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Fig. 3 | Journal for ImmunoTherapy of Cancer

Fig. 3

From: BTLA blockade enhances Cancer therapy by inhibiting IL-6/IL-10-induced CD19high B lymphocytes

Fig. 3

IL-6 and IL-10 could induce more BTLA+CD19hi B lymphocytes through the AKT and STAT3 signaling pathways. a Representative figures of flow cytometric analyses of the expression of BTLA on various kinds of immunocytes of splenocytes. Note: A1: T lymphocytes; A2: NK cells; A3: B lymphocytes; A4: subgroups of B lymphocytes (zone 1: BTLACD19hi; zone 2: BTLA+CD19hi; zone 3: BTLA+CD19low(lo); zone 4: BTLA+CD19lo). B lymphocytes, especially CD19hi B lymphocytes, had higher percentages expressing the BTLA molecule. (5 mice in this analysis) b Kinetic alterations in BTLA+CD19hi B lymphocytes in splenocytes of tumor-bearing mice after different days of tumor challenge. b1 Representative flow cytometric figures of percentages of BTLA+CD19hi B lymphocytes in splenocytes on indicated days. (5 mice in each group) b2 Bar figures exhibited the percentages of BTLA+CD19hi B lymphocytes in splenocytes on day 14 or day 35 after tumor challenge. The percentages of BTLA+CD19hi B lymphocytes were higher on day 35 (17.74 ± 0.71%) than on day 14 (11.76 ± 0.52%) (p = 0.009, Kruskal-Wallis test). (5 mice in each group) c Kinetic alterations in BTLA+CD19hi B lymphocytes in TACs of ascites from tumor-bearing mice after different days of tumor challenge. c1 Representative flow cytometric figures of BTLA+CD19hi B lymphocytes in TACs at indicated intervals. (5 mice in each group) c2 Bar figures of the percentages of BTLA+CD19hi B lymphocytes in TACs on day 14 or day 35 after tumor challenge. The percentages of BTLA+CD19hi B lymphocytes were higher on day 35 (48.94 ± 0.92%) than on day 14 (19.34 ± 0.88%) (p = 0.007, Kruskal-Wallis test). (5 mice in each group) d Alterations in the percentages of BTLA+CD19hi B lymphocytes in sorted B lymphocytes treated with IL-6, IL-10, or TGF-β, analyzed by flow cytometry. d1 Representative flow cytometric figures of the percentages of BTLA+CD19hi B lymphocytes in sorted B cells. (5 mice in each group) d2 Bar figures of the percentages of BTLA+CD19hi B lymphocytes in sorted B cells treated with respective cytokines. The percentages of BTLA+CD19hi B lymphocytes increased under treatment with IL-6 or IL-10 compared with TGF-β (p = 0.033, Kruskal-Wallis test). (5 mice in each group) e Various signaling molecules of sorted B lymphocytes treated with IL-6 and IL-10, detected by western blotting and flow cytometric analyses. e1 IL-6 (10 or 20 ng/mL) could stimulate phosphorylation of STAT3 and AKT in sorted B lymphocytes. (5 mice in each group) e2 Phosphorylation of STAT3 and AKT in sorted B cells also could be promoted by IL-10 (10 or 20 ng/mL). (5 mice in each group) e3 The inhibition of p-AKT by LY294002 showed inhibition of p-STAT3 (Lanes 3 and 9). However, the inhibition of p-STAT3 by BP-1-102 did not block activation of p-AKT (Lanes 4 and 10). Therefore, AKT activation was upstream of STAT3 in the IL-6/IL-10 signaling pathway. (5 mice in each group) e4 Percentages of BTLA+CD19hi B lymphocytes in sorted B lymphocytes pretreated with respective Ab or specific inhibitor and then incubated with respective cytokine, analyzed by flow cytometry. The percentages of BTLA+CD19hi B lymphocytes decreased when the B lymphocytes were pretreated with anti-BTLA Ab, LY294002 (AKT inhibitor), or BP-1-102 (STAT3 inhibitor) compared with PD98059 (ERK inhibitor). (5 mice in each group) f Anti-tumor effects of chemotherapy combined with various BTLA-related inhibitors. (F1) Diagrammatic representation of different treatment protocols using paclitaxel and various BTLA inhibitors. Note: Ga: paclitaxel 6 mg/kg; Gb: paclitaxel 6 mg/kg and LY294002 800 μg/mouse; Gc: paclitaxel 6 mg/kg and BP-1-102 40 μg/mouse; Gd: paclitaxel 6 mg/kg and anti-BTLA Ab 20 μg/mouse. (F2) Representative luminescence images of mice in various groups using the IVIS system on day 35 after tumor challenge. (5 mice in each group) (F3) Luminal analyses of tumor volumes in tumor-bearing mice with various regimens. Mice treated with paclitaxel and various BTLA-related inhibitors exhibited less luminescence than the paclitaxel-treated group (p < 0.001, Kruskal-Wallis test). Among mice receiving paclitaxel and various BTLA-related inhibitors, those receiving paclitaxel and anti-BTLA Ab 20 μg/mouse showed the least luciferase activity (p = 0.002, Kruskal-Wallis test). (5 mice in each group) (F4) Survival analysis of mice in various groups. Animals treated with chemotherapy and respective BTLA-related inhibitor lived longer than those treated only with paclitaxel (p < 0.001, log-rank test). All mice treated with paclitaxel and anti-BTLA Ab 20 μg/mouse, 80% of mice treated with paclitaxel and BP-1-102 40 μg/mouse, and 40% of animals treated with paclitaxel and LY294002 800 μg/mouse were alive 100 days after WF-3/Luc tumor challenge. (5 mice in each group) g Schematic diagram showing possible regulation and preclinical application of BTLA

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