Activated/memory T cells are able to infiltrate tumor spheroids
upon IDX treatment
(A) To enable an unbiased analysis of data, we selected live cells and
then used t-SNE to reduce dimensionality, followed by a graph-based
heat-map clustering analysis. Representative t-SNE analysis of
concatenated mononuclear cells (2,000 cells/sample) from “IN”
compartment done by pooling 8 wells/condition of infiltrating tumor
immune cells upon IDX treatment. Merged t-SNE plot (left panel) for drug
treated and untreated cocultures, with each cluster indicated by a color
(DMSO in pink; IDX2 in blue). Data were further clustered according to
criteria used to define known T cell subsets: CD3, CD4, CD8, CD62L,
CD27, CCR7, CD45RO, CD45RA and PD1. Automatic gating by Flowjo performed
multivariate clustering of cells based on self-organized map algorithm
and categorized all individual cells into 100 distinct clusters based on
the surface expression marker proteins. Most prominent differential
protein expressions CD3 a), CD62L b) and PD1 c) are highlighted in
representative rose-charts boxes. (B) Flow cytometry analyses of T cells
(respectively gated CD3+ ) as well as
CD4+, CD8+ an double-positive T
cells subsets (respectively gated
CD4+ CD8-,
CD4-CD8+ and
CD4+ CD8+ among
CD3+) percentages in the IN and OUT compartments, in
the presence or without IDX at 72 hours. (C) Flow cytometry analyses of
migratory T cells (respectively gated
CD62L+/CCR7+ of
CD3+) on CD4+,
CD8+ an double-positive T cells subsets, percentages
in the IN compartments, in the presence or without IDX at 72 hours. (D)
Flow cytometry analyses of exhaustion (PD1+) T cells
phenotypes percentages (respectively gated
CD4+ CD8+ of CD3+) on CD45RO+CD27+ (memory T cells)
and CD45RO-CD27+ (naïve T cells )
subsets, in the IN compartments, in the presence or without IDX at 72
hours. (E) Quantitation of flow cytometric data on the percentage of
PD+ and PD1- memory T cells and
naïve T cells in the presence of IDX relative to control (DMSO). *
p≤0.05, ** p<0.01 when compared to DMSO control.