Validation of the indicated regulators of TD B cell responses by sgRNA-mediated gene inactivation. (A and B) Flow cytometric analysis of two independent validation experiments performed by sgRNA-mediated inactivation of putative positive and negative regulators. The outline of the strategy and flow cytometric analysis of the validation experiments is explained in detail in Fig. 5 A. mCherry⁺ and mAmetrine⁺ sgRNA-transduced B cells were mixed at a ratio of 2.5:1 and transferred into recipient mice, followed by immunization with NP-KLH/alum. Flow cytometric analysis of splenocytes from recipient mice was performed 7 days after immunization. mCherry⁺ donor B cells expressed the control sgRNA (sg.Chr1) or sgRNAs targeting positive regulator genes (sg.Isg20, sg.Ccrl2, sg.Tmem198, and sg.Aldh1l2) or negative regulator genes (sg.Prg3 and sg.Stk3), while the mAmetrine⁺ donor B cells expressed only the control sgRNA. The percentages of mCherry⁺ and mAmetrine⁺ plasmablasts (TACI⁺CD138⁺) and GC B cells (GL7⁺CD95⁺) in total B cells (CD19⁺CD138^–/lo and CD19^loCD138⁺; green), in mCherry⁺ cells (red), or in mAmetrine⁺ cells (blue) are shown. The ratios of mCherry⁺ versus mAmetrine⁺ plasmablasts and GC B cells were quantified and calculated, as explained in C, and are shown in Fig. 6, B and C. (C) Calculations of the ratio of mCherry⁺ to mAmetrine⁺ plasmablasts in total B cells relative to the control. The corresponding results are shown in Fig. 6 B. First, the ratio (R) of the percentage of mCherry⁺ plasmablasts (sg.Control [sg.Chr1]) in total B cells versus the percentage of mAmetrine⁺ plasmablasts (sg.Control) in total B cells was determined for each control (c) mouse. Subsequently, the mean value of the control group (mean control group) was determined by dividing the sum of all Rc values by the total number of control mice analyzed. In parallel, the ratio (R) of the percentage of mCherry⁺ plasmablasts (for each sgRNA test gene) in total B cells versus the percentage of mAmetrine⁺ plasmablasts (sg.Control) in total B cells was determined for each experimental (e) mouse. We then normalized the Re value of each experimental mouse by dividing it by the mean value of the control group, which allowed to plot all Re values of the different mice in the same graph by setting the mean Rc value to 1 as shown in Fig. 6 B. The same calculation was used to determine the ratios for GC B cells (Fig. 6 C).