IDX administration induces differential expression of activation and homing markers in T cells infiltrates
To assess which particular T cells infiltrate the spheroids upon IDX treatment, we determined T cell subsets based on expression patterns of 10 cell surface markers through flow cytometry. We observed dissimilar patterns of infiltrating cells following IDX treatment (blue) as compared to control (pink, Figure 6A). T cell phenotypes (CD3, CD4, CD8), followed by memory (CD45RO) and naïve (CD45RA) subsets were characterized which were thereafter gated for lymph node homing receptors (CD62L and CCR7) and activation markers (CD27 and PD1). Among those biomarkers, we observed substantial different surface expressions for CD3, CD62L and PD1 upon treatment with IDX in the cocultures. To technically validate our findings, we explored the T cell panelvia automatic gating by performing multivariate clustering of cells based on a self-organized map (SOM) algorithm. Representative rose-charts of the clusters illustrated an up-regulation of CD3 expression (Figure 6A, a), and reduction in CD62L expression (Figure 6A, b). Surprisingly, by performing k-means clustering and generating SOM, we observed fluctuated expression of PD1 in cocultured PBMCs upon IDX2 treatment. Interestingly, an inverse correlation was observed, such that upregulated PD1 expression correlated to reduced CD45RO expression (Figure 6A, c).
By comparing CD3+ cell populations IN and OUT of the spheroids, we observed a significant increase in DP T cells in the tumor structure upon IDX2 treatment (Figure 6B). Additionally, tumor-infiltrating DP T cells displayed a pronounced reduction in CD62L, but not CCR7, expression compared to control (Figure 6C), suggesting that DP T cells could have a particular advantage toward spheroid infiltration following IDX treatment. Through gating analysis on CD3/CD4/CD8+ T cells following IDX-treated coculture, infiltrating DP T cells showed decreased proportions of CD45RO+ CD27+ memory cells and increased proportions for CD45RO-CD27+ naïve cells, as compared to control (Figure 6D, Supplementary Figure 3C). Finally, additionally to the above findings (Figure 6A, c), we detected a significant reduction in PD1+ expression in total infiltrating memory cells compared to DMSO controls (mean 48.57±5.117 vs. mean 62.06±3.066; p = 0.0086; Figure 6E). In contrast, we found significant up-regulation in PD1+ expression in naïve infiltrated cells upon IDX treatment as compared to DMSO (mean 62.10±7.049 vs. mean 43.57±3.870; p=0.0017). These results suggest that DP memory T cells are prone to infiltrate spheroids with a weak exhaustion profile upon IDX treatment.