Figure 4. T-bet⁺ and T-bet⁻ MBCs are selected from a shared pre-immune lineage but do not interconvert.

HA-specific splenic MBCs from Tbx21-ZsGreen reporters (day 100 post immunization) were sorted into T-bet⁻ and T-bet⁺ subsets, with naïve follicular (IgD⁺) B cell controls, for immunoglobulin heavy chain genomic sequencing. Human HA-specific splenic MBCs were similarly sorted into CD21⁺ and CD21⁻CD85j^hi subsets; CD21⁻CD85j^hi phenotype identifies human T-bet^hi B cells (Knox et al., 2017) subsets. (A) CDR3 lengths (in nucleotides) of in-frame sequences from murine T-bet⁻ and T-bet⁺ HA⁺ MBCs and naïve follicular (IgD⁺) B cell controls after all replicates were pooled. (B) CDR3 lengths of in-frame sequences from CD21⁺ and CD21⁻CD85j^hi HA⁺ MBC subsets were quantified (in nucleotides). Bulk splenocytes (largely naive follicular B cells) served as a control. (C) The number of clones that overlap between T-bet⁻ (blue) and T-bet⁺ (red) HA⁺ MBCs in mouse (M. mus, MM). (D) The number of clones that overlap between CD21⁺ (blue) and CD21⁻CD85j^hi (red) HA⁺ MBCs in humans (H. sap; HS). (E) Percentages of clones binned by the level of somatic mutation (expressed as the percent difference in nucleotide sequence to the nearest germline VH gene) in mouse T-bet⁻ and T-bet⁺ HA⁺ MBCs and naïve follicular B cells. (F) Percent of the heavy chain V-gene that is mutated from germline in CD21⁺ and CD21⁻CD85j^hi HA⁺ MBCs and bulk splenocytes in humans. (G) Representative lineage trees of shared clones between T-bet⁻ and T-bet⁺ HA⁺ murine MBCs, with inferred nodes (black), T-bet⁻ nodes (blue), and T-bet⁺ nodes (red). Trees were generated in ImmuneDB and visualized with ETE3 (see Methods). Lineages had to contain at least 10 copies of T-bet⁺ and T-bet⁻ and have at least 4 trunk mutations (shared SHMs) to be included. Numbers indicate the number of mutations compared to the preceding vertical node. The inferred node at the top of the tree indicates the nearest germline sequence. (H) Tbx21-ZsGreen^creERT2-Rosa26^LSL/tdTomato mice (Yu et al., 2015) were treated with tamoxifen to mark T-bet expressing cells with permanent, Rosa21-driven, tdTomato expression and the status of T-bet expression of marked B cells in the blood was tracked over 40 days. For panels (A), (C), (E), and (G), two independent experiments were carried out with at least 4 mice per group. Each gave similar results, and the results for the more recent experiment are shown. For panels (B), (D), and (F), the splenocytes from 4 adult subjects were sorted and sequenced. For genetic fate mapping (H), two independent experiments were carried out with at least 4 mice per group; one experiment is shown here.