Optimised phenotyping and t-SNE analysis for T cell products
Conventionally, T cell memory subtypes are identified through co-expression of homing receptors such as CD62L and CCR7 with leukocyte common antigen (CD45) isoforms RO and RA, but these memory types can also be classified by cytokine co-expression (Supplementary Figure 3). We characterised T cells from start material through to final product for memory / differentiation status using a combined surface marker/ intracellular cytokine panel, and used t-SNE analysis to simplify multi-parameter phenotyping.
The t-SNE dimensionality reduction identified a clear sequential spatial correlation in both the CD4 and CD8 differentiation from naive (CCR7+/CD45RO-) to TCM (CCR7+/CD45RO+) to TEM (CCR7-/CD45RO+) to TEMRA (CCR7-/CD45RO-) stages. Furthermore, fluorescent intensity heat maps applied over this same t-SNE plot (Figure 5B) indicated highest intensity of IFN-γ in the CD8+ TEM region; highest intensity of TNF-α in the CD8+ TEM and CD4+ TEM regions; and highest intensity of IL-2 in the CD8+ TCM and CD4+ TCM/TEM compartments, confirming the correlation of differentiation status with cytokine profile.
As CD8+ T cells form the principal component of the final cell therapy product, the CD8+ naïve, TCM, TEM and TEMRA populations were then sequentially analysed by expression of cytokines IFN-γ, TNF-α and IL-2. The mean percentage of each cytokine subpopulation from six buffy coat-derived PBMC donors stimulated with PMA/Ionomycin is expressed in pie chart format in Figure 5C. The majority of CD8+ naïve T cells are cytokine-null, with the remaining 26.2% mostly comprising IL-2 or TNF-expressing cells. The CD8+ TCM cells predominantly express all cytokines (IFN-γ+/TNF-α+/IL2+), though there is also a distinct TNF-α+/IL2+ population (27%) within this subset which represents early central memory development. Within the CD8 TEM compartment, there is developmental transition from central memory (retention of IFN-γ+/TNF-α+/IL2+) into more effector-like IFN-γ/TNF-α co-expressing cells which form almost 50% of the TEM subpopulation. The CD8 TEMRA population shows further transition, with IFN-γ+/TNF-α+ or IFN-γ+ only T cells forming the principal population, with a small residual population retaining some TCM (IFN-γ+/TNF-α+/IL2+) characteristics. There is also an increased presence (17%) of cytokine-null T cells suggesting that some cells may have become anergic, with no functional secretory response. Importantly, this data demonstrates the limitations of trying to discern T cell memory phenotype purely through surface marker expression alone. For example, even within the conventionally defined naïve cells (CCR7+ CD45RO+) which are typically described as cytokine null, there are evident small subpopulations within that secrete cytokines.
The use of t-SNE dimensionality reduction generates a single-image analysis of complex multi-parameter flow cytometric data which can be utilised to gain a clear representation of the quality and composition of the final T cell product in comparison with the starting mixed leukocyte material (Figure 5D).