Figure 3. Purifying selection against C5024T in memory T and B lymphocytes in vivo.

(A) Gates used to FACS isolate naive and memory T and B lymphocyte subsets from C5024T mice (see Methods). All subsets were isolated from the spleen, apart from B-1a cells, which were isolated from the peritoneal cavity. Tn, naive T cells; Tem, effector memory T cells; Tcm, central memory T cells. (B) Wilcoxon’s matched-pairs signed-rank test of percentage of C5024T heteroplasmy (%T) for isolated memory lineages compared to their naive counterparts (n = 6–17 per cell type). (C) %T for all naive and memory lymphocyte subsets in B isolated by FACS. (D) Percentage change in T (vs. ear) for naive and memory T and B cell lineages in B. (E) Schematic of the vaccination timeline and readouts. n = 10 C5024T and n = 10 age- and sex-matched WT animals (2 months of age) were immunized. One C5024T mouse was sacrificed during the experiment (week 7) due to development of a wound. (F) FACS isolation of vaccine-induced, class-switched S-specific IgG⁺ B cells from the draining lymph nodes of vaccinated C5024T mice, n = 6. A representative pseudocolor plot of the antigen-specific (Ag-specific) B cell population from total B-2 cells is shown. Subsets defined using hierarchical gating as naive (CD3^–CD19⁺B220⁺IgM^hiIgG^–S^–) or Ag-specific memory (CD3^–CD19⁺B220⁺IgM^–IgG⁺S⁺). (G) Wilcoxon’s matched-pairs signed-rank test of %T in Ag-specific IgG⁺ cells vs. naive cells (Bn). (H) ID₅₀ neutralizing titers for vaccinated mouse sera. C5024T (n = 9) shown in red, WT (n = 10) shown in blue. Assay limit of detection ID₅₀ = 45. A 1-tailed Mann-Whitney test was used to analyze the data in H. Nonparametric 2-tailed Wilcoxon’s matched-pairs signed-rank tests (B and G) or 2-tailed Mann-Whitney tests (C and D) were used to analyze the data.