Figure 5. Analytic phenotyping of T cell memory subpopulations . Differentiation profiles of the T cell compartment were identified by combination of surface marker and intracellular cytokine detection. Donor PBMCs freshly isolated from buffy coats were stimulated to induce cytokine expression, and CD8+ and CD4+ populations were gated into T cell memory subsets dependent on surface marker expression: naive (CCR7+/CD45RO-), TCM (CCR7+/CD45RO+), TEM (CCR7-/CD45RO+) and TEMRA(CCR7-/CD45RO-). Stochastic Neighbour Embedding (t-SNE) analysis was used to cluster cellular sub-populations. (A) Manual gating overlays of an exemplar demonstrate the distribution of CD8 and CD4 memory subtypes in the total lymphocyte population. (B) The data were also analyzed for individual cytokines by fluorescence intensity. Each subtype was then quantified for expression of single or multiple cytokines as outlined in the gating strategy. (C) The relative frequency of cytokine subpopulations from representative PBMC demonstrates changes in cytokine expression throughout T cell differentiation (mean of n=6). (D) Exemplar of t-SNE analysis of a T cell product from initial PMBC fraction through to final product indicating the accumulation of TCM / TEM CD8 T cells over time.