Fig. 2. Derivation of macaque CMV-specific CD8⁺ T cells from T_EM or T_CM subsets.

(A) Flow cytometry showing CD8⁺ T-cell subsets in macaque PBMC including T_CM (CCR7⁺CD95⁺), T_EM (CCR7⁻CD95⁺), and T_N (CCR7⁺CD95⁻). (B) Frequency of CD8⁺ T_CM, CD8⁺ T_EM, and CD8⁺ T_N (%) in peripheral blood CD8⁺ lymphocytes. Aliquots of PBMC obtained from 18 healthy macaques were stained with mAbs binding to CD8, CD3, CD95, and CCR7 and examined by flow cytometry after gating on CD3⁺CD8⁺ T cells. Mean and SD is shown. (C) CMV IE-specific T cells are present in distinct CD8⁺ T_M subsets. CFC assay for IFN-γ⁺CD8⁺ T cells specific for CMV in sort-purified CD8⁺CD62L⁺ and CD8⁺CD62L⁻ T-cell subsets obtained from 4 macaques. The absolute number of CMV-specific CD8⁺ T_CM or T_EM/μL peripheral blood was determined by calculating the absolute number of CD3⁺CD8⁺ T cells/μL of blood (% of CD8⁺ T cells/lymphocyte subset × lymphocyte count/μL blood/100). Subsequently, the absolute number of the T_EM, T_CM, and T_N subset was derived (% subset × number of CD3⁺CD8⁺/μL blood/100). Finally, we calculated the absolute number of CMV-specific CD8⁺ T cells/μL in each subset (% IFN-γ⁺ cells × absolute number of CD3⁺CD8⁺ T_CM+T_N/μL or CD3⁺CD8⁺ T_EM/μL blood/100). (D, E) CFC assay of macaque CMV-specific T-cell lines detects IFN-γ⁺CD8⁺ CMV-specific T cells in sort-purified CD8⁺CD62L⁺ cells containing T_CM (upper panels) and CD8⁺CD62L⁻ T_EM subsets (lower panels). Sort-purified subsets from 2 representative macaques were stimulated with autologous CMV peptide-pulsed antigen-presenting cells and assayed by CFC for production of IFN-γ after stimulation with medium alone (left panels), or CMV peptide antigen (right panels). Data are gated on CD3⁺CD8⁺ cells.