Naïve CD8 T cells are phenotypically heterogenous.

(A) tSNE analysis of PBL from Specific pathogen free (SPfree) and Specific pathogen experienced (SPexp) mice 5 days post last infection. Sequential infection of SPfree C57BL/6 mice with PR8-IAV, MCMV-Smith, VacV, att. Lm., and LCMV-arm in 5 d intervals was used to generate SPexp mice. Top row tSNE plots display clusters enriched in SPfree and SPexp mice and outlined populations define CD8 T cell populations. Bottom row tSNE plots display heatmap overlays of surface markers CD8α, CD11a, and Ly6C used to define CD8 T cell populations 1–3. (B) Frequencies of Ly6C- and Ly6C+ naïve CD8 T cells in WT, TCR transgenic P14, and OT-I C57BL/6 mice. Prior gating on CD8αhiCD11alo single-cell lymphocytes. (C) Ly6C expression on naïve CD8 T cells in 4- and 18–20-month-old WT C57BL/6 mice. (D) CD8 T cell related marker expression on Ly6C- and Ly6C+ naïve P14 CD8 T cells. Data are representative of at least three independent experiments with at least 5 mice per group. Error bars represent SEM. *p < 0.05, **p < 0.01, ****p < 0.0001.