Fig. 3: Gene signatures regulated by Bob1 in Tfh cells.

a Experimental protocol for RNAseq analysis using FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice and control FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice to obtain CD4^Cre/+Bob1^fl/fl Tfh cells and CD4^+/+Bob1^fl/fl Tfh cells, respectively. NP₄₉-CGG was administered intraperitoneally with CFA on day 0. As shown in the gating strategy, CD4^Cre/+Bob1^fl/fl and CD4^+/+Bob1^fl/fl Tfh cells (GFP^-B220^-CD3⁺CD4⁺CXCR5⁺PD-1⁺) were sorted from spleens on day 7 and subjected to RNAseq analysis. b Volcano plot showing differentially expressed genes (P < 0.05) with more than two-fold expression (log₂ fold change > 0.5) in CD4^Cre/+Bob1^fl/fl Tfh cells vs. CD4^+/+Bob1^fl/fl Tfh cells. The red and blue dots indicate the upregulated and downregulated genes, respectively, in CD4^Cre/+Bob1^fl/fl Tfh cells. c Gene set enrichment analysis (GSEA) of the transcriptomes of CD4^Cre/+Bob1^fl/fl and CD4^+/+Bob1^fl/fl Tfh cells. Gene sets include ATP synthesis coupled electron transport (GO: 0042773), oxidative phosphorylation (GO: 0006119), cellular respiration (GO: 0045333), mitochondrial respiratory chain complex assembly (GO: 0033108), NADH dehydrogenase complex (GO: 0030964), glucose import (GO: 0046323), negative regulation of immune system process; (GO: 0002683), αβ-T cell differentiation (GO: 0046632), αβ-T cell activation (GO: 0046631), and fatty acid biosynthetic process (GO: 0045723). Gene sets showed P < 0.05 and FDR < 0.25. NES, normalized enrichment score. d Relative mRNA expression levels of Pou2af1, Pou2f1, Pou2f2, Bcl6, Icos, Il4, Il21, Sostdc1, Padi4, and Mef2b in CD4^Cre/+Bob1^fl/fl and CD4^+/+Bob1^fl/fl Tfh cells examined by RT-qPCR analysis. Pou2af1, **P = 0.0012; Icos, *P = 0.0284; Il4, **P = 0.0058; Sostdc1, *P = 0.0462; Padi4, *P = 0.0478; Mef2b, **P = 0.0045. e, f Representative flow cytometric profiles and graphs of the expression of negative regulators in CD4^Cre/+Bob1^fl/fl and CD4^+/+Bob1^fl/fl Tfh cells as measured in (a). e Profiles of Lag3⁺ Tfh cells and graphs of the % of Lag3⁺ Tfh cells among the total Tfh cells. **P = 0.0056. f Profiles of Tigit⁺ Tfh cells and graphs of the % of Tigit⁺ Tfh cells among the total Tfh cells. **P = 0.0039. g Oxygen consumption rate (OCR) of CD4^Cre/+Bob1^fl/fl and CD4^+/+Bob1^fl/fl Tfh cells. The OCR was measured after supplementation with oligomycin (a complex V inhibitor), FCCP (a protonophore), and rotenone/antimycin A (complex I/III inhibitors). *P = 0.0303, *P = 0.0101, *P = 0.0101, *P = 0.0177, in order from left to right. RNAseq data in (b) were obtained from specimens of three experiments in each mouse group (n = 10–12 per experiment). Data from RT-qPCR analysis in (d) represents the mean ± SD by unpaired t-test (1 dot; n = 4–5), *P < 0.05, **P < 0.01. Graphs in (e, f) show the mean ± SD determined by the Mann-Whitney U-test (e, f, n = 9), **P < 0.01. Graphs in (g) show the mean ± SEM determined by the Mann–Whitney U-test (CD4^+/+Bob1^fl/fl Tfh cells; n = 5, CD4^Cre/+Bob1^fl/fl Tfh cells; n = 7). *P < 0.05. Data in (d–f) are shown from FoxP3^GFP/DTRCD4^+/+Bob1^fl/fl mice (open triangle) and FoxP3^GFP/DTRCD4^Cre/+Bob1^fl/fl mice (closed triangle). Similar results were obtained across two to three independent experiments (d–g).