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.