generation of inositol phosphates through hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] both in cultured fetal mouse thymic lobes and in the CD4+CD8+ immature thymocyte cell line, TM10G. Stimulation of the TcR complex with anti-CD3 mAb provoked an accumulation of inositol phosphates diagnostic of the occurrence of receptor-stimulated phosphoinositidase C (PLC) activation. Anti-TcRC beta mAb and SEB provoked smaller but similar responses. The PLC activation evoked by anti-CD3 mAb was suppressed by inhibitors of receptor tyrosine kinases and was unmodified by protein kinase C activation or elevation of cytosolic [Ca2+]. It thus appears that apoptosis triggered by TcR stimulation is associated with PLC activation by a receptor-regulated tyrosine kinase. Treatment of thymic lobes or TM10G cells with fluoroaluminate provoked apoptosis of a wider range of thymocyte subtypes and such stimulation also provoked an accumulation of inositol phosphates. The responses to fluoroaluminate were not prevented by inhibitors of tyrosine kinases, suggesting that unidentified GTP-binding proteins which couple to PLC activation may also be capable of initiating apoptosis by a route independent of the TcR. These results, when considered alongside previous studies of mature T cells, indicate that stimulation of immature thymocytes or of mature T cells through their TcR complex activates the PLC-catalyzed PtdIns(4,5)P2 hydrolysis signaling pathway, and thus that this signaling pathway may be implicated both in provoking apoptosis in immature T cells and in initiating proliferation in mature T cells.

Isolation of differentially expressed sequence tags from steroid-responsive cells using mRNA differential display

Transcriptional control of steroid-regulated gene networks by nuclear receptor proteins results in the coordinate expression of a limited number of target genes. Although much is known about the structure and function of steroid receptors, relatively few cell-specific steroid-regulated genes have been isolated and characterized. In this paper we describe results using mRNA differential display reverse transcriptase PCR (DDPCR) to identify and isolate short cDNA sequence tags from thymocyte and prostate cells under various hormone conditions. Using this technique we have isolated several differentially expressed sequence tags (DESTs) from the mouse thymocyte cell line WEHI 7.2. Two of these DESTs, GIG10 and GIG18, are rapidly induced by dexamethasone within 2 h of treatment. GIG10 is a novel sequence and GIG18 is the mouse homologue of a human expressed sequence tag isolated from activated B lymphocytes. We also used DDPCR to isolate DESTs from androgen-modulated rat ventral prostate tissue, one of which we characterized and found to correspond to the 3′ end of prostatic spermine binding protein mRNA, a known androgen-regulated gene. Modifications of the original DDPCR protocol, which we found can potentially decrease the frequency of isolating false-positive DESTs, are described and the merits of DDPCR, relative to other differential cDNA cloning strategies, are discussed.

The programmed cell death of an immature thymocyte cell line transgenic for an alpha beta TCR and the c-myc proto-oncogene.

The c-myc proto-oncogene linked to the mouse Thy-1 gene transcriptional unit predisposes mice to development of thymic tumors consisting predominantly of immature CD4+ CD8+ cells. In an attempt to immortalize immature T cells expressing a known T-cell antigen receptor (TCR), Thy-1/c-myc transgenic mice were bred to αβ TCR transgenic mice (F5), and CD4+ CD8+ cell lines were established from thymic tumors in double-transgenic mice. These cells expressed high-level heat-stable antigen (HSA) and were able to undergo programmed cell death upon induction with steroids and CD3 cross-linking, but not with cognate peptide. In addition, one line had rearranged and transcribed endogenous TCR c and genes, in spite of the fact that transgenic α and β genes were also expressed. Furthermore, we show that Thy-1/myc transgenic mice deficient in recombination activating gene-1 (RAG-1) do not develop tumors, in contrast to RAG-1-/- mice, which are also transgenic for both Thy-1/myc and the F5 TCR. This indicates that in order for thymocytes to be transformed by the Thy-myc transgene, they need to proceed to the double-positive stage.

