Thymocyte Cell Line Research Articles

Cooperation between Mutant IL7R and TLX3 Generates Mixed Myeloid/Lymphoid Leukemia from Thymocyte Progenitors

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite current pediatric treatment protocols that have resulted in 5-year overall survival rates up to 90%, treatments have substantial side effects. Therefore, it is important to identify more specific therapeutic targets and develop more effective and less toxic drugs and drug combinations. We have shown that overexpression of TLX3 in association with mutant IL7R (mutIL7R) is sufficient to generate aggressive leukemia from transduced CD4-CD8- (DN) thymocyte progenitors. To better understand mutIL7R/TLX3-mouse leukemia, we combined single cell RNA sequencing (scRNAseq), Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), DNA Next Generation Sequencing (NGS) and flow cytometry to explore the cooperation of mutIL7R and TLX3 in orchestrating leukemogenesis. We found that mutIL-7R+TLX3 induced murine mixed T/myeloid leukemia. Myeloid cell clones (CD11b+) expressed identical TCR rearrangements with T cell clones (CD8+), indicating that mutant IL-7R+TLX3 generates mixed leukemia from T cell progenitors. Moreover, the passaged leukemia lost the T-cell phenotype and acquired a pro-B-like phenotype, suggesting preferential expansion of pro-B cells (CD19+, B220+ IgM-), which were not detected in the founder leukemia presumably due to its low frequency. We next sought to determine whether the oncogene combination is sufficient to drive a similar immunophenotype profile in vitro. We showed that ectopic expression of mutIL7R and TLX3 in DN primary thymocytes pushed the cells to myeloid (CD11b+, Gr1+) and B-cell (B220) phenotype over a time course of 4, 8 and 20 days, while T-cell development was blocked, suggesting that TLX3 expression prevented differentiation into CD4+/CD8+ T-cells. To further characterize the mixed T/myeloid leukemia at the molecular level, we compared the leukemia expressed genes to a TLX-bound genes public dataset. We found that 42 TLX3 target genes are expressed in mutIL7R+TLX3 leukemia. Interaction network analysis suggested significant Myc stimulation. We then confirmed Myc upregulation at the protein level in a thymocyte cell line, indicating that mutIL7R alone induces Myc but requires TLX-3 cooperation to induce maximal Myc expression levels. Taken together, we generated a high-resolution dataset with detailed information about the transcriptional landscape and immunophenotype of leukemia generated by mutIL-7R+TLX3, a combination that is observed in patient samples, and suggests new therapeutic drug strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

Abstract B47: Overexpression of TLX3 or HOXA9 in association mutant IL7Rα are sufficient to generate T-ALL in vivo

Abstract Background: Mutations of the IL7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Rα is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Rα alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present, contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Rα plus TLX3 or HOXA9 overexpression indicate in vitro grow advantage, suggesting these two genes as potential collaborative candidates. Furthermore, patients with mutated IL7Rα were more likely to have TLX3 or HOXA subgroup leukemia. Objective: We sought to determine whether combination of mutant hIL7Rα plus TLX3 or HOXA9 overexpression is sufficient to generate T-cell leukemia in vivo. Methods: Double-negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7Rα plus TLX3 or HOXA9. The same combinations were tested for the hIL7Rα wild-type. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 7-13 days and accessed for driver oncogenes expression and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Results: Thymocytes transduced with muthIL7Rα-TLX3 showed overexpression of c-Myc and Pim-1. Nonetheless, there was no difference in the protein level expression for these two proteins in either thymocytes transduced with muthIL-7Rα-HOXA9 or in the control wthIL7Rα-TLX3. Mice injected with either muthIL7Ra-TLX3 or muthIL7Rα-HOXA9 cells, but not the controls (wthIL7Rα-TLX3 or wthIL7Rα-HOXA9), developed leukemia approximately 14 days post injection, characterized by GFP-expressing T-cells in blood, spleen, liver, lymph nodes, and bone marrow. Conclusion: Thymocytes transduced with muthIL7Rα-TLX3 showed c-Myc and Pim-1 upregulation. Cells expressing the combination muthIL7Rα-TLX3 or muthIL7Rα-HOXA9 were sufficient to trigger T-cell leukemia in vivo. Citation Format: Gisele O.L. Rodrigues, Julie Hixon, Hila Winer, Wenqing Li, Scott Durum. Overexpression of TLX3 or HOXA9 in association mutant IL7Rα are sufficient to generate T-ALL in vivo [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B47.

