Fig. 4. Naïve CD8 T cells require cell-to-cell contact to acquire activated/memory phenotype.
a Schematic view of the experimental approach showing that naïve and memory TCM CD8 T cells were sorted from healthy adults then labeled with CTV and CFSE respectively. CFSE-labeled TCM cells were stimulated with anti-CD3/CD28 beads (1:1 ratio). After overnight stimulation, beads were removed by plate magnet followed by collection of cell-free supernatant, which then was added to unmanipulated CTV-labeled naïve CD8 T cells. Following 7 days of incubation, activated/memory T cell phenotype was examined in CTV-labeled naïve CD8 T cells. b Schematic view of the transwell system experimental approach showing sorted naïve CD8 T cells were cultured on the top well of transwell system while activated memory TCM CD8 T cells were cultured at the bottom well. c Representative flow cytometry plots and d Box plot showing the incidence of CTV-labeled naïve CD8 T cells that acquired activated/memory phenotype in the presence of cell-free supernatant from stimulated TCM CD8 T cells compared to co-culture conditions (n = 4). e Representative flow cytometry plots and f Bar-graph showing the incidence of CTV-labeled naïve CD8 T cells that acquired an activated/memory phenotype (upregulation of CD45RO) in the presence of stimulated TCM CD8 T cells but separated from each other using the transwell system compared to co-culture conditions (n = 5). Blue closed circles depict conditions were naïve CD8 T cells were cultured at the top (n = 2) while green closed circles represent conditions were naïve CD8 T cells cultured at the bottom (n = 3). To examine if there was a carry-over effect of beads following beads removal, medium was added to CTV-labeled naïve CD8 T cells after beads removal (n = 3). **P < 0.01 *P < 0.05 Unpaired nonparametric Mann-Whitney test was used. Data are presented as means ± SEM.