American Society of Hematology

Figure 2

$Figure 2. Virus-specific cytotoxic T lymphocytes from CB are derived from the CD45RA+/CCR7+ naive T-cell population. (A) With the use of flow cytometry, T cells from 3 CB units were gated on CD3+ cells and then sorted for cells that were either double positive for both CD45RA and CCR7 or double negative for CD45RA and CCR7. Each of these populations was then stimulated with transduced autologous antigen-presenting cells. Indicated are percentage of CD45RA−/CCR7− T cells versus percentage of CD45RA+/CCR7+ T cells that were selected. (B) After 3 stimulations, the phenotype of the CTL lines derived from cells that were either double positive for both CD45RA and CCR7 (CD45RA+/CCR7+) or double negative for CD45RA and CCR7 (CD45RA−/CCR7−) was determined by flow cytometry. (C) Virus-specific activity of the CTL lines was determined after 3 stimulations by the IFN-γ ELISPOT assay in response to direct stimulation with CMVpp65 PepMix (CMVpp65), CMVIE1 PepMix (CMVIE1), Adenovirus hexon PepMix (Adv hexon), irradiated autologous EBV-LCL at an E/T ratio of 4:1 (EBV) or PMA-ionomycin (PMA-I). ELISPOT analysis of a T-cell line derived from the CD45RA and CCR7 double-positive fraction versus a T-cell line derived from the double-negative fraction from a representative CB unit from a CMV-seronegative mother is shown. Mean values (± SDs) of triplicate experiments are reported.$

Virus-specific cytotoxic T lymphocytes from CB are derived from the CD45RA⁺/CCR7⁺ naive T-cell population. (A) With the use of flow cytometry, T cells from 3 CB units were gated on CD3⁺ cells and then sorted for cells that were either double positive for both CD45RA and CCR7 or double negative for CD45RA and CCR7. Each of these populations was then stimulated with transduced autologous antigen-presenting cells. Indicated are percentage of CD45RA⁻/CCR7⁻ T cells versus percentage of CD45RA⁺/CCR7⁺ T cells that were selected. (B) After 3 stimulations, the phenotype of the CTL lines derived from cells that were either double positive for both CD45RA and CCR7 (CD45RA⁺/CCR7⁺) or double negative for CD45RA and CCR7 (CD45RA⁻/CCR7⁻) was determined by flow cytometry. (C) Virus-specific activity of the CTL lines was determined after 3 stimulations by the IFN-γ ELISPOT assay in response to direct stimulation with CMVpp65 PepMix (CMVpp65), CMVIE1 PepMix (CMVIE1), Adenovirus hexon PepMix (Adv hexon), irradiated autologous EBV-LCL at an E/T ratio of 4:1 (EBV) or PMA-ionomycin (PMA-I). ELISPOT analysis of a T-cell line derived from the CD45RA and CCR7 double-positive fraction versus a T-cell line derived from the double-negative fraction from a representative CB unit from a CMV-seronegative mother is shown. Mean values (± SDs) of triplicate experiments are reported.

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