Immune checkpoint profiling of CD56-CD3+ T cells
In order to visualize the co-expression of TIGIT, PD-1, Tim-3, DNAM-1 and NKG2D in T cells at the single-cell level within the CD56-CD3+ T cell populations of the study groups, a high-dimensional t-SNE analysis was performed. The results showed a different pattern of immune checkpoint co-expression between CC, HG, LG and HD groups (Figure 4 A). In contrast with the single marker cytometry (Figure 1) that showed a loss of CD3+ T cells in patient groups, when we analyzed PD-1 expression in these cells, a significant increase was observed in all patient groups. That is, of the fewer T cells encountered in the patient groups, PD-1 expression was increased on these cells compared to T cells in HD. Next, analysis of the expression showed that, similar to the NK cells, expression of TIGIT and Tim-3 was not significant, but when double (PD-1+TIGIT+) and triple positive (PD-1+TIGIT+Tim-3+) cells were analyzed, these populations were found to be significantly increased (up to 1.4 fold and 1.9 fold, respectively) in patient groups (Figure 4 B-G). A correlation test was performed to assess the association between PD-1 and TIGIT or PD-1 and Tim-3 in T cells from HD and CC. Those analyses did not show a significant correlation between the groups (data not shown).
Focusing on the activating receptors NKG2D and DNAM-1, our results were less clear. Here we found NKG2D (Figure 5 A) was significantly increased in LG and HG groups, while we did not observe any difference in the CC group. However, we did observe a population of PD-1+ T cells that were NKG2D positive and significantly (over 2 fold in HG) increased in all patient groups (Figure 5 B). Interestingly, when we evaluated PD-1 positive cells for lack of NKG2D expression, we saw that these PD-1+ NKG2Dneg cells were also significantly increased in HG and CC patients versus healthy controls, though not to the same extent (Figure 5 C). Similar to what was observed with the NK cells, DNAM-1 was found to be expressed in about 95% of the T cells, thus expression of DNAM-1 showed no significant change between HD and HG or CC; there was a small but significant decreased in LG patients. However, we found a significant increase of cells that were positive for both DNAM-1 and TIGIT markers in CC (Figure 5 D-E).