Fig. 8. Competitive disadvantage of GCBCs after targeted CPT2 mRNA reduction.

a, Experimental outline of competitive GC development using in vitro-transduced B cells with shRNA retroviral vectors. B cells from CD45.1 or CD45.2 Balb/c mice were transduced with retroviral shRNA vectors targeting the mRNAs encoding CD8 (control gene; CD45.1 in blue) or CPT2 (CD45.2 in green) after stimulation with CpG DNA. Retrovirally transduced cells express the fluorescent reporter protein Ametrine. Transduced cells were co-transferred into NP-unresponsive CD45.1/2 AM14^tg+ Vk8R^+/– mice that had been CGG carrier-primed 4 days earlier. Recipients were immunized i.p. with NP-CGG in alum and analyzed 13 days later. b, Gating strategy to identify CD8- and CPT2-transfected live Ametrine⁺ non-GC (CD38⁺ CD95⁻) and GCBCs (CD38⁻ CD95⁺) singlets within the same animal. Complete gating strategy is shown for GCBCs only but was also applied for non-GCBCs. c, qRT-PCR of sort-purified GCBCs 7 days post immunization. Shown are 3 replicates of CPT2 mRNA levels of 2 individual mice (indicated as circles or triangles) normalized to GAPDH. d, Competitive ratio of control CD8- to CPT2-targeted B cells in non-GC, GCBCs and input cells. The ratio is the number of shRNAmiR-CD8a transduced CD45.1/1 Ametrine positive B cells divided by the number of shRNAmiR-CPT2 CD45.2/2 transduced Ametrine positive B cells after gating on each compartment. Dashed line represents input transduction ratio at the time of cell transfer. Data are from two independent experiments depicted as circles or triangles; data points in grey represent mice with less than 200 Ametrine positive cells recovered. Statistical comparison with and without data points depicted in grey resulted in the same degrees of statistical significance. Bars represent mean +/− SEM; ***p ≤ 0.001; ****p ≤ 0.0001 by unpaired, two-tailed t-test.