The JAK Inhibitor Ruxolitinib Is Effective in Treating T Cell Acute Lymphoblastic Leukemia with Gain of Function Mutations in IL-7R Alpha

Acute lymphoblastic leukemia (ALL) results from transformation of immature B or T cells, and is the most common pediatric cancer. Though the current cure rate of ALL is 80-90%, it is important to understand the underlying biology of ALL in order to develop refined therapies for patients who fail to respond to conventional chemotherapy as well as to reduce its toxicity. The IL-7 receptor (IL-7R) signaling pathway is necessary for the proliferation and survival of T cells. Together with collaborators, we have shown that 9% of patients with T cell acute lymphoblastic leukemia (T-ALL) have gain of function mutations in IL-7R alpha. These mutations promote homodimerization of IL-7R alpha subunits, resulting in constitutive activation of this pathway via Janus Kinase 1 (JAK1). As the JAK-STAT pathway is downstream of the IL-7 receptor, we hypothesized that JAK inhibitors could be used to treat T-ALL patients with IL-7R mutations. To demonstrate this, we first established a cell line model of T-ALL driven by constitutive IL-7R signaling. The model cells were established by transforming the D1 thymocyte cell line with a mutated IL-7R alpha derived from a patient sequence. These cells termed "D1_hIL7R_P1" are also GFP+, which allows us to monitor the proliferation of the cells in vivo.I have showed that D1_hIL7R_P1 cells delivered intravenously result in an aggressive leukemia with morbidity within 18-21 days. Ruxolitinib, a JAK1 inhibitor, inhibits survival, proliferation, and STAT5 activation of D1_hIL7R_P1 cells in vitro. To treat this leukemia in vivo, I administered ruxolitinib for 5 days at a dose of 150 mg/kg to mice starting 8 days after D1_hIL7R_P1 engraftment. Tissues were then harvested for analysis of GFP+ cells as a measure of leukemic burden via flow cytometry. Ruxolitinib reduced leukemic cells from 15% in the blood (as demonstrated in untreated mice) to 5%, from 30% to 10% in the spleen, and 40% to 20% in the lungs. With these promising results, I have acquired T-ALL patient samples that can be xenografted into NSG mice. TALL#5 cell line successfully engrafts in 30 days and can be detected in the bone marrow, spleen, and peripheral blood by flow cytometry. Additionally, TALL#5 expresses human IL-7R alpha and is sensitive to ruxolitinib treatment in vitro. I have also identified two T-ALL cell lines, DND41 and KOPTK1, which express high levels of IL-7R alpha. These cell lines are also potential candidates for studying the effects of ruxolitinib on T-ALL in NSG mice. TALL#5, DND41, and KOPTK1 will be engrafted into mice, and after leukemia is established, mice will be treated with ruxolitinib. If successful, this will give us insight into the effectiveness of JAK inhibitors in treating ALL, and which patients can be recruited for future clinical trials. DisclosuresOff Label Use: Ruxolitinib is a JAK1/2 inhibitor that is FDA approved for myelofibrosis. My experiments involve pre-clinical studies looking at the efficacy of ruxolitinib in leukemic mice..

Abstract 2287: A model system to treat T-cell acute lymphoblastic leukemia with JAK inhibitors

Abstract The IL-7 Receptor signaling pathway is necessary for the proliferation and survival of T cells. Together with collaborators, we have shown that 9% of patients with T cell acute lymphoblastic leukemia (T-ALL) have gain of function mutations in IL-7R exon 6. These mutations promote homodimerization of IL-7Rα subunits, resulting in constitutive activation of this pathway via JAK1. Approximately 20% of T-ALL patients fail to respond to conventional chemotherapy, so there is a need for additional options for these patients. We hypothesized that JAK inhibitors could be used to treat T-ALL patients, particularly those with IL-7R mutations. We established a cell line model of T-ALL driven by constitutive IL-7R signaling which grew as a leukemia in RAG1-/- mice. The model cells were established by transforming the D1 thymocyte cell line with a mutated IL-7Rα derived from a patient sequence. These cells termed D1_hIL7R_P1 are also GFP+ which allows us to monitor the proliferation of the cells in vivo. D1_hIL7R_P1 cells delivered intravenously result in an aggressive leukemia with morbidity within 18-21 days. Currently we are studying the chemotherapeutic effects of two JAK inhibitors, Ruxolitinib and Tofacitinib on T-ALL. The maximum tolerated doses for Tofacitinib and Ruxolitinib are 50 mg/kg and 150 mg/kg, respectively. Ruxolitinib appears to be the most effective, showing a reduction of leukemogenesis detected in blood and tissues after just three days of oral treatment. Additionally, we have obtained samples derived from pediatric T-ALL patients. Once growth kinetics in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice is established, we will proceed to study the efficacy of the JAK inhibitors with human leukemia cells. Citation Format: Emilee Senkevitch, Julie Hixon, Wenqing Li, Scott Durum. A model system to treat T-cell acute lymphoblastic leukemia with JAK inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2287. doi:10.1158/1538-7445.AM2015-2287

