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.
  1. Result
  2. 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.