Figure 2. Ex vivo differentiated T_CM and T_EM exhibit extracellular markers, intracellular molecules and killing capacity characteristic of T_CM and T_EM.

A&B. CD44 and CD62L staining of OT-I in vitro derived T_CM (black lines) and T_EM (grey lines) cultured as per materials and methods. Isotype control shown in filled grey histograms. C–E. Intracellular Interferon-γ, tumour necrosis factor-α, granzyme B expression in in vitro differentiated T_CM and T_EM following stimulation with PMA/ionomycin. T_CM shown as black lines, T_EM as grey lines and unstimulated cells shown as filled grey histograms. F. Intracellular expression of the T-box transcription factor T-bet. T_CM shown as black lines, T_EM as grey lines and naïve cells shown as filled grey histograms. All vertical axes in histograms are frequency of cells as percentage of total cells analysed. Flow cytometry plots represent one three individual experiments conducted with pooled lymphocytes from OT-I mice. G&H. Micrograph of OT-I T_EM (G) and T_CM (H). Photos represent one of two individual experiments using pooled lymphocytes. Scale bar = 20μm. I & J. In vitro cytotoxic killing assays of T_CM (black lines) and T_EM (grey lines) demonstrating chromium release following 2.5 hours (K) and 5 hours (L) of exposure to SIINFEKL-pulsed (sold lines) and non-pulsed targets (broken lines). **p<0.01 by one-way ANOVA test with Bonferroni post-hoc analysis. Target lysis calculated by [(sample − spontaneous)/(maximum − spontaneous)] × 100%.