Induction of S100A4, S100A6, and galectin-1 during the lineage commitment of CD4+CD8+ thymocyte cell line is suppressed by 2,3,7,8-tetrachlorodibenzo-p-dioxin

To study the mechanisms underlying the linage commitment of CD4+CD8+ thymocytes and the skewed differentiation of CD4+CD8+ into CD4-CD8+ thymocytes induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we stimulated with antigen DPK cells, a CD4+CD8+ thymic lymphoma cell line which can differentiate into CD4+CD8- thymocytes and performed a comparative proteomic analysis of DPK cells stimulated with antigen or not. Among the 10 up-regulated or induced proteins upon antigenic stimulation, S100A4, S100A6, and galectin-1 were highly up-regulated. Kinetic studies revealed that expression of S100A4, S100A6, and galectin-1 was dramatically increased as early as 10min after antigen stimulation, similar to that of cKrox and Runx3, transcription factors intimately associated with the lineage commitment. Among four thymocyte subpopulations of the thymus examined, S100A4, S1006, and galectin-1 were most prominently expressed in CD4+CD8+ thymocytes, but not at all in CD4-CD8+ and CD4-CD8- thymocytes. In the spleen, expression of S100A4, S1006, and galectin-1 was greater in CD4 than in CD8 splenocytes. When TCDD was added to antigen-stimulated DPK cells, antigen-induced up-regulation of S100A4, S1006, and galectin-1 were remarkably inhibited, probably partly accounting for the skewed differentiation of CD4+CD8+ into CD4-CD8+ thymocytes induced by TCDD.

The tumor suppressor Ikaros transcription factor is crucial for the anti‐proliferative Vasoactive Intestinal Peptide Receptor‐1 expression in developing thymocytes

The Leukemia and Lymphoma Society states more children die of leukemia yearly than any other cancer, therefore effective treatments are crucial. In the absence of the tumor suppressor Ikaros (IK), mice develop T cell leukemia with 100% penetrance. We demonstrated that IK regulates a gene termed vasoactive intestinal peptide receptor‐1 (VPACR‐1), when overexpressed in NIH‐3T3 cells. In T cells, VPACR‐1 suppresses interleukin‐2 and subsequent proliferation. Therefore, we hypothesize that IK tumor suppressor activities are manifested by maintaining high levels of VPACR‐1. Thus, an IK null, double negative stage 3 (DN3) thymocyte cell line, JE131, showed a significant reduction in VPACR‐1 compared to wild type (WT) thymic tissue. To obtain an accurate comparison to JE131 cells, thymic tissue was purified by AutoMACS and FACS for the DN3 subpopulation with a similar phenotype to JE131 cells (CD44Lo/CD25Hi/CD4−/CD8−). By qPCR, WT DN3s expressed VPACR‐1 ≤20% that of total DN cells, however this low level is ≥16X that of JE131 cells, and strongly supports IK as a positive potentiator for thymocyte VPACR‐1 expression. Future studies will reintroduce IK into JE131 cells by lentiviral transduction to rescue VPACR‐1 expression and support VPACR‐1 as an IK gene target. Another major goal is to map VPACR‐1 expression levels in all DN populations. Supported by NIH/NIDDK Career Award (KO1 5K01DK64828‐2) and COBRE at NDSU.

