Various types of CD4 and CD8 T_Ms show a similar polarized tissue distribution. (A) Tissue distribution of CD4 and CD8 T_Ms of various originalities. Top: CD4 and CD8 T_Ms (gated as Thy1.2⁺CD4⁺ and Thy1.2⁺CD8⁺, respectively) generated in the Thy1.1 congenic mice 2 months after transfer of a mix of equal number (10 × 10⁶) of B6 CD4 and CD8 T_Es; middle: memory-phenotype (MP) CD4 T_Ms (gated as CD4⁺CD25⁻CD44^hi, CD25 staining was included to gate off Tregs) and CD8 T_Ms (gated as CD8⁺TCRβ⁺CD44^hi, TCRβ staining was included to gate off the CD8⁺ nonαβ T cells present in some tissues) spontaneously generated in 1-year-old B6 mice (similar results were observed for B6 mice aged from 3 months to 1 year); and bottom: OVA antigen-specific OT2 T_Ms (gated as CD4⁺CD25⁻TCRVβ5⁺CD44^hi, CD25 staining was included to gate off Tregs) and OT1 T_Ms (gated as CD8⁺TCRVβ5⁺CD44^hi) generated in OT2 or OT1 transgenic mice 2 months after infection with 1 × 10⁸ TU DC-directed lentivirus expressing OVA antigen. Data are presented as mean ± SEM (n = 4). (B) Tissue distribution of CD4 and CD8 T_CMs and T_EMs. Various tissues were harvested from 1-year-old B6 mice and analyzed for the presence of MP CD4 and CD8 T_CMs and T_EMs (gated as CD4⁺CD25⁻CD44^hiCD62L⁺, CD4⁺CD25⁻CD44^hiCD62L⁻, CD8⁺TCRβ⁺CD44^hiCD62L⁺, or CD8⁺ TCRβ⁺CD44^hiCD62L⁻, respectively) using flow cytometry. Histogram plots (top) and quantification of T_Ms (bottom) are shown. Data are presented as mean ± SEM (n = 4). T_CMs indicates central memory T cells; and T_EMs, effector memory T cells. (C) Visualization of the CD4 T_Ms formed in abino B6 mice each receiving 1 × 10⁶ MFG-labeled CD4 T_Es that had been differentiated in vitro into T_H0, T_H1, T_H2, or T_H17 cells using BLI. Representative BLI images collected 2 months after adoptive transfer are shown (n = 4). Schematic showing the individual tissue localization in mice is provided for reference. (D) Functional analysis of MP CD4 T_Ms residing in PPs of 1-year-old B6 mice. PP cells (pool of 4 mice) were stimulated with PMA + Ionomycin in vitro for 6 hours and analyzed for the cytokine production of CD4 T_Ms (gated as CD4⁺CD44⁺) using flow cytometry. Contour plots representative of 3 independent experiments are shown.