Phenotypic characterization of splenic CD8⁺and CD4⁺T cells in RAG1^−/−recipients. (A-B) To identify naive and memory CD8⁺ T cells, splenocytes were stained for CD3, CD8, CD62L, and CD44 expression. Shown are representative flow cytometry profiles of naive (CD44⁻CD62L⁺), central memory (CM; CD44⁺CD62L⁺), and effector memory (EM; CD44⁺CD62L⁻) T cells within the CD3⁺CD8⁺ T cells from young and aged nontransplanted mice and RAG1^−/− recipients of DY and DO HSCs. (C) Quantification of naive CD3⁺CD8⁺ T cells within the total CD3⁺CD8⁺ population (Y, n = 8; O, n = 7; DY, n = 16; DO, n = 19). (D) Proliferation of CD3⁺CD8⁺ T cells isolated from young and old animals and RAG1^−/− recipients of DY and DO HSCs was analyzed by expression of the nuclear protein Ki-67. Percentage of naive (CD44⁻) and memory (CD44⁺) CD3⁺CD8⁺ T cells, which are Ki-67⁺, are depicted (Y, O, n = 4; DY, n = 5; DO, n = 9). (E) CD3⁺CD8⁺ T cells isolated from nontransplanted young and old B6.SJL mice and RAG1^−/− hosts transplanted with DY or DO HSCs were stained for the exhaustion marker PD-1, CD244.2, TIM-3, and LAG-3. Representative flow cytometric profiles of individual stains are depicted. (F) Percentages of the different inhibitory receptors within CD3⁺CD8⁺ T cells are shown (Y, n = 8; O, n = 7; DY, n = 16; DO, n = 19). (G-H) Splenic naive CD4⁺ T cells from recipients of DY and DO HSCs and nontransplanted young and aged mice were identified as CD44⁻CD62L⁺ cells within the CD3⁺CD4⁺ T-cell population. Representative dot plot profiles of the flow cytometric analysis are depicted. (I) Quantification of naive CD3⁺CD4⁺ T cells within the total CD3⁺CD4⁺ population (Y, n = 8; O, n = 7; DY, n = 16; DO, n = 19). *P < .05; **P < .01; ***P < .001; ****P < .0001 2-tailed unpaired Student's t-test, mean + SEM.