Fig 2: MiR-155 is required for promoting mitochondrial fitness in GC-B cells.a Experimental design for all the experiments for Fig. 1. b Percentage of MitoSOX+ GC-B cells five days after HEL3 × -SRBC immunization. Pooled from three independent experiments (WT n = 12; KO n = 12). Unpaired student’s t-test, two-tailed. c Percentage of active-Caspase 3+ GC-B cells at seven days after HEL3 × -SRBC immunization. Pooled from two independent experiments (WT n = 18; KO n = 19). Unpaired student’s t-test, two-tailed. d The PAGA algorithm generates a topology-preserving map of single cells along with the sequential changes of gene expression. PAGA graphs were constructed using a k-nearest neighbors’ algorithm to show relationships between clusters. The Ball-and-stick representation displays PAGA connectivity among GC-B cell clusters identified though scRNA-seq. Ball size represents cluster size, and edge thickness indicates connectivity between clusters. Normalized gene expression for Cxcr4 or Myc is shown in the PAGA graph. e Predicted relationship between the clusters identified in scRNA-seq of GC-B cells. f Violin plots showing gene signature scores for “Mitochondrion” and “OXPHOS”. The bar represents median value. Wilcoxon rank sum test, two-sided, n.s., not significant. g, Enrichment map visualizing GSEA results of the bulk RNA-seq data from WT and KO cMyc+ LZ GC-B cells. GC-B cells were sorted five days after HEL3 × -SRBC immunization. WT-enriched pathways (q < 0.0001) were used. Nodes represent gene sets, and edges represent mutual overlap. Highly redundant gene sets were grouped as clusters (left). ES; enriched score, FDR; false discovery rate. Unless otherwise stated, mean ± SEM is indicated.

Fig 3: The miR-155-Kdm2a interaction regulates mitochondrial ROS and apoptosis in GC-B cells.a Schematics representation of the 8-mer miR-155-binding site in the 3’UTR of the Kdm2a (boxed) and the introduced base changes (red letters) to disrupt miR-155 binding in Kdm2a mutant mice. b Representative flow cytometric histograms showing KDM2A vs. RCN (top). RCN, relative cell number. Relative MFI values of KDM2A in LZ and DZ GC-B cells are shown (bottom). GC-B cells were derived from WT and MUT mice seven days after SRBC immunization. Unpaired student’s t-test, two-tailed. Pooled from three independent experiments (WT n = 4; MUT n = 4). c Number of LZ and DZ GC-B cells from WT and MUT mice within the µMT adoptive transfer system. Unpaired student’s t-test, two-tailed. Pooled from two independent experiments (WT n = 7; MUT n = 7). d Representative flow cytometric plots of MitoTracker DeepRed vs. MitoSOX in GC-B cells from WT and MUT mice within the µMT adoptive transfer system (top). Percentage of MitoSOX+ GC-B cells (bottom). Unpaired student’s t-test, two-tailed. Pooled from two independent experiments (WT n = 7; MUT n = 7). e Representative flow cytometric plots of B220 vs. active-Caspase 3 in GC-B cells from WT and MUT mice within the µMT adoptive transfer system (top). Percentage of active-Caspase 3+ GC-B cells (bottom). Unpaired student’s t-test, two-tailed. Pooled from two independent experiments (WT n = 7; MUT n = 7). Unless otherwise stated, mean ± SEM is indicated. n.s., not significant.

Fig 4: MiR-155 primarily controls KDM2A expression in high affinity cMyc+ LZ GC-B cells.a Heat map showing the transcript levels of the enzymes involved in the demethylation of histone H3K36me2. The bulk RNA-seq dataset from WT and KO cMyc+ LZ GC-B cells was used. FDR values are shown. b Sequence alignment of a part of the 3’UTR of Kdm2a. The 8-mer miR-155 binding site is highlighted in a box. c Immunofluorescence staining of splenic section from a WT mouse seven days after SRBC immunization, showing IgD (white), MYC (red) and KDM2A (blue). Scale bar, 50 µm. Representative section images from two experiments (WT n = 4; KO n = 4). d Scatter plots show the MI of MYC and KDM2A within segmented GC cells in spleen sections from immunized WT and KO mice. The Pearson correlation coefficient (r) is presented with a 95% confidence interval in red. A.U., arbitrary unit. e Relative MFI of KDM2A in cells from four GC-B cell subpopulations. GC-B cells were derived from WT or KO donor B cells seven days after HEL3 × -SRBC immunization. Pooled from two independent experiments (WT n = 11; KO n = 9). Unpaired student’s t-test, two-tailed. f Relative MFI of KDM2A in cells from four GC-B cell subpopulations. GC-B cells were derived from WT or KO donor B cells seven days after HEL3 × -SRBC immunization. Low and high affinity were determined based on HEL3 × binding within Igκ+ GC-B cells. Pooled from two independent experiments (WT n = 11; KO n = 9). RCN, relative cell number. Paired student’s t-test, two-tailed. Unless otherwise stated, mean ± SEM is indicated. n.s., not significant.