T cell activation assays were performed using primary CD31+ CD8T cells purified from PBMC or SF (negative selection with Rosette Sep, Stem Cell Tech). Jurkat and JRT3 cells (ATCC) were used as internal system controls. Activation bioassays with 2 μg/ml anti-TCRαβ (1P26, Biolegend) or 5 μg/ml anti-CD3 (OKT3, Orthoclone) followed established procedures (15 (link), 20 (link)). For stimulations using anti-CD31 (WM59, eBioscience) and recombinant CD38-Ig (R&D Systems), optimal concentrations of 1 and 10 μg/ml, respectively, were empirically determined. Stimulating agents were either cross-linked in solution with species-specific anti-Ig, or immobilized in tissue culture plates following published procedures (15 (link), 20 (link)). All activation assays included Ig isotype and media only controls.
At the indicated time periods, cells were harvested and immunostained for CD25, CD69, phospho-tyrosine (4G10, Millipore), and granzyme/CD107a (BD). The latter two molecules were determined from permeabilized cells using Cytoperm reagent (BD). For granzyme detection, Golgi Plug/Stop reagent (BD) was added to cultures during the last 6 hours of incubation. Markers of cell activation were measured by multicolor flow cytometry as described above. The culture supernatants were also harvested, and the cytokine content measured by the Luminex system (6 (link), 15 (link)).
Proliferation assays on primary T cells in PBMC or SF were performed using standard bioassays using 5- (& 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (6 (link)). Subsets of dividing or non-dividing cells were identified by immunostaining for CD4, CD8, CD28, CD31, and γH2AX after 5 days in culture.
For the analysis of in vitro senescence cultures, purified CD28+ T cells from PBMC were subjected to repeated stimulations with irradiated allogeneic PBMC and EBV-transformed lymphoblastoid cells, anti-CD3 (OKT3), and recombinant interleukin (IL)-2 (Proleukin, Chiron) as described previously (21 (link)). Cell phenotypes of the cultures were determined by flow cytometry at 7 days after each stimulation cycle.
For T cell population doubling analysis, cultures of T cells from PBMC or SF were monitored according to the procedure of Koetz et al (22 (link)).
At the indicated time periods, cells were harvested and immunostained for CD25, CD69, phospho-tyrosine (4G10, Millipore), and granzyme/CD107a (BD). The latter two molecules were determined from permeabilized cells using Cytoperm reagent (BD). For granzyme detection, Golgi Plug/Stop reagent (BD) was added to cultures during the last 6 hours of incubation. Markers of cell activation were measured by multicolor flow cytometry as described above. The culture supernatants were also harvested, and the cytokine content measured by the Luminex system (6 (link), 15 (link)).
Proliferation assays on primary T cells in PBMC or SF were performed using standard bioassays using 5- (& 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE) (6 (link)). Subsets of dividing or non-dividing cells were identified by immunostaining for CD4, CD8, CD28, CD31, and γH2AX after 5 days in culture.
For the analysis of in vitro senescence cultures, purified CD28+ T cells from PBMC were subjected to repeated stimulations with irradiated allogeneic PBMC and EBV-transformed lymphoblastoid cells, anti-CD3 (OKT3), and recombinant interleukin (IL)-2 (Proleukin, Chiron) as described previously (21 (link)). Cell phenotypes of the cultures were determined by flow cytometry at 7 days after each stimulation cycle.
For T cell population doubling analysis, cultures of T cells from PBMC or SF were monitored according to the procedure of Koetz et al (22 (link)).
