Short-Chain Fatty Acids Promote Immunotherapy by Modulating Immune Regulatory Property in B Cells

Figure 5

PA induces TTP degradation through the AKT/T-bet pathway. (a) Naive human B cells were treated with CpG and PA in the culture overnight. Protein extracts of B cells were precipitated with an anti-TTP Ab. The IP products were analyzed by mass spectrometry (MS). The amino acid sequences are two representative peptides of GZMB amino acid sequence identified by MS. (b) Immunoblots show a complex of TTP and GZMB in IP products. (c–f) Naïve B cells were exposed to PA in the culture overnight. (c, d) Violin plots show GZMB mRNA, and immunoblots show GZMB protein levels in the B cells. (e, f) Immunoblots show phosphor AKT (e) and T-bet (f) in the B cells. (g) Naïve B cells were treated with the procedures as denoted on the axis. Boxplots show GZMB mRNA levels. GDC: GDC-0068, an AKT inhibitor (5 nM); T.bet.d: T-bet-deficient B cells (prepared by RNAi); cRNAi: B cells were treated with control RNAi; PA: 10 μM. (f) Immunoblots show a TTP/GZMB complex in B cells after the treatment with PA. (g) Immunoblots show the colocalization of TTP and ubiquitin in B cells after treatment with PA. (ANOVA+Dunnett’s test), compared with the saline group. Each dot in boxplots presents data obtained from one sample.

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