Discussion
Immunological checkpoint–based anti-tumor therapy has attracted extensive attention. Reversing exhaustion by blocking PD-1 is a promising target for cervical cancer treatment. Although the blockade of PD-1 and its ligand may be of use in the treatment of cervical cancer, there also exist other checkpoint molecules, such as TIGIT and Tim-3 that play an important role in T and NK cell exhaustion [17, 18]. However, only a few studies have explored the expression of these immune checkpoints on T cells from cervical cancer patients [10, 11], and the expression of these three receptors on CD56dim and CD56brightNK cells from cancer patients is still unknown.
The expression of peripheral blood PD-1+ NK cells has already been reported in healthy donors and patients with ovarian carcinoma [19]. In that study, PD-1 expression was found to be restricted to the mature NK cell population (NKG2AnegKIR+CD57+CD56dim NK cells), which is characterized by high CD16, perforin, and granzyme expression [20]. However, and similar to our results, a more recent study Liu, Y et all observed PD-1 to be expressed in both CD56bright and also CD56dimNK cells, from the blood or tumor infiltrates, in patients with digestive cancers including esophageal, liver, colorectal, gastric and biliary cancer [21].
In our study, we found a rare population of PD-1+CD56bright NK cells increased in the blood of cervical cancer patients. This population also co-expressed TIGIT and or Tim-3. Although CD56dim NK cells are the typically cytotoxic NK cell subtype, some studies have shown that following activation, CD56bright NK cells are equally or even more cytotoxic [22-24]. This might suggest that the expression of different immune checkpoint molecules on CD56bright NK cells in cervical cancer patients might play an important role in the inhibition of cytotoxicity in some tumor environments.
Of course, the traditional role of CD56bright NK cells has to be taken into consideration. These cells are classically considered the cytokine producing subset of NK cells. With respect to this, the roll of immune checkpoint expression on IFN-γ production has been investigated in peripheral blood PD1+CD56bright NK cells from asymptomatic pediatric thoracic transplant patients with lymphoproliferative disorders [25]. This group observed that those cells down‐modulate the activating receptors NKp46 and NKG2D while up‐regulating PD‐1. These phenotypic changes were associated with decreased production of IFN‐γ, an effect that was abrogated after disruption of the PD‐1 inhibitory pathway. However, the authors suggest that these defects were partially PD‐1 independent. This then suggests that it is important to evaluate the impact of other immune checkpoints such as TIGIT and Tim-3 in the functional activity of this subset of NK cells.
It is also important to note that great variety was observed in the controls with respect to the PD-1 population in NK cells; controls recovering from transient viral infections might explain this variation. Indeed, it has been noted that chronic asymptomatic HCMV infections may explain persistently high PD-1 levels in normal controls [19].
Returning to the CD56dim NK cells, our data showed both a significant increase in this population in cervical cancer patients in comparison with controls, and increases in PD-1 and TIGIT and/or Tim-3 co-expression. Here it is notable to observe that looking at only TIGIT or only Tim-3 we did not see significant increases. This might induce one to believe that these checkpoint markers are not important in cervical cancer and precursor lesions. However, when one examines double and triple expression of all three checkpoint molecules together the importance of all three become evident.
The increase of CD56dim NK cells in patients with cervical cancer was accompanied by a decrease in the percentage of T cells. We observed a significant negative correlation when we compared both lymphocyte populations in the cancer group. This suggests, that at the peripheral level at least, there is a compensatory relationship between cytotoxic cells; that is, that a loss of one cytotoxic population is balanced by an increase in another.
The role that checkpoint molecules may play in complementary, but not identical cytotoxic populations is still not very well understood. Here we observed in cervical cancer patients that while PD1+ NK cells were less than 50% of the total NK cells (Range= 4.21-47.8%), PD-1 expression on T cells could reach 77% (Range=39.01-77.48%) of the CD3 cells. In contrast, we observed that TIGIT or Tim-3 is expressed much more highly on NK cells (Range= 62.02-95.51%; 11.19-88.97%, respectively) in comparison with T cells (Range= 32.97-71.92%; 2.69%-51.74%) in cervical cancer patients. These results are consistent with the experiments performed on checkpoint molecules in NK and T cells from tumor-bearing mice and patients with colon cancer [18], where TIGIT expression was observed to be higher on NK cells and was associated with NK cell exhaustion. This same paper also found that PD-1 and CTLA-4 were expressed mainly by tumor-infiltrating T cells and were barely expressed by tumor-infiltrating NK cells. Thus, in accordance with that report, it is possible that peripheral NK cell activity might be mainly regulated by TIGIT and Tim-3 in cervical cancer patients.
When comparing the percentage of immune checkpoints on T cells from cervical cancer patients and healthy donors, we found a significant increase in the percentage of PD-1+ and TIGIT+ T cells in the cancer group. We also observed a greater proportion of T cells in cervical cancer patients with a double (PD-1+TIGIT+ and PD-1+Tim-3+) or triple positive (PD-1+TIGIT+Tim-3+) co-expression of immune checkpoint molecules. As noted above with NK cells, focusing on double and triple positive cells allowed us to see significant populations that were not visible when focusing on single positive groups.