Recessive mutations in a common pathway block thymocyte apoptosis induced by multiple signals.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls genes necessary to initiate glucocorticoid-induced thymocyte apoptosis. We have performed a genetic analysis of thymocyte cell death by isolating and characterizing a panel of GR+ dexamethasone-resistant mutants of the murine WEHI7.2 thymocyte cell line. These apoptosis-defective (Apt-) mutants were used to identify previously unknown early steps in the apoptotic pathway. The Apt- mutants contain nonglucocorticoid receptor, recessive mutations in genes that represent multiple complementation groups. These mutations block apoptosis induced by dexamethasone, gamma irradiation, and c-AMP treatment before the point where Bcl-2 exerts its protective effect. We propose that different signals share a common apoptotic pathway, and that the induction of apoptosis involves multiple precommitment steps that can be blocked by recessive mutations.

Transition from TCR-beta dimer to TCR-alpha beta-expressing cells by introduction of an alpha-chain in an immature thymocyte cell line.

The molecular basis for the surface expression of TCR-beta chain in the absence of association with TCR-alpha, -gamma, or -delta chain by an immature thymocyte cell line was investigated. The TCR-beta chain expressed by this cell line was not encoded by any unique DNA sequence, nor was it inserted into the membrane via a glycosyl-phosphatidylinositol linkage. Transfection of two other beta-chains derived from mature T cell clones resulted in the surface expression of dimers of the transfected beta-chains in both cases. Immunoprecipitation of the beta-dimer-CD3 complex demonstrated that the association of the beta-dimer with the CD3 complex, especially the CD3 zeta chain, was so weak that they dissociated under the detergent conditions in which the TCR-CD3 complex of mature T cells is kept intact. Transfection of TCR-alpha chain resulted in the expression of a TCR-alpha beta-CD3 complex and the disappearance of beta-dimers. In accordance with the changes in TCR complex components, the association between TCR-alpha beta and CD3 complex became stable and the cells transduced signals more efficiently. The results demonstrate that the expression of TCR-beta as part of an incomplete TCR-CD3 complex is developmentally regulated and the expression of TCR-alpha chain results in normal configuration and function of TCR complex.

Glycophorin A interacts with interleukin-2 and inhibits interleukin-2-dependent T-lymphocyte proliferation

Sialoglycolipids shed by tumor cells have been implicated in tumor-induced inhibition of T-lymphocyte responses to interleukin-2 (IL-2). In the present study, we have used glycophorin A, the major sialoglycoprotein of the human erythrocyte membrane, to investigate whether shedding of glycoproteins might also contribute to immunosuppression. Glycophorin A inhibited IL-2-stimulated proliferation of the IL-2-dependent cell lines HT-2 and CTLL-2 in a dose-dependent manner. Time course studies on synchronized cell populations indicated that the glycoprotein acted early in the activation process. On the other hand, glycophorin A had essentially no effect on IL-1-mediated stimulation of the IL-1-sensitive thymocyte cell line EL-4 NOB-1. Gel nitration FPLC demonstrated that IL-2 was able to bind to glycophorin aggregates under physiological conditions. Reconstituted vesicles containing glycophorin were also shown to bind IL-2. In addition, both soluble glycophorin aggregates and lipid vesicles containing glycophorin blocked binding of IL-2 to high-affinity cellular IL-2 receptors. Taken together, these results suggest that shedding of tumor sialoglycoproteins with oligosaccharide chains similar to glycophorin A might contribute to negative modulation of IL-2-mediated immune responses.

Introduction of the human interleukin-6 (IL-6) receptor in murine IL-3- dependent hematopoietic cells restores responsiveness to IL-6

