Panel A shows scatter plot of germinal center B cell numbers in draining lymph nodes at steady state and day 14; vertical axis percentage of germinal center B cells and horizontal axis mouse genotypes and conditions. Panel B shows scatter plot of germinal center B cell proportions at day 28; vertical axis percentage of germinal center B cells of B220 positive cells and horizontal axis mouse genotypes. Panel C shows scatter plot of RBD specific germinal center B cell proportions at day 28; vertical axis percentage of RBD positive cells and horizontal axis mouse genotypes. Panel D shows flow cytometry plots illustrating gating strategy for plasma cells with CD138 and CD98 markers; no quantitative axes required. Panel E shows scatter plot of plasma cell proportions; vertical axis percentage of plasma cells of live dump negative cells and horizontal axis mouse genotypes. Panel F shows scatter plot of RBD positive germinal center B cell numbers across time; vertical axis number of cells and horizontal axis days post immunization. Panel G shows scatter plot of immunoglobulin M fluorescence intensity in germinal center B cells; vertical axis immunoglobulin M geometric mean fluorescence intensity and horizontal axis mouse genotypes. Panel H shows scatter plot of immunoglobulin M fluorescence intensity in RBD specific germinal center B cells; vertical axis immunoglobulin M geometric mean fluorescence intensity and horizontal axis mouse genotypes. Panel I shows pie charts representing clonal distribution of antibody sequences from germinal center B cells across genotypes; no axes shown. Panel J shows dot plot of variable heavy chain gene usage frequencies; vertical axis percentage mutations and horizontal axis gene groups and mouse genotypes. Panel K shows bar graph of variable light chain gene usage frequency; vertical axis frequency percentage and horizontal axis immunoglobulin lambda variable genes across mouse genotypes.
Prime immunization with RBD. Related to Figs. 2 and 3. (A) Absolute numbers of GC B cells in draining LN at steady state (left) and 14 days after RBD immunization (right). (B) Proportions of GC B cells in B220+ B cells at day 28 after immunization. (C) Proportions of RBD-specific cells in GC B cells at day 28 after immunization. (D) Flow cytometric gating strategy for PCs. (E) Proportions of PCs in live Dump− cells. (F) Numbers of RBD+ GC B cells at day 7, day 14, day 21, and day 28 after RBD immunization. (G) IgM GMFI in CD95+ B220+ GC B cells. (H) IgM GMFI in RBD-specific CD95+ B220+ GC B cells. (I) Pie charts depicting the distribution of antibody sequences obtained 14 days after prime immunization from GC B cells from four mice/genotype. Inner circle numbers indicate the number of sequences analyzed for each mouse. White section indicates nonexpanded sequences; colored or black pie slices are proportional to the number of clonally related sequences. The outlined black line indicates the percentages of cells in a clonal family. (J) Iglv gene usage frequency in genotypes. VH genes split by the mean frequency in mM-only are shown. Top 10 highest frequency (hi) and remaining low-frequency VH genes (lo) are plotted. Each dot represents a VH gene. Bars indicate the median. (K) Dot plot showing the number of VH nucleotide somatic hypermutations in sequences from top 10 most frequent V genes in mM-only mice from Fig. 5 F (Hi) compared with sequences from all remaining low-frequency V genes. Each dot indicates VH sequence from one cell. Bars indicate the median. Each dot represents a single mouse. Bars indicate the mean ± SD. *P < 0.05, **P < 0.01 in one-way ANOVA. D, day; GMFI, geometric mean fluorescence intensity.