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.