To create experimental systems that facilitate studies aimed at the responsiveness of hematopoietic progenitors to interleukin (IL)-6 in combination with IL-3, we introduced the human IL-6 receptor (hIL-6R) into the IL-3-dependent cell line 32D. For this purpose, a retroviral vector containing the hIL-6R cDNA was constructed. 32D parental cells did not respond to IL-6, neither alone nor in combination with increasing concentrations of IL-3, and did not express detectable numbers of IL-6R as determined by 125I-IL-6 binding. 32D/hIL-6R cells expressed high-affinity IL-6 binding and responded synergistically to IL-6 in combination with suboptimal amounts of IL-3 in DNA synthesis assays. In addition, IL-6 promoted the short-term survival of IL-3- responsive clonogenic 32D/hIL-6R cells. On the other hand, although introduction of hIL-6R resulted in the formation of high-affinity IL-6 receptor structures in the IL-2-dependent thymocyte cell line CTLL, CTLL/hIL-6R cells did not respond to IL-6 in synergy with IL-2. We conclude that 32D cells possess the intracellular machinery permissive for IL-6 signal transduction. Murine IL-3-dependent cell lines with ectopic IL-6 receptors can serve as models for dissecting the molecular basis of IL-6 responses in primitive hematopoietic cells.

Introduction of the Human Interleukin-6 (IL-6) Receptor in Murine IL-3-Dependent Hematopoietic Cells Restores Responsiveness to IL-6

To create experimental systems that facilitate studies aimed at the responsiveness of hematopoietic progenitors to interleukin (IL)-6 in combination with IL-3, we introduced the human IL-6 receptor (hIL-6R) into the IL-3-dependent cell line 32D. For this purpose, a retroviral vector containing the hIL-6R cDNA was constructed. 32D parental cells did not respond to IL-6, neither alone nor in combination with increasing concentrations of IL-3, and did not express detectable numbers of IL-6R as determined by 125I-IL-6 binding. 32D/hIL-6R cells expressed high-affinity IL-6 binding and responded synergistically to IL-6 in combination with suboptimal amounts of IL-3 in DNA synthesis assays. In addition, IL-6 promoted the short-term survival of IL-3-responsive clonogenic 32D/hIL-6R cells. On the other hand, although introduction of hIL-6R resulted in the formation of high-affinity IL-6 receptor structures in the IL-2-dependent thymocyte cell line CTLL, CTLL/hIL-6R cells did not respond to IL-6 in synergy with IL-2. We conclude that 32D cells possess the intracellular machinery permissive for IL-6 signal transduction. Murine IL-3-dependent cell lines with ectopic IL-6 receptors can serve as models for dissecting the molecular basis of IL-6 responses in primitive hematopoietic cells.

Persistent productive HIV-1 infection of a CD4- human fetal thymocyte line.

Human fetal thymuses were obtained from abortuses of HIV-1 seronegative females. Thymocytes were isolated and cultured for 2 days with PHA. Thereafter, the culture was divided and half of the cells were exposed to the HIV-1 RF isolate for 4 h. After this incubation period, the HIV-1 exposed and nonexposed control cells were cultured in RPMI 1640 supplemented with IL-2 for 30 days and subsequently maintained in RPMI without the addition of growth factors. Long term culture of both HIV-1 exposed and control thymocytes has yielded two cell lines that have been maintained for more than 3 yr without the addition of growth factors. Flow cytometry using mAb that recognize T cell differentiation markers was used to analyze cell phenotypes. The HIV-1 exposed thymocyte cell line (E88/RF) was shown to be HIV-1 infected by p24 ELISA, reverse transcriptase activity, immunocytochemistry, in situ hybridization, polymerase chain reaction, electron microscopy, and to produce infectious particles by a syncytial forming assay. The non-HIV-1-exposed thymocyte cell line (T412) has remained negative by all criteria for HIV-1 infection. Flow cytometry showed the T412 cells to be positive for the T cell markers CD45, CD38, and CD4 but negative for all other markers tested. The E88/RF cells are positive for CD45 and CD38 but negative for CD4 and other markers. These data report the isolation of two human fetal thymocyte cell lines; one uninfected and susceptible to HIV-1 infection, and the other persistently and productively infected with HIV-1 with little cytopathology. These findings suggest that HIV-1 can persistently infect early T cells and may alter T cell differentiation.

The alternative splicing of the CD45 tyrosine phosphatase is controlled by negative regulatory trans-acting splicing factors.

