2.8. Statistics.
We analyzed data using GraphPad Prism6. To determine whether parametric
or non-parametric statistical analyses were required, D’Agostino and
Pearson omnibus normality tests were conducted. Statistical significance
was based on p=0.05.
- Result
- Increased percentages of PD1, PD-L1 and related cytokines in
serum in the development of CC patients compared with NCs.
We investigated the expression of PD1, PD-L1, IL-2, IL-6, IL-10, TGF-β
and INF-γ in serum in CC, CIN, Chronic Cervicitis, HPV+ patients and NC
group simulated the development of CC(Fig.1A-H ). Through
comparative analysis, we found that in each detection group, except for
the INF-γ group, the expression level of CC group was the highest, and
the difference was significant compared with the NCs, which was
statistically significant. In addition to CC group, CIN, Chronic
Cervicitis, and HPV+ group, independent t-test analysis was also
conducted respectively with NC group, and the results showed
differences, some with significant statistical differences, such as
Figure1A, B, C, G and H. Some showed no significant difference, as shown
in Figure1D, E and F. One-way ANOVA was performed for each test group,
and the differences were statistically significant. Therefore, we
concluded that, except for INF-γ, PD1, PD-L1, IL-2, IL6, IL-10 and TGF-β
expression in CC was significantly higher than that in normal healthy
people, in which IL-2, IL-6, IL-10, PD1 and PD-L1 showed a significant
upward trend in the occurrence and development of CC, and the expression
levels were all higher than that in the NC group, with significant
differences.
The expression of tregs and PD1 in peripheral blood in CCs
higher than NCs.
Peripheral blood samples of CC, CIN, Chronic Cervicitis, HPV+ and NC
group were collected, PBMC was isolated, CD4, CD25, CD127 and PD1
antibodies were stained, and analyzed by flow
cytometry(Fig.2A-C ). The main objective was to investigate
whether the expression level of PD1 in
tregs(CD4+CD25+CD127-/low),
PD1 in total cells and tregs changed during the development of CC. The
expression of these three indexes was statistically different by one-way
ANOVA analysis(Fig.2D-F ). The independent sample T-test
analysis showed that the expression of CC was significantly increased
compared with that of the NC group, and the difference was statistically
significant, while the Chronic Cervicitis and HPV+ group had no
significant difference(Fig.2D-F ).
The expression level of PD1, PD-L1 and related cytokines in CC
patients before and after surgery.
Follow-up tracking five in the surgical treatment of CC patients, from
preoperative to within six months after the operation. Plasma was
collected in stages and detected by ELISA for PD1, PD-L1, IL-2, IL-6,
IL-10, TGF-β and INF-γ (Fig.3a-h ). According to the curve
analysis, the expressions of PD1, PD-L1, IL-2, IL-6, IL-10, and TGF-β
gradually decreased and tended to be flat with the time after the
operation. However, the expression of INF-γ did not change significantly
in relation to time. Therefore, we conclude that the occurrence and
development of cervical cancer are associated with increased expression
levels of PD1, PD-L1, IL-2, IL-6, IL-10, TGF-β and no definitive
association with INF-γ was found in this study.
In situ localization of related cytokines,tregs , PD1 and
PD-L1 in CC tissues.
Two parts of CC tissue samples were selected, one was tumor tissue, and
the other was normal tissue at the edge of the surgically removed
tissue. Firstly, histoimmunochemical staining was performed to detect
CD4, CD25, Foxp3, PD1, PD-L1, IL-2, IL-6, IL-10, TGF-β and INF-γ,
respectively, to understand the expression position and level of these
indicators in CC tissues(Fig.4 ). According to
histoimmunochemical analysis, a large number of CD4+,
CD8+ and Foxp+ lymphocyte
infiltration in CC tissue(Fig.4Ⅰ,Ⅲ,Ⅳ ), PD-L1, TGF-β shows
diffuse distribution(Fig.4Ⅵ,Ⅹ ), IL-6 mainly expressed in
extracellular(Fig.4Ⅷ ), IL-10 and INF-γ mainly expressed in the
cell membrane (Fig.4Ⅸ,Ⅺ ). Secondly, we performed multicolor
immunofluorescence staining to detect CD4, CD25, CD8, Foxp3, PD1, PD-L1,
DAPI, separately. Fig.5A-B shows that
CD8+Foxp+ and
CD4+Foxp+ tregs are diffusently
expressed in tumor tissues. As shown in Fig.5C-D , PD1 was
expressed in Foxp+ cells, and it was found that a very
small number of Foxp+ cells were coincident with
PD-L1+ cells, that is, a small number of
Foxp+ cells could express PD-L1. Comparison with the
control tissues showed that the expression levels of
CD4+, CD8+,
CD4+PD1, CD8+PD1,
Foxp3+PD1 and Foxp+PD-L1 in tumor
tissues were all higher than those in the control tissues analyzed by
imageJ software and the differences were statistically significant.Fig.6 shows the combined images of six fluorescence channels
taken under the same field of view. It was found a large number of cell
clusters with irregular nuclei in the three regions of A, B and C in the
figure, preliminarily judging the cervical tumor cells, which were
highly consistent with the regions where PD-L1 was expressed, further
confirming the conclusion that a large number of tumor cells expressed
PD-L1. In addition, a large number of CD8+ cells in
tumor cell clusters field infiltration, and a small amount of
CD4+ infiltration among them, the majority of
CD4+ cells in tumor cells surrounded gathered.