In our T cell samples, we observed a wide range PD-1 expression levels on individual cells. Other groups have reported on this diversity of expression, and based on those works we began by subdividing our PD-1 positive T cells into six different groups, which we eventually condensed down to three broad gates: PD-1 low, PD-1 intermediate (int) and PD-1 high (hi). Our results show that the difference between patients and controls is most stark and significant when comparing PD-1 intermediate or PD-1 high expressing cells. Others have also noted that there might exist a difference between low or basal PD-1 expression, and higher levels, indicating more complete exhaustion[26, 27] [16]. For example, different levels of PD-1+ have been reported on CD8 T cell subsets expressing LAG-3 and PD-1 after chronic stimulation within tolerizing environments [28]. In this work, the authors found that CD8 T cells could differentiate into either LAG-3+PD-1int, LAG-3negPD-1int, or PD-1high. These subsets were found to show distinct phenotypes and functional properties as well; CD8 T cells expressing the highest levels of PD-1 (PD-1hi) were not capable of producing IFN-γ, while in contrast, cells expressing an intermediate level of PD-1 could produce IFN-γ.
Therefore, we focused our analysis of the PD-1+ T cells into two PD-1 population: high and intermediate; as the PD-1low population was very common in controls, we did not continue our analysis with this population. Our data demonstrated, for the first time, to our knowledge, that in cervical cancer patients the percentage of PD-1int and PD-1high CD3+ cells is significantly higher in patients as compared with healthy donors. Interestingly, and also not reported elsewhere, we also saw a greater percentage of CD3+ cells with intermediate and high expression of PD-1 co-expressing TIGIT and Tim-3 as well. Together these results suggest that in peripheral blood from cervical cancer patients it is possible to find T cell populations expressing PD-1 at different levels and co-expressing other immune checkpoint molecules (TIGIT and Tim-3). These types of cells show a pattern of immune checkpoint receptor expression previously observed in tumor-infiltrating CD8 T cells from patients with non-small-cell lung cancer, where distinct expression levels of PD-1 show a different spectrum of exhaustion [16].
It is important to note that the DNAM-1 receptor plays a role in T cell and NK cell-mediated cytotoxicity against tumors via its interaction with CD155 and CD112 [29]. When comparing healthy donors and cervical cancer patients we did not see significant changes in the expression of DNAM-1 on NK cells and T cells (DNAM-1 expression was uniformly high, above 90% of cells -we should note, however, that a decreased percentage of T cells expressing this receptor was observed in the LG group, but this same group also had lower T cell expression of TIGIT, suggesting lower activation of these cells in these patients). It has been reported that the expression of DNAM-1 is downregulated during chronic HIV infection [30]. It is also known that the same ligands that activate cells via DNAM-1 (CD155 and CD112) may also lead to inhibition via binding with TIGIT [29]. For this reason, we investigated the co-expression of both these receptors on NK cells and T cells. What we found was a significant increase in the percentage of DNAM-1+ TIGIT+CD3+ cells (and a decrease of the DNAM-1+ TIGITnegCD3+ population, data not shown) in our cervical cancer group. This might indicate that emerging TIGIT is playing a dominant role in some of these previously activated DNAM-1+ T cells. For CD8 T cells, the TIGIT / DNAM-1 axis has emerged as an inhibitory / stimulatory receptor pair of importance for CD8 T cell function, similar to CTLA-4 and CD28 [31].
It also must be noted that the analysis of these cells in the tumor environment, not just peripheral blood, is absolutely essential. In a recent study [27], Pesce, Silvia et all. compared the NK cell phenotype in patients affected by peritoneal carcinomatosis versus healthy donors and observed that the features of the tumor-associated PD-1+ NK cell subset are different from those of the subset present in healthy donors. Another study showed an increase in PD-1 expression on infiltrating T cells from CIN grade II-III patients versus CIN 0 patients [10]. A recent study on multiple checkpoint molecules in T cells from blood and tumors from different samples found PD-1, TIGIT and Tim-3, among other receptors, to be markedly increased in tumor infiltrating cells compared to peripheral blood [32].
The increase in soluble PD-L1 in CC was notable. We observed a sharp increase in only 6/24 CC patient samples. This suggests that liberation of soluble PD-L1 may be a phenomenon seen only in certain most advanced patients where escape from immuno surveillance has been modulated by the PD-1/ PD-L1 system, and that these patients are those most likely to respond positively to PD-1 or PD-L1 blockade. When we evaluated the levels of sPD-L1 with respect to T cell PD-1 percentages we noted that these six patients also were among the highest of the PD-1 positive patients, and also tended to have elevated double and triple positive cells. It is also possible that the presence of sPD-L1 may indicate patients that would not respond well to some forms of PD-L1 blockade antibody therapy as it has been reported that secreted PD-L1 variants can mediate resistance to PD-L1 blockade by acting as a decoy [33]. Continuing with the idea that sPD-L1 or the presence of highest PD-1 or TIGIT and Tim3 levels might serve as a marker for advancement of the tumor development, we classified our samples with respect to tumor subtype and grade. We did not find any association, thus indicating that the expression of these cell types and sPD-L1 is not directly related with the development of the tumors, but instead may identify select patients with tumors at different stages that would be uniquely amenable to current and emerging therapies targeting PD-1, TIGIT and Tim-3.