Differing PD-1 expression identifies sub-populations with higher significance
Additionally, we assessed whether PD-1 positive T cells expressed PD-1 at different levels according to the PD-1 MFI (Figure 6 A). We found that PD-1 low T cells were not significantly increased between controls and patients, while two other gates that we labeled intermediate (PD-1int) or high level (PD-1hi) identified PD-1 positive populations that were significantly increased in patients with cancer and precursor lesions (Figure 6 B, F).
We also investigated whether PD-1int (Figure 6 C-E) and PD-1hi (Figure 6 G-I) T cells co-expressed TIGIT and Tim-3. We saw a significant increase of the PD-1intTim-3+ population in the CC group in comparison with the HD group (Figure 6 D). Interestingly, we also found a significantly higher percentage of PD-1int and PD-1hiCD3+ cells co-expressing TIGIT. The percentages of those triple positive (PD-1-TIGIT-Tim-3) PD-1int and PD-1hi CD3+ cells were also increased in cancer patients (Figure 6 E, F), which might suggest an exhausted T cell phenotype. It was notable that the stratification of the PD-1 positive T cells allowed visualization of more significantly separated populations of cells with checkpoint markers between HD and CC, and higher levels of significance. PD-1hi, and both PD-1hi TIGIT+ and PD-1hiTIGIT+Tim-3+cell populations were also significantly increased in LG, and LG and HG respectively (Figure 6 F-I).