PKA dependent regulation of Foxo1 and downstream targets in murine S49 thymic lymphoma cells

Previous investigations revealed the presence of a norepinephrine (NE)/beta‐2‐adrenergic receptor/cAMP/PKA‐dependent mRNA decay system in T lymphocytes. The Fox family of transcription factors is involved in regulating lymphocyte physiology. Foxo1 is associated with the suppression of T cell proliferation and apoptosis. We explored whether treatment with PKA‐selective cAMP analogues (8Br‐cAMP or 8CPT‐cAMP), Forskolin, and NE could regulate BALB/c murine thymocyte cell lines, S49wt and S49kin‐ (PKA deficient), by affecting the genetic expression of Foxo1 and downstream targets Bim, Cyclin G2, and Fasl. Real‐time PCR studies revealed a 2–3 fold upregulation of Foxo1 mRNA in S49wt, but not kin‐cells with NE, 8Br‐cAMP or 8CPT‐cAMP 2 hrs after treatment. Foxo1 activation related to increase Bim, Cyclin G2, and Fasl mRNA expression in S49wt cells. Treatment with an Epac‐selective cAMP analogue had no effect. These results suggest that cAMP regulation of Foxo1 and downstream targets is acting via PKA, as opposed to the alternative Epac GEF pathway. These data imply PKA modulates increased Foxo1 mRNA expression and Fox gene‐mediated regulation of lymphocyte physiology.ted regulation of lymphocyte physiology. Supported by the NIH.

Dual Specificity Phosphatase (DUSP) activity regulates ERK activation and positive selection of thymocytes (87.16)

Abstract Regulation of MAP Kinase phosphorylation is thought to be important for proper translation of TCR signals into thymocyte cell fates. However, the role of phosphatase activity in thymic development has not been extensively characterized. DUSP-6, also known as Map Kinase Phosphatase-3 (MKP-3), is expressed in the thymus and can bind and dephosphorylate ERK. To explore the role of MKP in thymocytes, we expressed MKP-3 or a catalytically inactive form of MKP-3 (DM-MKP3) in an immature thymocyte cell line, 16610 D9. Lower amounts of phosphorylated ERK (p-ERK) were found in D9 cells that over express MKP-3, while D9 cells that over express DM-MKP3 exhibited higher amounts of p-ERK after TCR stimulation in comparison to vector control cells. Thus, over expression of a catalytically inactive ERK phosphatase results in inhibition of all ERK specific MKP activity, and acts as a dominant negative MKP. Utilizing real time PCR, we also observed elevated Egr-1 mRNA levels after TCR stimulation in D9 cells expressing DM-MKP3 compared to control D9 cells. Egr-1 is an ERK dependent immediate early gene known to support positive selection of thymocytes. Finally, we over expressed DM-MKP3 in vivo, and observed enhanced positive selection. Together these results indicate that MKP control of ERK activation after TCR stimulation is critical in regulation of thymocyte cell fate decisions. This work is funded by grants from the NIH and the ACS.

Norepinephrine mediated PKA-dependent regulation of Forkhead transcription factors in murine S49 thymoma cells (35.42)

Abstract Previous investigations have revealed the presence of a norepinephrine/β2-adrenergic receptor/cAMP/PKA-dependent mRNA decay system in T lymphocytes. The Fox family of transcription factors has been implicated in regulating lymphocyte physiology. Foxm1b is reported to be a master gene related to cellular proliferation and Foxo1 has been associated with the suppression of proliferation and promotion of apoptosis. Therefore, we explored whether a PKA-selective cAMP analogue (8Br-cAMP) and NE could regulate BALB/c murine thymocyte cell lines, S49wt and S49kin- (PKA deficient), by affecting the genetic expression of Foxm1b and Foxo1. Real-time PCR studies revealed a 2–3 fold upregulation of Foxo1 mRNA in S49wt, but not kin- cells with 150uM NE or 600uM cAMP as early as 2 hrs after treatment. Foxm1b mRNA was downregulated in S49wt cells by greater than 2 fold by 8Br-cAMP after 6 hrs (at 24 hrs). Treatment with an Epac-selective cAMP (500uM) analogue resulted in no change in Foxo1 or Foxm1b expression in S49 wt or S49 kin- cells. These results associate PKA in modulating both increased Foxo1 mRNA expression and decreased Foxm1b expression and consequent fox gene-mediated regulation of lymphocyte physiology. Supported by NIH.

