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. Author manuscript; available in PMC: 2012 Apr 1.

Published in final edited form as: J Med Primatol. 2010 Nov 2;40(2):88–103. doi: 10.1111/j.1600-0684.2010.00451.x

Fig. 6 — (A) Multiparameter flow cytometry of macaque PBMC obtained 4 weeks after a ΔCD19⁺ T_E infusion. Transferred ΔCD19⁺ T cells were identified in PBMC after staining with mAbs to CD3, CD8, and CD19. (B) The expression of T_M marker on CD3⁺CD8⁺ΔCD19⁺ T cells was determined by flow cytometry after co-staining with CD62L, CCR7, CD28, CD127 or CD95. (C) Aliquots of macaque PBMC were obtained 8 weeks after infusion of a T_CM-derived ΔCD19⁺CD8⁺ T_E clone. CCR7⁺CD95⁺ T_CM and CCR7⁻CD95⁺ T_EM subsets either in the CD3⁺CD8⁺ΔCD19⁻ or CD3⁺CD8⁺ΔCD19⁺ cells were identified as described in (A) and (B). Cells were then stained for intracellular expression of Ki-67. Inset values show the frequency (%) of Ki-67⁺ T cells. (D) Analysis of intracellular Ki-67 in PBMC obtained from 2 macaques 6-8 weeks after infusion of a T_CM-derived ΔCD19⁺CD8⁺ T_E clone, and in samples of BM and LN obtained from 3 macaques 2 weeks after the infusion. PBMC: mean; BM and LN: mean ± SD. (E) Function of re-isolated ΔCD19⁺ T_E obtained from T_CM or T_EM subsets. Aliquots of post-infusion PBMC were sort-purified in CD8⁺ΔCD19⁺CD62L⁺ and CD8⁺ΔCD19⁺CD62L⁻ subsets, restimulated in vitro using anti-CD3/CD28 mAbs, and examined in a chromium release assay for recognition of unpulsed (□) or peptide-pulsed target cells. E/T (■) 2.5:1, () 1.25:1. The transferred ΔCD19⁺ T_E clone served as control. (F) Aliquots of the infused ΔCD19⁺ T_E clone (■) and re-isolated ΔCD19⁺ T_E either obtained from T_CM () or T_EM (□) were stimulated with medium or CMV peptide-pulsed antigen-presenting cells, and examined by CFC for production of IFN-γ, TNF-α, and IL-2 after gating on CD3⁺CD8⁺ T cells.

Inline graphic — (A) Multiparameter flow cytometry of macaque PBMC obtained 4 weeks after a ΔCD19⁺ T_E infusion. Transferred ΔCD19⁺ T cells were identified in PBMC after staining with mAbs to CD3, CD8, and CD19. (B) The expression of T_M marker on CD3⁺CD8⁺ΔCD19⁺ T cells was determined by flow cytometry after co-staining with CD62L, CCR7, CD28, CD127 or CD95. (C) Aliquots of macaque PBMC were obtained 8 weeks after infusion of a T_CM-derived ΔCD19⁺CD8⁺ T_E clone. CCR7⁺CD95⁺ T_CM and CCR7⁻CD95⁺ T_EM subsets either in the CD3⁺CD8⁺ΔCD19⁻ or CD3⁺CD8⁺ΔCD19⁺ cells were identified as described in (A) and (B). Cells were then stained for intracellular expression of Ki-67. Inset values show the frequency (%) of Ki-67⁺ T cells. (D) Analysis of intracellular Ki-67 in PBMC obtained from 2 macaques 6-8 weeks after infusion of a T_CM-derived ΔCD19⁺CD8⁺ T_E clone, and in samples of BM and LN obtained from 3 macaques 2 weeks after the infusion. PBMC: mean; BM and LN: mean ± SD. (E) Function of re-isolated ΔCD19⁺ T_E obtained from T_CM or T_EM subsets. Aliquots of post-infusion PBMC were sort-purified in CD8⁺ΔCD19⁺CD62L⁺ and CD8⁺ΔCD19⁺CD62L⁻ subsets, restimulated in vitro using anti-CD3/CD28 mAbs, and examined in a chromium release assay for recognition of unpulsed (□) or peptide-pulsed target cells. E/T (■) 2.5:1, () 1.25:1. The transferred ΔCD19⁺ T_E clone served as control. (F) Aliquots of the infused ΔCD19⁺ T_E clone (■) and re-isolated ΔCD19⁺ T_E either obtained from T_CM () or T_EM (□) were stimulated with medium or CMV peptide-pulsed antigen-presenting cells, and examined by CFC for production of IFN-γ, TNF-α, and IL-2 after gating on CD3⁺CD8⁺ T cells.