Checkpoint expression is enriched in CD8 versus CD4 T cells
We next investigated the distribution of the checkpoint markers on LG CD4 and CD8 T cells (Figure 7). As noted above, CD3+percentages were decreased in all study groups when compared with HD. Examining just the LG samples, we observed that the CD4:CD8 ratio was 1.4:1, which is in line with what we have seen in healthy samples. When we analyzed the different checkpoint markers we began to see significant overrepresentation in CD8 cells. With respect to PD-1 we saw this ratio drop to 1.14:1; with TIGIT this dropped to 0.71:1 and with Tim-3 =0.65:1 (Figure 7 A). These were all significant changes compared to the raw CD4:CD8 ratios. Next, we evaluated the co-expression of PD-1 with TIGIT or Tim-3, and the co-expression of PD-1 with both TIGIT and Tim-3. We observed that the percentage of triple positive CD8 T cells was again significantly higher than CD4 cells (Figure 7 B). We also analyzed the expression of these markers selectively in PD-1 intermediate and high T cells. Here we found that the above observations were much more pronounced, with much higher ratios of CD8 versus CD4 T cells. The most notable effect was observed in PD-1 high cells that were also TIGIT positive: these cells exhibited about a 3:1 CD8:CD4 ratio, demonstrating an enrichment of CD8 about 4 fold over the ratio found in total T cells (Figure 7 C-H).
Soluble PD-L1 is increased in cervical cancer and is not correlated with disease stage
We performed an ELISA on serum samples from CC (n=21), HG (n=9), LG (n=24) and HD (n=24) donors and observed a significant increase between HD and CC and, HD and LG (Figure 8). Of note was the fact that a minority (6/24) of the cervical cancer patients had sharply higher sPD-L1 levels (1.5 fold to 2.5 fold increase over the median HD level). We next performed a correlation test to demonstrate the positive association between high sPD-L1, and T cell PD-1 expression. While these six patients also had some of the higher levels of T cell PD-1 expression, we did not find a significant correlation (data not shown).
As a final analysis, we evaluated our CC group according to stage, and attempted to associate our most “exhausted” T or NK cells (or sPD-L1) with stage of the tumor. We found no association. When we ranked our samples according to highest quartile of triple positive or double cells, we found equivalent numbers of highest soluble triple or double positive T cells or NK cells in both the patients with IB cancer, and those with invasive stage IV cancers.
We propose as a model that cytotoxic cells, either CD8 T cells, or CD56dim NK cells or educated CD56bright NK cells, begin to express different checkpoint molecules during the response to persistent HPV infection and cervical cancer. In the course of the progression of the disease the expression of these molecules will accumulate and identify the most afflicted or exhausted cells. Identification of patients with such characteristics may highlight those uniquely able to respond to combined antibody therapies.