Negative regulation of RORgt by direct binding to Egr3 (35.41)

Abstract The orphan hormone receptor RORgt is a transcription factor that is expressed in lymphoid tissue inducer cells, double positive thymocytes, and Th17 cells. Analysis of RORg-deficient mice has revealed that this factor is critical for normal function of all three of these cell types. It is therefore of interest to understand how RORgt function is regulated. We have found that RORgt function can be negatively regulated by Egr3, a zinc finger transcription factor that is induced by a variety of stimuli, including TCR signaling. Ectopic expression of both RORgt and Egr3 in a thymocyte cell line prevents RORgt from binding to target gene promoters, and blocks target gene expression. We have employed multiple strategies to demonstrate that the inhibition of RORgt function is due to direct binding of RORgt by Egr3. Immunoprecipitation of either Egr3 or RORgt brings down the other protein, even when both proteins are obtained by in vitro translation. A mammalian two-hybrid system also indicates that the two proteins physically interact. Mutational analysis of RORgt indicates that the domain responsible for steroid receptor coactivator recruitment is required for binding to Egr3. These studies demonstrate a novel function for Egr3, and suggest a mechanism for regulation of thymocyte and Th17 cell differentiation. This work is funded by a grant from the American Cancer Society.

Human Ikaros Function in Activated T Cells Is Regulated by Coordinated Expression of Its Largest Isoforms

The Ikaros gene is alternately spliced to generate multiple zinc finger proteins involved in gene regulation and chromatin remodeling. Whereas murine studies have provided important information regarding the role of Ikaros in the mouse, little is known of Ikaros function in human. We report functional analyses of the two largest human Ikaros (hIK) isoforms, hIK-VI and hIK-H, in T cells. Abundant expression of hIK-H, the largest described isoform, is restricted to human hematopoietic cells. We find that the DNA binding affinity of hIK-H differs from that of hIK-VI. Co-expression of hIk-H with hIk-VI alters the ability of Ikaros complexes to bind DNA motifs found in pericentromeric heterochromatin (PC-HC). In the nucleus, hIK-VI is localized solely in PC-HC, whereas the hIK-H protein exhibits dual centromeric and non-centromeric localization. Mutational analysis defined the amino acids responsible for the distinct DNA binding ability of hIK-H, as well as the sequence required for the specific subcellular localization of this isoform. In proliferating cells, the binding of hIK-H to the upstream regulatory region of known Ikaros target genes correlates with their positive regulation by Ikaros. Results suggest that expression of hIK-H protein restricts affinity of Ikaros protein complexes toward specific PC-HC repeats. We propose a model, whereby the binding of hIK-H-deficient Ikaros complexes to the regulatory sequence of target genes would recruit these genes to the restrictive pericentromeric compartment, resulting in their repression. The presence of hIK-H in the Ikaros complex would alter its affinity for PC-HC, leading to chromatin remodeling and activation of target genes.

Regulation of the Expression of Caspase-9 by the Transcription Factor Activator Protein-4 in Glucocorticoid-induced Apoptosis

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at -1.55 and -1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases -1140 and -944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Interleukin-7 Inactivates the Pro-apoptotic Protein Bad Promoting T Cell Survival

Interleukin-7 (IL-7) is a cytokine that is required for T cell development and survival. The anti-apoptotic function of IL-7 is partly through induction of Bcl-2 synthesis and cytosolic retention of Bax. Here we show that the Bcl-2 homology 3 domain-only protein, Bad, is involved in cell death following IL-7 withdrawal from D1 cells, an IL-7-dependent murine thymocyte cell line. IL-7 stimulation resulted in the inactivation of Bad by phosphorylation at Ser-112, -136, and -155. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated previously in Bad phosphorylation. In response to IL-7, the PI3K/Akt pathway induced phosphorylation at Ser-136 and -155, but Ser-112 was partly independent of the PI3K/Akt pathway, indicating an as yet unknown pathway in this response. Following IL-7 withdrawal, dephosphorylated Bad translocated from cytosol to mitochondria, bound to Bcl-2, and accelerated cell death. Thus, the inactivation of Bad contributes to the survival function of IL-7.

The novel murine CD4+CD8+ thymocyte cell line exhibits lineage commitment into both CD4+ and CD8+ T cells by altering the intensity and the duration of anti-CD3 stimulation in vitro.

