(A) CMV IE-specific T cells are present in T_CM and T_EM subsets of CD8⁺ peripheral blood lymphocytes. PBMCs from macaque 02258 were stained with mAbs to CD28, Fas, and CD8 to identify CD28⁺CD8⁺Fas^lo T_N, CD28⁺CD8⁺Fas^hi T_CM, and CD28^–CD8⁺Fas^hi T_EM fractions (left panel) and assayed by cytokine flow cytometry after stimulation with a CMV peptide (right panels). (B) Sorted CD62L⁺CD28⁺CD8⁺Fas^lo T_N (top panel), CD62L⁺CD28⁺CD8⁺ Fas^hi T_CM (middle panel), and CD62L^–CD28^–CD8⁺Fas^hi T_EM (lower panel) T cells were stimulated with autologous CMV IE peptide-pulsed monocytes and assayed for cytolytic activity against peptide-pulsed (filled triangles) or unpulsed target cells (open circles). (C) Isolation of T_CM- and T_EM-derived CMV-specific CD8⁺ T cell clones. CD8⁺CD62L^– and CD8⁺CD62L⁺ T cell subsets were cultured with peptide-pulsed monocytes. At day 7, T cell clones were isolated by limiting dilution, transduced to express ΔCD19 or CD20, and expanded for adoptive transfer. (D) Design of the retroviral vector constructs. MPSV-LTR, myeloproliferative sarcoma virus retroviral long terminal repeat; ψ⁺, extended packaging signal; PRE, woodchuck hepatitis virus posttranscriptional regulatory element; ΔCD19, truncated macaque CD19 cDNA; CD20, full-length macaque CD20 cDNA. (E) Selection of ΔCD19- and CD20-modified CD8⁺ T cell clones. Transduced T cells were enriched for ΔCD19 or CD20 expression using immunomagnetic beads and analyzed by flow cytometry to assess purity (right panels). Unmodified T cells (left panels) served as control. The percentages of CD8⁺ T cells positive for CD19 or CD20 are indicated.

(A and B) ΔCD19-modified T_EM-derived (A) and T_CM-derived (B) clones were transferred to macaque 02269 in separate infusions at a cell dose of 3 × 10⁸/kg, and samples of PBMCs, BM, and LNs were collected before (pre) and at the indicated times following infusion. The frequency of transferred CD19⁺ T cells was determined by flow cytometry and by PCR for vector sequences. Left: Percentage of CD19⁺CD8⁺ T cells by flow cytometry in PBMCs, BM, and LNs before and after infusion of the T_EM-derived (A) and T_CM-derived (B) clones. Cells were gated on CD3⁺CD8⁺ T cells. Right: Absolute numbers of CD19⁺CD8⁺ T cells/μl of blood determined by flow cytometry (gray bars; left y axis) and vector-positive T cells/10⁶ PBMCs (filled diamonds; right y axis). (C and D) ΔCD19-modified T_CM-derived and CD20-modified T_EM-derived clones were transferred to macaque 02258 in separate infusions at a cell dose of 6 × 10⁸/kg. Aliquots of blood, BM, and LNs obtained at the indicated times were analyzed by flow cytometry and PCR to detect transferred CD19⁺CD8⁺ (C) or CD20⁺CD8⁺ (D) cells, respectively. Left: Percentage of CD8⁺ T cells that expressed ΔCD19 (C) or CD20 (D) in PBMCs, BM, and LNs. Cells were gated on CD3⁺CD8⁺ T cells. Right: Absolute number of marked CD8⁺ T cells/μl of blood (gray bars; left y axis) and vector-positive T cells/10⁶ PBMCs (filled diamonds; right y axis).

