Figure 2.
Inflammatory signals from activated T cells drive TBX21 expression in CLL cells via NF-κB. (A) Log2-transformed TBX21 gene expression in CLL cells (n = 5) cultured alone, after coculture with CD40L-expressing fibroblasts, or in vitro–activated autologous T cells. P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (B-C) Flow cytometric analysis of T-bet levels in CLL cells. (B) CLL peripheral blood mononuclear cells (PBMCs) (n = 7) or (C) purified CLL cells (n = 11) were stimulated with various cytokines and combinations thereof. The induction of T-bet expression was compared with that in the medium control. P values were obtained by 1-sample t tests and Wilcoxon signed-rank tests and by controlling the FDR using the BH method. (D) Flow cytometric analysis of T-bet levels in CLL cells of patients before ibrutinib treatment and after 3 and 6 months of ibrutinib treatment (n = 8). P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (E) TBX21 gene expression in CLL cells of patients before acalabrutinib treatment and after 1 and 6 months of acalabrutinib treatment (n = 20). P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (F-G) Flow cytometric analysis of T-bet levels in CLL cells after stimulation of CLL PBMCs with various cytokines and combinations thereof. (F) Cells (n = 7) were stimulated in the presence of vehicle control or ibrutinib. Quantification displays log2FC in comparison to the vehicle control. P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (G) Purified CLL cells (n = 8) were stimulated in the presence of the vehicle control or the NF-κB inhibitor IKK-16. Quantification displays log2FC in comparison with vehicle control. P values were obtained using the Friedman test and controlling the FDR using the BH method. (H) T-bet levels of 2 individual TCL1 CLL clones harboring hyperactive NF-κB signaling (Nfkbie–/–) compared with WT controls, as analyzed by flow cytometry (n = 3 technical replicates). ns, not significant. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Inflammatory signals from activated T cells drive TBX21 expression in CLL cells via NF-κB. (A) Log2-transformed TBX21 gene expression in CLL cells (n = 5) cultured alone, after coculture with CD40L-expressing fibroblasts, or in vitro–activated autologous T cells. P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (B-C) Flow cytometric analysis of T-bet levels in CLL cells. (B) CLL peripheral blood mononuclear cells (PBMCs) (n = 7) or (C) purified CLL cells (n = 11) were stimulated with various cytokines and combinations thereof. The induction of T-bet expression was compared with that in the medium control. P values were obtained by 1-sample t tests and Wilcoxon signed-rank tests and by controlling the FDR using the BH method. (D) Flow cytometric analysis of T-bet levels in CLL cells of patients before ibrutinib treatment and after 3 and 6 months of ibrutinib treatment (n = 8). P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (E) TBX21 gene expression in CLL cells of patients before acalabrutinib treatment and after 1 and 6 months of acalabrutinib treatment (n = 20). P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (F-G) Flow cytometric analysis of T-bet levels in CLL cells after stimulation of CLL PBMCs with various cytokines and combinations thereof. (F) Cells (n = 7) were stimulated in the presence of vehicle control or ibrutinib. Quantification displays log2FC in comparison to the vehicle control. P values were obtained by RM 1-way ANOVA and controlling the FDR using the BH method. (G) Purified CLL cells (n = 8) were stimulated in the presence of the vehicle control or the NF-κB inhibitor IKK-16. Quantification displays log2FC in comparison with vehicle control. P values were obtained using the Friedman test and controlling the FDR using the BH method. (H) T-bet levels of 2 individual TCL1 CLL clones harboring hyperactive NF-κB signaling (Nfkbie–/–) compared with WT controls, as analyzed by flow cytometry (n = 3 technical replicates). ns, not significant. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

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