Discussion
The data from many articles show that the increased tumor infiltrating
lymphocyte cells(TILs) is associated with increased PD1/PD-L1 levels in
diverse tumors suggesting that these factors are biologically
linked(30-33).
Firstly, in order to analyze the expression of PD1, PD-L1 and related
cytokines in the occurrence and development of cervical cancer, this
study was divided into five groups: normal control group(NC),
HPV-positive group(HPV+), chronic cervicitis group, cervical
intraepithelial neoplasia group(CIN) and cervical cancer group(CC), to
simulate the development process of cervical cancer caused by continuous
infection of high-risk HPV. And plasma samples were collected from each
group, and the expressions of IL-2, IL-6, IL-10, TGF-β, INF-γ, Foxp3,
PD1 and PD-L1 were detected by ELISA. As shown in Figure 1: (1)With the continuous progress of high-risk HPV infection, the expression
of PD1 and PD-L1 in peripheral blood gradually increased, and compared
with NC, p<0.05 (independent t-test analysis), the difference
was statistically significant. (2) Except for INF-γ, the
expression level of other cytokines was higher than that of NC,
especially CC. (3) INF-γ results are different, there are no
INF-γ detected in the healthy control group, and HPV positive group is
much higher than other groups as the peak parabolic decline trend. At
the same time, the peripheral blood of the five groups was collected for
flow cytometry analysis to analyze the expression of
CD4+CD25+CD127-/lowand PD1 in the peripheral blood. It was found that the number of
CD4+CD25+CD127-/low,
PD1 and
CD4+CD25+CD127-/lowPD1+cells in the peripheral blood was significantly higher than that in the
NC group, with statistical difference, while there was no significant
difference or individual difference in the other groups. Secondly, the
patients with cervical cancer were followed up before and after surgery
to understand the changes in the expression levels of various factors in
the peripheral blood of patients. Results As shown in Figure 3:(1) The levels of PD1 and PD-L1 were significantly changed
before and after surgery, and gradually leveled off after surgery.(2) In addition to INF-γ, the expression of other related
cytokines also showed a trend of decreasing and gradually leveling off
after surgery, which was consistent with the experimental data presented
in Figure 1 . Finally, histoimmunochemical tests of tumor
tissues surgically removed from patients with cervical cancer showed a
large number of lymphocyte infiltration, such as CD4+and CD8+, and Foxp3+ cells,
including regulatory T-lymphocyte infiltration of
CD4+Foxp3+ and
CD8+Foxp3+. PD1, PD-L1 and related
cytokines were also abundantly expressed in the cancer tissues. The
pattern was as follows: (1) PD1 was mainly expressed in
infiltrating lymphocytes. (2) The distribution characteristics
of PD-L1 and CD8+ cells were diffusively distributed
throughout the whole field of vision, especially clustered in tumor
cells. (3) The interesting phenomenon is that we found that the
expression of CD4+ and Foxp3+ cells
was around the tumor cell population, but partially scattered in the
tumor cell population.
From what has been discussed above, we draw the conclusion: (1)the occurrence and development of cervical cancer are closely related to
the expression of PD1, PD-L1, and IL-2, IL-6, IL-10, the expression of
TGF-β, that is, with the development of the disease, the expression is
on the rise, while the relationship between the expression of INF-γ is
not clear from the current experimental data, which needs further study.(2) Increased CD4+ lymphocytes were found in
the peripheral blood of patients with CC, including
CD4+CD25+CD127-/lowcells, and the expression level of PD1 was increased in
CD4+CD25+CD127-/lowcells. (3) In CC tissues, there are a large number of
infiltrating lymphocytes, mainly CD8+ cells, and a
large number of them infiltrate into the tumor cell aggregation area,
while CD4+ cells gather around the tumor cells. At the
same time, the corresponding hypothesis was proposed: (1)Although an increase in
CD4+CD25+CD127-/lowcells was detected in the CC group, no significant increase was detected
in other groups. We know that
CD4+CD25+CD127-/lowcells are naturally regulated T cells, and we hypothesized that with the
development of CC, the body can be induced to produce adaptive
regulatory T cells (aTreg), such as Tr1(type 1 regulatory T cell), Th3(T
helper 3) cells. The secretion of IL-10 by Tr1 cells and TGF-β by Th3
cells can further promote the proliferation of CD8+regulatory T cells, further promote the secretion of IL-6, IL-10, INF-γ,
and play an immunosuppressive role. (2) CD4+and CD8+ T cells can infiltrate tumor areas, and it
may be that T cells dominated by CD8+ cells play an
anti-tumor role. The distribution of Foxp3+ regulatory
T cells is similar to that of CD4+ T cells, mainly
surrounding tumor cell population, and may play a role of regulating
tumor immunity mainly by secreting some cytokines, such as IL-10 and
TGF-β, etc. The above assumptions lay the foundation for the further
design of experimental scheme, and at the same time depends on further
experiments to explore the truth.