Immune checkpoint profiling of CD56bright and CD56 dim NK cells
In order to identify unique populations of cells in our patient samples, we utilized t-SNE analysis of our populations and markers (Figure 2 A). In CD56dim NK cells, we found that PD-1 was significantly increased in LG, HG and CC compared to controls (Figure 2 B). Here we observed that a subset of the patients had PD-1 levels much higher than the median of the controls, up to five and six and fold higher in the case of some of the cell populations. This distribution was not observed on the other immune checkpoints investigated. As PD-1 expression, albeit in T-cells, is already well characterized in cervical cancer patients, we used the t-SNE analysis to identify expression patterns that appeared linked with high PD-1 expression in cervical cancer. PD-1 and Tim-3 appeared to give similar t-SNE patterns, and it appeared a subset of the PD-1 positive cervical cancer cells might also be positive for TIGIT and perhaps other subsets might be positive for NKG2D and DNAM-1 (explored below). Focusing first on the inhibitory checkpoint molecules, we saw that, with the exception of an increase in Tim-3 in LG patients, changes in overall TIGIT and Tim-3 were not significantly different between patients and controls (Figure 2 C, D). However, when cells were gated (Supplemental Figure 1) on double and triple positive co-expression, i.e. PD-1+TIGIT+, PD-1+Tim-3+ and PD-1+TIGIT+ Tim-3+populations, significant increases were observed (Figure 2 E-G). Co-expression of NKG2D, but not DNAM-1, was also increased in patient groups (Supplemental Figure 2).
While absolute percentages of the CD56bright NK cells were not changed between groups, a differential expression pattern of immune checkpoints in the CC, HG, and LG groups (Figure 3 A-G), in particular on PD-1 and Tim-3 molecules, was found, with an over 2.6 fold PD-1 and Tim-3 increase, and an approximately 3 fold triple positive increase compared to HD. No significant changes were observed on the co-expression of NKG2D or DNAM-1 (data not shown).