CD45, a receptor-type protein tyrosine phosphatase involved in lymphocyte activation, consists of five isoforms generated by alternative mRNA splicing. The pattern of CD45 splicing depends upon cell type, state of activation, and thymic development. We previously showed that mouse B and thymocyte cell lines transfected with a human CD45 minigene construct appropriately splice the alternative exons, demonstrating the presence of trans-acting tissue-specific splicing factors. To study the regulation of CD45 alternative splicing, cells having different splicing patterns were transiently fused, and mRNA was analyzed using reverse transcription-polymerase chain reaction. Human B cells, normally producing only the largest CD45 mRNAs, could splice out the alternative exons after fusion with mouse thymocytes. In contrast, the splicing pattern of human T cell lines (smaller CD45 isoforms) was unaltered by fusion with mouse B cells. This suggests that cells expressing the smallest CD45 isoform contain negatively acting trans-factor(s) that allow the alternative exons to be skipped, and that the full length isoform containing all three alternative exons is the default pattern of splicing. In agreement, incubation of thymocytes or T cell subsets with cycloheximide increased mRNA corresponding to the larger isoforms. CD45 isoform expression can now be interpreted in terms of the presence or absence of negative regulatory trans-acting splicing factors.

Human thymic epithelial cells in serum-free culture: nature and effects on thymocyte cell lines.

Thymic epithelial cells (TEC) have been cultured for several months and/or for 4 to 5 transfers in a growth factor-defined serum-free medium without concurrent growth of other cell types. The use of monoclonal antibodies and αMAM-6 indicated that the majority of TEC were of medullary origin. The vast majority of cells were positive for LFA-3 and class I, and class II expression, was low or absent. Supernatants from the cultures were shown to contain IL-1ß, IL-6, and M-CSF. Coculture of cloned subpopulations of thymocytes and TEC showed effects of TEC and of secreted ILs on thymocyte proliferation. High percentages of TEC were able to bind DN, DP, or SP thymocyte populations, partly via CD2-LFA-3 adhesion. Thus, it is possible to culture TEC without unknown serum factors and with maintenance of functional activities.

CD4 and CD8 are positive regulators of T cell receptor signal transduction in early T cell differentiation.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.

The identification of the β2‐microglobulin binding antigen encoded by the human CDID gene

Human cluster of differentiation (CD1) is a family of cell surface glycoproteins composed of a 43-49-kDa heavy chain non-covalently associated with beta 2-microglobulin. Five human CD1 genes have been detected and cloned. Three genes (CD1A, -B and -C) encode the serologically defined CD1a, -b and -c antigens. Thus two genes remain, CD1D and CD1E, whose protein products have not been characterized so far. This report describes how a beta-galactosidase-CD1D fusion protein was used to raise specific antisera and a monoclonal antibody against the CD1D gene product. The monoclonal antibody defines a cell surface molecule expressed on a cortical thymocyte cell line and is composed of a 49-kDa heavy chain associated with beta 2-microglobulin, which is serologically distinct from CD1a.

A murine thymocyte clone expressing γδ T cell receptor mediates natural killer-like cytolytic function and TH1-like lymphokine production

The murine CD4 − CD8 − (double negative, DN) thymocyte cell line and clones expressing T cell receptor γδ chains in association with CD3 complex have been established and characterized. This line and a representative clone (DN7.12.11) which appear to derive from the minor population of CD3 + DN thymocytes can be stimulated to proliferate and to produce lymphokines by antiCD3 or anti-Thy-1 antibodies or calcium ionophore plus phorbol ester. Autocrine proliferation is dependent on binding of interleukin (IL)2 to functional IL2 receptor. Upon stimulation, these cells produce IL2 and IFN-γ but not IL4, resembling conventional CD4 + TH1 cells in this regard. The cloned line also mediates spontaneous cytolysis against a variety of tumor targets without regard for the presence of conventional major histocompatibility complex molecules on the target cell surface. Blocking and modulation experiments suggest that target recognition by the γδ/CD3 complex is not involved in the spontaneous lysis, resembling natural killer (NK) cells. The results suggest that γδ +DN T cells are able to have mature functions such as NK-like cytotoxicity and lymphokine secretion as peripheral γδ + T cells. They also provide a possible role of γδ + DN thymocytes in establishing a intrathymic environment for differentiation and selection of αβ-expressing T cells.