(A) Persistence and migration of a ΔLNGFR-modified T_EM-derived T cell clone in PBMCs. The ΔLNGFR⁺ T cell clone was transferred at a cell dose of 6 × 10⁸/kg. Aliquots of blood were collected at the indicated times and analyzed by PCR for the frequency of transferred ΔLNGFR⁺ T cells. Shown are the absolute numbers of vector-positive T cells/10⁶ PBMCs (gray bars) before and at intervals after infusion. (B) A necropsy was performed on day 10 after the T cell infusion. Samples of DNA were isolated from various tissues as indicated and examined for the presence of the transferred vector-positive cells (black bars) using a real-time PCR assay. DNA isolated from ΔLNGFR⁺ T cells was serially diluted 1:10 in water prior to amplification with the appropriate primer set. The real-time PCR assay to detect β-actin (gray bars) was used as a control to detect genomic DNA. The threshold cycle is shown for each sample.

(A) IFN-γ production. PBMCs obtained from macaque 02269 before and 14 days following infusion of a ΔCD19⁺ CMV-specific T cell clone were stimulated with medium, PMA/ionomycin, or peptide antigen and examined by cytokine flow cytometry. Data are gated on CD3⁺CD8⁺ cells and are representative of results from macaque 02258. (B) Transferred T cells that reexpress CD62L lack direct cytotoxicity but acquire cytotoxic function after stimulation. Left: PBMCs obtained 14–70 days after infusion of a T_CM-derived CD19⁺CD8⁺ clone to macaque 02258 were pooled, sorted into CD19⁺CD62L^–CD8⁺ and CD19⁺CD62L⁺CD8⁺ fractions (purity >80%), and examined for lysis of autologous unpulsed (white bars) or peptide-pulsed target cells (black bars) (E/T ratio, 5:1). The cultured T_CM-derived ΔCD19⁺CD8⁺ clone served as positive control for lysis. Right: Sorted CD19⁺CD62L^–CD8⁺ and CD19⁺CD62L⁺CD8⁺ T cells were stimulated using anti-CD3 and anti-CD28 mAbs for 14 days and then assayed for lysis of peptide-pulsed target cells (E/T ratio, 5:1). (C) Granzyme B expression. PBMCs and the transferred T_CM-derived clone were stained with mAbs to CD62L, CD8, and CD19 as well as intracellular granzyme B. Cells were analyzed by flow cytometry after gating on CD62L⁺CD8⁺, CD62L^–CD8⁺, CD19⁺CD62L⁺CD8⁺, and CD19⁺CD62L^–CD8⁺ cells. (D) Proliferation. PBMCs obtained from macaque 02269 on days 14–70 after infusion were sorted into CD19⁺CD62L⁺CD8⁺ (left panel) and CD19⁺CD62L^–CD8⁺ subsets (right panel), labeled with CFSE, and stimulated with peptide-pulsed APCs as described in Methods. After 5 days, CFSE dilution was assessed by flow cytometry after gating on CD19⁺CD3⁺CD8⁺ cells. M3 gate, proportion of cells that have undergone more than 5 divisions.

(A) Expression of CD62L, CCR7, CD28, CD127, granzyme B, and perforin (bold line) on individual T_CM- and T_EM-derived clones from 2 macaques. The dotted line indicates results from staining with an isotype control. Inset values represent the MFI. The data are shown for pairs of T_E clones used for adoptive transfer in macaques 02269 and 02258 and is representative of data for all clones used for adoptive transfer. (B) Cytotoxic activity of each pair of T_EM-derived (filled triangles) and T_CM-derived (filled squares) clones was examined at an effector-to-target ratio (E/T ratio) of 20:1 using autologous peptide-pulsed target cells. The peptide sequences were KKGDIKDRV (macaque 02269), ATTRSLEYK (macaque 02258), and EEHVKLFFK (macaque A99171). (C) In vitro growth of CMV-specific CD8⁺ T cell clones. T_EM-derived (filled triangles) and T_CM-derived (filled squares) clones used for adoptive transfer were stimulated with anti-CD3 and anti-CD28 mAbs, γ-irradiated feeder cells, and IL-2 (50 U/ml), and cell growth was measured by counting viable cells. (D) Telomere length in T_CM- and T_EM-derived CMV-specific T cell clones. The median telomere length of duplicate samples was measured by automated flow-FISH in peripheral blood T lymphocytes, in the infused T cell clones (iT_EM and iT_CM), and in each of 2 additional randomly selected T_EM- and T_CM-derived T cell clones from each macaque.

