miR-28 regulates the GC reaction. (A) miR-28 expression was assessed by qRT-PCR in naïve B cells (CD19⁺GL7⁻IgA⁻), GC B cells (CD19⁺GL7⁺), and switched B cells (post-GC, CD19⁺GL7⁻IgA⁺) from Peyer’s patches (top) (n = 2) and in spleen GC B cells (CD19⁺Fas⁺GL7⁺) on the indicated days after immunization of C57/BL mice with sheep red blood cells (bottom) (n = 2) (gating strategy is shown in supplemental Figure 1A-B). (B) Spleen B cells were labeled with violet cell tracer, stimulated with lipopolysaccharide and IL-4, and transduced with control or pre-miR-28–containing retroviral constructs. CSR to IgG1 was assessed by FACS analysis of GFP⁺ cells 3 days after transduction. Left, representative FACS plot; right, quantification (n = 5). Lower panels: number of cell divisions was quantified with cell tracer by FACS analysis 3 days after transduction. Left, representative histogram (open line, control-transduced cells; shaded histogram, miR-28–transduced cells). Number of cell divisions is indicated. Right, quantification (n = 5). (C-D) Mouse chimeras transplanted with bone marrow cells transduced with miR-28 SPG or empty vector were analyzed by FACS 10 to 14 days after immunization. (C) Left, representative plots of GC (top) and plasma (bottom) cells; right, quantifications after normalization to the mean of control mice in each individual experiment. Phosphate-buffered saline (PBS): nonimmunized chimeric mice. Analyses were performed gating on CD45.1⁺ GFP⁺ cells. Data are from 6 (GC) and 4 (plasma cell) independent transplantation and immunization experiments. (D) miR-28 SPG or empty vector chimeras were generated using bone marrow cells from R26-Tomato^ki/+; AID^Creki/+ mice to track GC-experienced cells. Left, representative plots of Tomato⁺IgG1⁺ and Tomato⁺IgG1⁻ memory and Tomato⁺ CD138⁺ plasma B cells are shown; right, quantifications. Analyses were performed gating on Tmt⁺ GFP⁺ cells. Data are from 2 independent transplantation and immunization experiments. Each symbol (C-D) represents an individual mouse. *P < .05, **P < .01, unpaired Student t test (A-D).