Panel A: Experimental schematic shows naïve B-cell activation using 40LB feeder cells, interleukin-4 stimulation, fluorescence-activated cell sorting, western blotting, messenger ribonucleic acid sequencing, and induced germinal-center B-cell differentiation timeline. Panel B: A Western blot analysis shows the expression levels of eIF3e and beta-Actin across different days for control and cKO groups. Panel C: A scatter plot displays the fold change in cell number relative to input over four days, comparing control and cKO groups. Panel D: A flow cytometry histogram shows CellTrace Violet intensity, indicating cell proliferation over three days. Panel E: A scatter plot shows the percentage of Annexin V and 7-AAD positive cells, indicating cell death over four days. Panel F: Flow cytometry histograms and scatter plots with bars show the mean fluorescence intensity (MFI) of CD80, OX40L, ICAM-1, and ICOSL over four days. Panel G: Flow cytometry histograms and scatter plots with bars show the MFI of MHC 2 and GL7 over four days. Panel H: Bar graphs display the results of Metascape analysis, showing upregulated and downregulated biological processes in eIF3e-deficient day 3 iGCB cells. Panel I: A scatter plot shows the log2 fold changes in protein and RNA expression levels in eIF3e-deficient B cells relative to control at iGCB day 3, highlighting genes with altered translation efficiency. Panel J: A volcano plot shows differentially expressed genes identified by RNA-seq in cKO versus control B cells cultured for three days.
eIF3e represses expression of costimulatory and adhesion molecules in B cells. (A) Experimental outline for B cell in vitro activation assay. The in vitro activated B cells were sorted for RNA-seq, WB, and flow cytometry analysis (FACS). (B–E) Analysis of eIF3e expression (B), cell proliferation (C), and death (E) at indicated time points. Cell proliferation was calculated by dividing the number of activated cells by the number of input cells. (D) CellTrace Violet intensity assay of cell proliferation. Naïve B cells were labeled with CellTrace Violet, cultured in the in vitro activation system in A for 3 days, and analyzed by flow cytometry for CellTrace Violet intensity. (F and G) Flow cytometry analysis of cell surface proteins at indicated time points. Representative FACS histograms (also shown in Fig. S3 D, column 4) and statistical analyses of MFI are shown. (H) Metascape analysis of genes upregulated (top) and downregulated (bottom) at the protein level in eIF3e-deficient day 3 iGCBs relative to control, as determined by LC-MS/MS (Q <0.05 and |AVG.Log2.Ratio| >0.3). (I) Scatter plots showing log2FCs in protein and RNA expression levels in eIF3e-deficient B cells relative to control at iGCB day 3. The 343 highlighted genes exhibit altered translation efficiency, with interested targets marked by gene names. These 343 translationally regulated genes were classified into distinct functional categories: Opposite (RNA and protein abundance changed in biologically opposing directions), Intensified (protein FC exceeded RNA log2(FC) by >0.5 log2 units), and Buffered (log2(FC) in protein response was blunted by >0.5 compared with the RNA shift). (J) Volcano plot showing DEGs (|FC| >1.5, adjusted P <0.05) identified by RNA-seq in cKO versus control B cells cultured in the in vitro activation system in A for 3 days. Statistical analysis of cell proliferation (C), cell death (E), and MFI (F and G) was performed using paired two-tailed Student’s t test for the control and cKO CD19+ iGCBs. ns, not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. Each dot represents one mouse. Three independent experiments (B–G) or three biological replicates (H–J) were performed. WB, western blot; MFI, mean fluorescence intensity. Source data are available for this figure: SourceData F4.