A CD4(+)CD8(+) double-positive thymocyte cell line, 257-20-109 was established from BALB/c mice thymocytes and used to analyze the requirements to induce CD4 or CD8 single-positive (SP) T cells. CD4SP cells were induced from 257-20-109 cells by anti-CD3 stimulation in the presence of the FcR-positive macrophage cell line, P388D1. During stimulation, maturation events, such as the down-regulation of CD24 and the up-regulation of CD69, H-2D(d), CD5, and Bcl-2, were recognized. Furthermore, these CD4SP cells appeared to be functional because the cells produced IL-2 and IL-4 when activated with phorbol ester and calcium ionophore. In contrast, CD8SP cells could be induced by stimulation with fixed anti-CD3 after removal of stimulation. To investigate the extent of signals required for CD4SP and CD8SP, the cells stimulated under either condition for 2 days were sorted and transferred to different culture conditions. These results suggested that the fate of lineage commitment was determined within 2 days, and that CD4 lineage commitment required longer activation. Furthermore, the experiments with subclones of 257-20-109 demonstrated that the lower density of CD3 did not shift the cells from CD4SP to CD8SP, but only reduced the amount of CD4SP cells. In contrast, when the 257-20-109 cells were stimulated by the combination of fixed anti-CD3 and anti-CD28, the majority of the cells shifted to CD4SP, with an enhancement of extracellular signal-regulated kinase 1 phosphorylation. Our results indicate that the signals via TCR/CD3 alone shifted the double-positive cells to CD8SP cells, but the reinforced signals via TCR/CD3 and costimulator could commit the cells to CD4SP.

The mitochondrial phase of the glucocorticoid-induced apoptotic response in thymocytes comprises sequential activation of adenine nucleotide transporter (ANT)-independent and ANT-dependent events.

In thymocytes, dexamethasone initiates cytochrome c-dependent processing of caspase-9 and the activation of caspase-3 to trigger apoptotic damage. Using murine thymocytes or a thymocyte cell line WEHI 7.1, we show that this pathway is inhibited by dominant-negative caspase-9, the anti-apoptotic protein Bcl-2, or by blocking components of the mitochondrial permeability transition pore complex (PTPC). We use DIDS (dithiocyanatostilbene-2,2-disulfonic acid), a pharmacological modifier of VDAC (voltage-dependent anion channel) function or ectopic expression of hexokinase-II, to examine the role of the VDAC--a mitochondrial outer membrane protein--in this apoptotic pathway. This approach implicated the VDAC in dexamethasone-mediated cytochrome c release, processing of caspase-9 and caspase-3, the loss of mitochondrial transmembrane potential (Deltapsim), nuclear damage and cell lysis. Inhibiting the adenine nucleotide transporter (ANT), a protein on the mitochondrial inner membrane, also blocks dexamethasone-induced apoptosis, but the ANT regulates caspase-3 processing and nuclear damage but not the mitochondrial efflux of cytochrome c. Collectively, the data identify two separable, but connected events in dexamethasone-induced mitochondrial damage in thymocytes. The first event is an increase in permeability of the mitochondrial outer membrane leading to VDAC-regulated efflux of cytochrome c and initial processing of caspase-9 followed by ANT-dependent caspase-3 processing and apoptotic damage to cells.

IL-7 induces tyrosine phosphorylation of clathrin heavy chain

IL-7 induction of protein tyrosine phosphorylation was examined in an IL-7-dependent thymocyte cell line, D1, which was generated from a p53 −/− mouse. Anti-phosphotyrosine antibody was used both to immunoprecipitate and Western blot, and showed that IL-7 induced tyrosine phosphorylation of a protein with a molecular weight of approximately 200 kDa. The P200 band was purified by reversed-phase high-performance liquid chromatography. Amino acid sequencing by mass spectrometry revealed three peptides identical to rat clathrin heavy chain (CHC) 1 (192 kDa), and this was confirmed by blotting with an anti-clathrin antibody. Stimulation of normal pro-T cells by IL-7 showed an increased tyrosine phosphorylation of clathrin heavy chain. Tyrosine phosphorylation of clathrin heavy chain was strongly induced by IL-7 and to a lesser extent by IL-4, while no effect could be observed with the cytokines IL-2, IL-9 and IL-15, whose receptors share the γ c chain. Phosphorylation of clathrin heavy chain was found to be sensitive to Jak3 inhibitors but not to Src inhibitors. Clathrin is involved in internalization of many receptors, and its phosphorylation by IL-7 stimulation may affect the internalization of the IL-7 receptor.

A statin-based inhibitor of lymphocyte function antigen-1 protects against ischemia/reperfusion-induced leukocyte adhesion in the colon.

