Fig. 5: Adoptively transferred engineered B cells can undergo CSR and clonal expansion upon immunization.

a Isotype specific anti-idiotypic ELISA measuring 3BNC117 isotypes in mice sera collected after boost immunizations. # left = pv = 0.0278, # right = pv = 0.0309, ##pv = 0.0014 for Dunnett’s multiple comparisons and ***pv = 0.0003 and * left = pv = 0.0343 and * right = pv = 0.0461 for two-tailed t-test. Comparisons performed to sera of mice receiving adoptive transfer of non-engineered B cells and boost immunized with the YU2.DG gp120 antigen. b Analysis by flow cytometry of IgA and CD45.1 expression among GC cells after prime and boost immunizations, gating on live, lymphocytes, GL-7⁺, B220⁺. ## upper = pv = 0.0087 and ##pv = 0.0041 for two-way ANOVA, *pv = 0.0074 for two-tailed t-test and indicated p value is for one-sample t-test c Ratio of non-synonymous to synonymous mutations in the different samples. #pv = 0.0143 for two-way ANOVA between the prime and boost cohorts. *pv = 0.0169, **pv = 0.0088, ***pv = 0.0002 for two-tailed t-test and indicated p value is for one-sample t-test. d Quantitation of the clonal expansion by measuring polarity: the relative cumulative share of the ten most abundant clones. # pv = 0.0496; two-way ANOVA. e Pie charts of mice immunized with the YU2.DG gp120 antigen and having at least one clone representing more than >10% of the mutant repertoire. The most abundant clones in each mouse were colored. Shades of red indicate clones that were not found in the ten most abundant clones of other mice (Supplementary Fig. 10). Shades of blue indicate shared clones. Indicated clones are the K46R, A89T, and D97Y. f Quantitation of amino acid (AA) substitutions per clone in mice immunized once or twice with the YU2.DG or THRO4156.18 gp120 antigens. ##pv = 0.005, #pv = 0.0177 for two-way ANOVA and **pv = 0.0043, *pv = 0.0167 for Tukey’s multiple comparison. For c, d, f n = 3 for all except THRO Boost samples in which n = 2, each dot represents a mouse.