Figure 5.
TCL1A enhances TCR downstream signaling. (A) Enforced TCL1A expression in human HH mature T-leukemia cells enhances (earlier and higher levels) the phospho-activation of AKT (pAKTSer473) and pERK1/2Tyr202/204 upon CD3/CD28 crosslinking (TCL1A-dose relatedness by titrated doxycycline in this inducible iHH-TCL1A system). Surface CD3 and CD28 were expressed at similar degrees in both HH−/+ TCL1A sublines (supplemental Figure 8B). Bar charts indicate densitometric quantification of immunoblots. (B) Single-cell and time-resolved Ca2+ flux after TCR stimulation in Jurkat cells and their stable transfectants of TCL1A (fluo-4 loading). Stronger and more extended flux signals were detected in the presence of TCL1A, which was particularly noted for CD3/28 coligation, but also for single crosslinking of either TCR component. (C) Basal and TCR stimulation induced surface expression of the activation markers CD38 and CD69 on Jurkat and iHH cells is increased in the context of TCL1A overexpression (unpaired Student t test, SEM). D) Titration of TCL1A expression and TCR activation in iHH-TCL1A T cells and recording of IL-2 release (ELISA). Multi-level combinations: TCL1A (no, low, high doxycycline dosages) each with CD3 (low, 0.1 µg/mL; high, 1.0 µg/mL) or/and each with CD28 (low, 0.2 µg/mL; high, 2.0 µg/mL) crosslinking antibodies. In a sensitizer-like fashion, TCL1A enhanced IL-2 secretion (earlier reach of isoconcentrations) upon submaximal levels of anti-CD3 (also supplemental Figure 8E). Analyses used fitting kinetic models with a maximum likelihood routine. Observations at 24 hours are not shown for better visibility. Illustrations of each time point are shown in supplemental Figure 8E. (E) Differential gene expression in Hut78-TCL1A T cells (over Hut78-empty-vector controls) without or with TCR stimulation detected by RNAseq. Volcano plot resulting from the extraction of TCR-induced genes in the TCL1A condition (over empty-vector control) with cutoffs of absolute Log2 fold-changes ≥ 1.5 and FDR values < 0.01. GSEA (gene set enrichment analysis) of these genes (10 downregulated and 27 upregulated) shows, among others, a prominent enrichment for IL-2/STAT5 and IL-6/JAK/STAT3 pathway clusters. More details in supplemental Figure 8F and supplemental Table 8. (F) The presence of TCL1A enhances TCR-induced NFAT-coupled GFP expression in the A5 T-cell hybridoma reporter system (unpaired Student t test, SEM). (G) IL-2-dependent murine CTLL-2 cells and their transduction with TCL1A or a GFP control followed by treatment with IL-2. (i-ii) Induced levels of pERK1/2Thr202/Tyr204 and pAKTS473 (flow cytometry, 3 experiments) are higher in the TCL1A expressing subline (unpaired Student t test, SEM). (iii) CTLL-2 cells execute a higher growth response (total cell number) under stimulation with increasing IL-2 concentrations upon ectopic TCL1A expression. TCL1A did not confer IL-2 independence.

TCL1A enhances TCR downstream signaling. (A) Enforced TCL1A expression in human HH mature T-leukemia cells enhances (earlier and higher levels) the phospho-activation of AKT (pAKTSer473) and pERK1/2Tyr202/204 upon CD3/CD28 crosslinking (TCL1A-dose relatedness by titrated doxycycline in this inducible iHH-TCL1A system). Surface CD3 and CD28 were expressed at similar degrees in both HH−/+ TCL1A sublines (supplemental Figure 8B). Bar charts indicate densitometric quantification of immunoblots. (B) Single-cell and time-resolved Ca2+ flux after TCR stimulation in Jurkat cells and their stable transfectants of TCL1A (fluo-4 loading). Stronger and more extended flux signals were detected in the presence of TCL1A, which was particularly noted for CD3/28 coligation, but also for single crosslinking of either TCR component. (C) Basal and TCR stimulation induced surface expression of the activation markers CD38 and CD69 on Jurkat and iHH cells is increased in the context of TCL1A overexpression (unpaired Student t test, SEM). D) Titration of TCL1A expression and TCR activation in iHH-TCL1A T cells and recording of IL-2 release (ELISA). Multi-level combinations: TCL1A (no, low, high doxycycline dosages) each with CD3 (low, 0.1 µg/mL; high, 1.0 µg/mL) or/and each with CD28 (low, 0.2 µg/mL; high, 2.0 µg/mL) crosslinking antibodies. In a sensitizer-like fashion, TCL1A enhanced IL-2 secretion (earlier reach of isoconcentrations) upon submaximal levels of anti-CD3 (also supplemental Figure 8E). Analyses used fitting kinetic models with a maximum likelihood routine. Observations at 24 hours are not shown for better visibility. Illustrations of each time point are shown in supplemental Figure 8E. (E) Differential gene expression in Hut78-TCL1A T cells (over Hut78-empty-vector controls) without or with TCR stimulation detected by RNAseq. Volcano plot resulting from the extraction of TCR-induced genes in the TCL1A condition (over empty-vector control) with cutoffs of absolute Log2 fold-changes ≥ 1.5 and FDR values < 0.01. GSEA (gene set enrichment analysis) of these genes (10 downregulated and 27 upregulated) shows, among others, a prominent enrichment for IL-2/STAT5 and IL-6/JAK/STAT3 pathway clusters. More details in supplemental Figure 8F and supplemental Table 8. (F) The presence of TCL1A enhances TCR-induced NFAT-coupled GFP expression in the A5 T-cell hybridoma reporter system (unpaired Student t test, SEM). (G) IL-2-dependent murine CTLL-2 cells and their transduction with TCL1A or a GFP control followed by treatment with IL-2. (i-ii) Induced levels of pERK1/2Thr202/Tyr204 and pAKTS473 (flow cytometry, 3 experiments) are higher in the TCL1A expressing subline (unpaired Student t test, SEM). (iii) CTLL-2 cells execute a higher growth response (total cell number) under stimulation with increasing IL-2 concentrations upon ectopic TCL1A expression. TCL1A did not confer IL-2 independence.

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