1. Statins are mainly used to control hypercholesterolemia; however, recent studies have also ascribed anti-inflammatory effects to the statins. LFA703 is a novel statin-derived compound, which potently inhibits lymphocyte function antigen-1 (LFA-1, CD11a/CD18) but does not affect HMG-CoA reductase activity. 2. The objective of this study was to examine the anti-inflammatory mechanisms of LFA703 in ischemia/reperfusion (I/R)-induced leukocyte-endothelium interactions in the colon. For this purpose, the superior mesenteric artery was occluded for 30 min and leukocyte responses were analyzed in colonic venules after 120 min of reperfusion in mice using inverted intravital fluorescence microscopy. 3. First, the inhibitory mechanisms of LFA703 on leukocyte adhesion were investigated in vitro using a mouse CD4+8+ thymocyte cell line. Immunoneutralization of LFA-1 and ICAM-1 abolished leukocyte adhesion, whereas inhibition of VLA-4 had no effect in this in vitro assay. Indeed, it was found that LFA703 dose-dependently reduced LFA-1-dependent leukocyte adhesion to mouse endothelial cells in vitro with an IC50 of 3.2 microm. 4. I/R caused an increase in leukocyte rolling and adhesion in colonic venules. Immunoneutralization of LFA-1 significantly reduced I/R-induced leukocyte adhesion by 89% in colonic venules. In contrast, I/R-provoked leukocyte rolling was insensitive to inhibition of LFA-1 function. 5. Administration of 30 mg kg-1 of LFA703 decreased reperfusion-induced leukocyte adhesion by more than 91%, while the level of leukocyte rolling was unchanged, suggesting that LFA703 effectively blocked LFA-1-dependent firm adhesion of leukocyte in the colon. However, LFA703 did not decrease the expression of LFA-1 on circulating leukocytes. 6. This study demonstrates that LFA-1 is indeed a critical adhesion molecule in mediating postischemic leukocyte adhesion in the colon. Moreover, this is the first study showing that a statin-based synthetic compound has the capacity to abolish LFA-1-dependent leukocyte adhesion in I/R. These novel findings may have great implications in the clinical treatment of conditions associated with I/R-induced tissue injury, such as organ transplantation, trauma and major surgery.

Expression profiling of a transformed thymocyte cell line undergoing maturation in vitro identifies multiple genes involved in positive selection

Biochemical and genetic studies of thymocyte maturation would be facilitated by the development of cultured cell lines that reflect stages of positive selection. We have derived a CD4 +CD8 +TCR + T-lymphoid cell line (M20) from a murine thymic tumor induced by a retrovirus carrying the v-myc oncogene (M-MuLV(myc)). M20 subclones undergo several aspects of positive selection in response to co-culture with a thymic stromal cell line (St3), including down-regulation of CD4 and CD8, and up-regulation of CD5 and TCR. M20 possesses a functional TCR complex, and ligation of this complex produces changes similar to co-culture with St3 stroma. Expression profiling of M20 cells in this system identified 23 genes previously shown to be important in thymocyte maturation, as well as several novel candidate genes. This system provides a new model to elucidate the molecular mechanisms of thymocyte maturation and TCR-mediated cell signaling in double-positive thymocytes.

Serial analysis of gene expression in murine fetal thymocyte cell lines.

FTL-1, -3 and -10 are three murine day 14 fetal thymocyte cell lines produced in order to model developmental stages within early (CD3-CD4-CD8-) thymocyte differentiation. In this study, we used the serial analysis of gene expression (SAGE) method to perform a systematic analysis of transcripts present in these three cell lines. A total of 77,313 SAGE tags were sequence identified from the three cell lines, representing 24,645 unique transcripts. Differentially expressed mRNA transcripts representing different gene classes were identified, including T cell functional genes, cytokine receptors, adhesion molecules and transcription factors. These results may serve as a model of the transcriptome of early thymocyte differentiation. A large number of unknown expressed sequence tags were also found to be differentially expressed. In order to validate the SAGE data, selected differentially expressed transcripts identified by SAGE were analyzed by quantitative RT-PCR in normal murine double-negative stage DN1-4 thymocytes. Expression of the transcription factors RUNX2 and PHD finger protein 2 and of the IGF type 1 receptor was shown to have differentially regulated expression patterns in sorted DN1-4 cells. These genes, and others identified by this analysis, are likely to play important roles in the development of T cells.