The CD8/CD4 T-cell ratio is increased by combination treatment of IDX and cis, potentially potentiating the tumor-killing ability of T cells
On the basis of the above findings, we selected a cis concentration of 1.2 µM (thereafter referred as Cis1.2) and IDX concentrations of 1 and 2 µM (thereafter referred as IDX1 and IDX2) for the subsequent experiments. We first confirmed that the selected doses elicited <5% toxicity toward immune cells (Supplementary Figure 2A). Next, we evaluated the effects of IDX, cis or IDX+cis combination treatments on immune lineage markers (CD4 T helper cells, CD8 T cytotoxic cells, CD14 monocytes, CD19 B cells and CD56 NK cells) within the cocultures. We first performed an unsupervised high-dimensional data analysis using the t-distributed stochastic linear embedding (t-SNE) algorithm (Figure 3A). cis1.2-treated cancer cells showed similar t-SNE coordination in clusters that overlapped with untreated cancer cells (DMSO). A unique pattern was identified when PBMCs were cocultured with IDX1-treated cancer cells, where both numbers of CD4+and CD8+ subsets were substantially diminished. This effect was restored when cells were treated with IDX2, which was further upregulated upon IDX+cis combined treatment (Figure 3B). We then focused on cells expressing CD4 and CD8. Figure 3C illustrates that treatment with IDX2 alone and IDX2+Cis1.2 significantly induced the frequency of CD8+ cells as compared to DMSO. Specifically, CD8/CD4 ratio in the cocultures increased upon treatment with IDX1 and IDX2+Cis1.2 (Figure 3D).
Having shown that treatment with IDX alone or IDX+cis in cancer cells could drive T cell induction, we next sought to determine whether the tumor-primed-T cells could elicit effector functions. PBMCs post-cocultured with treated cancer cells were collected and further expanded with IL-2 to achieve enriched primed-T cells. Based on the cytotoxicity assay, primed-T cells which were previously educated by IDX1/IDX2+cis1.2-treated cancer cells were capable of killing tumor cells in an effector-to-target ratio-dependent manner (Figure 3E). These data clearly indicate that IDX and cis treatment alone or in combination not only directly mediate tumor cell apoptosis, but can also prime T cells toward acquiring an increased killing ability, confirming the critical role of these treatments in making the tumor more susceptible to T-cell-mediated killing.