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: MiR-155 regulates H3K36me2 levels in dividing LZ GC-B cells.a Enrichment map visualizing GSEA results of bulk RNA-seq data from WT and KO cMyc+ LZ GC-B cells. KO-enriched pathways (q < 0.1) were used. b The relative abundance of histone PTMs, including histone H3.1 K36, H3.3 K36, H3.1 K27 and H3.3 K27 in LZ GC-B cells from WT and KO mice. Donor-derived GC-B cells were sorted five days after HEL3 × -SRBC immunization for analysis. Triplicate samples were used for both WT and KO backgrounds. Unpaired student’s t-test, two-tailed. c Splenic sections of C57BL/6 mice, in the presence and absence of miR-155, seven days after SRBC immunization were stained for GL7 (green), MYC (red) and H3K36me2 (blue). Yellow wedges represent cells that are positive for MYC but negative for KDM2A. Scale bar, 50 µm. Representative images from two experiments (WT n = 4; KO n = 4). d Relative H3K36me2 levels determined by calculating the ratio of H3K36me2 MI to MYC MI from Supplementary Fig. 2c. The top 5% of MYC intensity values, representing the highest MYC expressing cells, were selected from each batch of two independent experiments. The bar represents mean. Unpaired student’s t-test, two-tailed. A.U., arbitrary unit. e MFI of H3K36me2 in cMyc+ LZ GC-B cells. Pooled from three independent experiments (WT n = 8; KO n = 9). Unpaired student’s t-test, two-tailed. Unless otherwise stated, mean ± SEM is indicated. n.s., not significant.

Fig 4: 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.