Fig. 5: SLAMF5 regulates the transcription factor c-Maf.

B cells derived from EAE-induced Vert-x mice were sorted for Bregs (CD19⁺GFP⁺) and treated for 24 h with SLAMF5-blocking or IgG-control antibodies. RNA was then extracted from the cells and analyzed. a, b mRNA-seq analysis of differentially expressed genes in anti-SLAMF5 compared to IgG-treated cells performed in two independent replicates. a Heatmap showing differentially expressed genes, with the log2 normalized counts standardized for each gene to a mean of zero. The hierarchical clustering of the genes is shown. The expression profile is accompanied by a colored bar indicating the standardized log2 normalized counts. Blue: downregulated; red: upregulated genes. b Pathway enrichment analysis of 277 genes that were found to be significantly changed (absolute change > 2; P value < 0.05) was done using the EnrichR platform; the graph shows upregulated (red) and downregulated (blue) pathways (IgG n = 2; SLAMF5-blocking n = 2, each sample is a pool of cells derived of three mice). c qPCR validation analysis of c-Maf mRNA in SLAMF5-blocked Vert-x sorted Bregs (CD19⁺GFP⁺) (IgG n = 5; SLAMF5-blocking n = 5. Each dot represents a pool of two to three mice, four independent experiments). d AhR mRNA levels in Vert-x sorted Bregs following SLAMF5-blocking. Data are shown as x-fold of the treatment group compared to IgG-control (IgG n = 5; SLAMF5-blocking n = 5. Each dot represents a pool of two to three mice, four independent experiments). e, f EAE was induced in WT mice. On days 7, 9, and 11, the mice were injected i.v. with 30 µg SLAMF5-blocking Ab or IgG. On day 15, spleens were collected and analyzed for c-Maf expression by FACS. e Representative histogram showing c-Maf^high expression of Bregs or non-Bregs. f Bar chart showing c-Maf levels in Bregs and non-Bregs. (non-Bregs: IgG n = 10; SLAMF5-blocking n = 15, Bregs: IgG n = 5; SLAMF5-blocking n = 10, two independent experiments). C-Maf expression was done under the Breg or non-Breg gate as shown in Supplementary Fig. 2c. Data expressed as mean ± s.d. (c, d, f). Ratio paired Student’s t test with 95% confidence levels two-tailed (c, d), unpaired Student’s t test with 95% confidence levels two-tailed (f). Pathway analysis was done using EnrichR and showing combined score (combined = ln(P value) x z-score. P value is derived from Fisher’s exact test, z-score is derived of a modified Fisher exact test.