Inhibition of endogenous miR-217 expression in B cells impairs the GC response. (A) miR-217-Sponge (miR-217SPG) retroviral construct. Four miR-217 complementary sites, separated by 4-nt spacers, were placed downstream of GFP in the MGP vector. (B-C) GC response of mouse chimeras reconstituted with control or miR-217SPG retrovirally transduced bone marrow precursors analyzed 14 days after NP-CGG immunization. (B) Representative FACS analysis of B220+ GC cells (Fas+GL7+) in (upper) lymph nodes and (lower) spleen and of switched (IgG1+) lymph node cells after NP-CGG immunization. Plots are gated on CD45.1+GFP+ cells. (C) Quantification of GC and switched B cells in mouse chimeras reconstituted with control (open circles) or miR-217 SPG (in red) retrovirally transduced bone marrow precursors. Nonimmunized mice injected with PBS (triangles) were included as immunization controls. Each symbol represents an individual mouse. Data are normalized to the mean response of control mice of 2 independent experiments. Statistical significance was determined by a 2-tailed Student t test vs control immunized mice (P = .12 for LN GCs, P = .05 for SP GCs, and P = .04 for switched B-cell generation). (D) NGS quantification of mutations in (left) JH4 and (right) Sµ sequences in GFP+CD45.1+ GC (Fas+GL7+) B cells isolated from pooled Peyer’s patches of 8 control (open bars) and 8 miR-217SPG (red bars) mouse chimeras. Data are from 2 (JH4) and 1 (Sµ) independent experiments. At least 190 000 kb was sequenced per genotype. Statistical significance of the mean total mutation frequency at each position was determined by a paired Student t test (P = 3.7 × 10−5 for JH4 SHM and P = 4 × 10−4 for Sµ SHM).