(A) Aliquots of T_EM- and T_CM-derived clones used for adoptive transfer were plated at the end of a 14-day stimulation cycle in medium alone (open triangles), IL-2 (1 ng/ml) (filled diamonds), or IL-15 (1 ng/ml) (filled squares), and viability was determined using Trypan blue dye exclusion. Data are representative of 13 T_CM- and 11 T_EM-derived clones from 3 macaques. (B) Expression of IL-15Rα, IL-2Rβ, and IL-2Rγ on T_CM-derived (bold lines) and T_EM-derived (black lines) clones transferred to macaque 02269 was measured by flow cytometry on days 13–14 after stimulation (dotted lines, isotype control mAb). Data are representative of clones administered to macaques 02258 and A99171. (C) Bcl-xl and Bcl-2 expression on 3 T_EM-derived (white bars) and T_CM-derived (gray bars) clones transferred to macaques 02269, 02258, and A99171 analyzed 14 days after stimulation. Mean ± SD of the MFI is shown on the y axis. (D) Apoptosis of T_EM- and T_CM-derived clones in vivo. The percent of CD19⁺CD8⁺ T cells in PBMCs that were Annexin V⁺ and/or PI⁺ 1 day after infusion of ΔCD19⁺ T_EM-derived or T_CM-derived T cell clones to macaque A99171 was determined directly and after 24-hour culture. Samples were gated on CD8⁺CD19^– (white bars) and CD8⁺CD19⁺ T cells (black bars). Hatched bars show the T cells used for infusion. (E) Persistence and migration of T_EM-derived and T_CM-derived T_E clones in macaque A99171. PBMC, BM, and LN cells obtained at indicated times were analyzed by flow cytometry to detect transferred CD19⁺CD8⁺ T_E in samples gated on CD3⁺CD8⁺.

(A) Expression of CD62L on T_EM-derived (upper panels) or T_CM-derived (lower panels) T_E clones after adoptive transfer. Samples of PBMCs were obtained before and on day 3 after the infusion of T_CM- and T_EM-derived clones and analyzed by flow cytometry after gating on CD3⁺CD8⁺ T cells. The percentage of T cells that expressed the ΔCD19 or CD20 marker gene and CD62L is shown in the upper right quadrant of each panel. (B–E) A major fraction of CD8⁺ T cells that persist after adoptive transfer acquire phenotypic markers of T_CM and reside in LNs. Aliquots of PBMCs, LNs, and BM were obtained from macaque 02258 at days 14 and 56 after infusion of the ΔCD19-modified T_CM-derived clone. The expression of phenotypic markers of T_CM, including CD62L (B), CCR7 (C), CD28 (D), and CD127 (E) on the subset of transferred CD19⁺ T cells was determined by flow cytometry after gating on CD3⁺CD8⁺ cells.

(A) T-APCs pulsed with IE peptide are lysed by IE-specific CD8⁺ T cells. T-APCs generated from macaque A99171 were pulsed with peptide (filled squares) or media alone (open squares), labeled with ⁵¹Chromium, and used as targets for the autologous IE-specific CD8⁺ T cell clone that was used for adoptive transfer. (B) Expansion of endogenous and transferred CMV-specific T cells by infusion of T-APCs. A dose of 1 × 10⁷ T-APCs/kg was administered to macaque A99171 nine weeks after infusion of a CD19⁺ T_CM-derived clone. The number of CD3⁺CD8⁺ T cells and CD19⁺CD8⁺ T cells/μl of blood was measured by flow cytometry prior to and on indicated days following the T-APC administration. The number of endogenous IE-specific CD8⁺ T cells was measured by cytokine flow cytometry at the same time points after gating on CD19^–CD3⁺CD8⁺ cells. The data show the fold increase in the absolute numbers of CD3⁺CD8⁺ T cells (white bars), CD19⁺CD8⁺ T cells (gray bars), and CD19^– IE-specific CD8⁺ T cells (black bars) after the T-APC infusion.