A large mutated IgM B cell pool with memory marker expression accumulates with age in the spleen and shows T cell and TLR dependence. (A) Gating strategy used to defined the total IgM⁺CD73⁺CD80⁺ subset: a CD73⁺CD80⁺ gate including most EYFP⁺ cells is set on the B220⁺IgM⁺GL7⁻EYFP⁺ population (upper right), and then used to define the percentage of non-EYFP⁺ cells in this gate (lower right). (B) Evolution of the total IgM⁺CD73⁺CD80⁺ compartment with time (based on a gate set as defined in A). Ig gene mutations (rearranged intronic J_H4 sequences) are shown in Table S1. (C) Frequency of IgM⁺CD73⁺CD80⁺ B cells in T cell and TLR-deficient backgrounds, compared with age-matched, 4-mo-old, wild-type, and AID-Cre-EYFP mice (with a preset gate on EYFP⁺B220⁺GL7⁻IgM⁺ cells from a parallel analysis). *, P < 0.05; **, P < 0.01, one-way ANOVA with Holm-Sidak correction. Mean values are indicated. Cumulative data of all analyzed animals are indicated. (D) Representative FACS profiles of naive (B220⁺IgM⁺EYFP⁻GL7⁻CD73⁻CD80⁻), B220⁺GL7⁻CD73⁺CD80⁺ EYFP⁺ and EYFP⁻ subsets (gates represented in A [right], including for naive B cells).