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B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas
Human Immunology 75 (2014) 1203–1209
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B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas Xin Wang a, Tingting Wang a, Man Xu a,⇑, Ling Xiao b, Yi Luo b, Wenlian Huang a, Yan Zhang a, Weipu Geng a a b
Department of Pathology, Molecular Medicine and Tumor Research Center of Chongqing Medical University, Chongqing, China Department of Obstetrics and Gynecology, The First Affiliate Hospital of Chongqing Medical University, Chongqing, China
a r t i c l e
i n f o
Article history: Received 14 February 2014 Accepted 6 October 2014 Available online 13 October 2014 Keywords: B7-H4 T lymphocyte Cytokine Cell proliferation Cervical carcinoma
a b s t r a c t Objective: To investigate B7-H4 expression and its correlation with the number of infiltrating T lymphocytes and cytokine production by those lymphocytes in human cervical cancer and to determine the effect of recombinant B7-H4 on the active peripheral blood T cells of the patients in vitro. Methods: B7-H4 expression was detected in 67 cases of cervical cancer using immunohistochemical staining. Tumor-infiltrating CD8+T, CD4+T, and FOXP3+ (Forkhead Box P3) T lymphocytes and their levels of IFN-c and TGF-b1 production were determined by immunofluorescent double-staining. After the peripheral blood T lymphocytes of patients were co-cultured with B7-H4, proliferation, apoptosis, and cell subtypes were analyzed using flow cytometry. Cytokines in the supernatant were detected by ELISA. Results: B7-H4 was expressed in 46% (31/67) of the cases of cervical cancer. The number of infiltrating CD8+T lymphocytes and their IFN-c production in positive B7-H4 expression cervical cancers was significantly lower than in negative B7-H4 cases (P < 0.01, P < 0.05), but there was no significant difference between cases positive and negative for B7-H4 with respect to infiltrating FOXP3+T and CD4+T cells or TGF-b1 production. After co-culture with B7-H4 for 48 h, the patients’ activated T lymphocytes were arrested at G1/G2 phase. The Ki67 positive rates of CD4+T and CD8+T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, and they were lower than in the blank group. The proportion of CD4+T and CD8+T cells decreased, but CD4+T/CD8+T and the proportion of CD4+CD25+Foxp3+T cells increased. In addition, concentrations of IL-10 and TGF-b1 in the supernatant of co-cultured T cells increased significantly (P < 0.05, P < 0.05), but that of IFN-c decreased. B7-H4 had no significant effect on apoptosis of the T cells. Conclusion: B7-H4 is overexpressed in human cervical cancers, and it is associated with lower numbers of tumor-infiltrating CD8+T lymphocytes and therefore less IFN-c production. In vitro, B7-H4 inhibits the proliferation of CD4+T and CD8+T but promotes the proliferation of Tregs and the secretion of IL-10 and TGF-b1. B7-H4 plays an important role in depressing the anti-tumor immunity of CD8+T cell in microenvironments of cervical cancer. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Cervical cancer is a malignant tumor common in Chinese women; the morbidity, the proportion of young patients, and the number of early stage cancer cases are increasing recently [1,2]. Persistent infection of high-risk human papilloma-virus (HPV) is a key inducing factor of this cancer. In most cases, HPV infection is self-limiting and can be eradicated by immune response in cervical microenvironment. The decrease in immune surveillance and clearance plays an important role in cervical cancer carcinogenesis ⇑ Corresponding author.
and development [3]. Recent studies have shown immunosuppression of tumor microenvironment to be associated with certain factors, including expression of inhibitory ligand B7s, recruitment of regulatory T cells and tumor-induced myeloid-derived suppressor cells, and up-regulation of transforming growth factor-beta (TGFb), galectin-1, indoleamine 2,3-dioxygenase, and arginase [4]. B7 family molecules, including B7-H1 (PD-L1), B7-DC (PD-L2), and B7-H4 (B7S1), combine with corresponding receptors of activated T lymphocytes and negatively regulate T cell immunity. This plays an important role in down-regulating anti-tumor immunity in cancer microenvironments [5]. Overexpression of PD-L1 and B7-DC has been observed in cervical cancer cells, and these were
http://dx.doi.org/10.1016/j.humimm.2014.10.002 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
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associated with decreasing CD8+T cell infiltration. B7-H4 is a transmembrane protein. It is expressed in human tissues in the cancers of the lung, esophagus, stomach, liver, pancreas, breast, ovary, brain, prostate, and kidney [6,7]. It combines with surface receptors of the activated T cell, thus regulating T cells’ growth, cell cycle, and immune function. It is associated with cancer progression and poorer patient outcomes [8]. The soluble form of B7-H4 has also been detected in blood samples from cancer patients. Recently, B7-H4 was found in cervical cancer cells and infiltrating cells in the matrix. It was associated with distant spread, lymph node metastasis, and poor prognosis for cervical cancer patients [9,10]. However, the relationship between B7-H4 expression and the immune status of cervical environment has not yet been explored. In cervical cancer tissue, the infiltrating T lymphocytes include CD4+T cells, CD8+T cells, and FOXP3+ regulatory T cells (Treg), and the CD8+/FOXP3+T cell ratio is an independent and prognostic factor of cervical cancer patients [11]. Data have shown B7-H4 overexpression to be associated with immunosuppression in the tumor microenvironment and with poor prognosis in patients [7,8,10]. In order to assess B7-H4 expression in cervical cancer and its effect on T-cell immunity in the cervical microenvironment, immunohistochemical and immunofluorescent staining were here used to detect B7-H4 expression in 67 cases of human cervical cancers and analyze its association with the infiltration of FOXP3+T, CD4+T, and CD8+T cells and their TGF-b1 and IFN-c production in tumors. Flow cytometry and ELISA were used to assess the effects of recombinant human B7-H4 on peripheral blood T cells’ proliferation, apoptosis, and cytokine production in cervical cancer patients. 2. Materials and methods 2.1. Specimens Here, 67 cases of cervical carcinoma (60 cases of squamous cell carcinoma and 7 cases of adenocarcinoma) were collected from pathology department of the first affiliate Hospital of Chongqing Medical University from 2010 to 2012. The average patient age was 43.12 y (median 43 y; range, 32–65 y). Patients who underwent pre-operative treatment, such as chemotherapy or radiotherapy, were excluded. Then, 30 samples of high grade cervical intraepithelial neoplasia (CIN II-III) and 30 samples of normal cervix were obtained from biopsy or uterine hysterectomy due to leiomyoma, and 4 lm sections of paraffin-embedded tissues were collected for immunohistochemical staining and immunofluorescent staining.
2.3. Immunofluorescent double-staining Immunofluorescent double-staining was performed to detect FOXP3+T, CD4+T, and CD8+T cells and TGF-b1- and IFN-c-producing cells in cervical tissues. Primary antibodies were mouse anti human FOXP3 (1:100, 14-4777-82, eBiosciences, U.S.), mouse anti human CD4, rabbit anti-human CD8, and rabbit anti-human TGF-b1 (MAB-0251, RMA-0514, RAB-0238, Maxim, China) and mouse antihuman IFN-c (1:200, ab82447, Abcam, U.K.). The secondary antibodies were goat anti mouse IgG-FTTC and goat anti-rabbit IgGRBITC (1:1200, bs-0296G-FTTC, bs-0295G-RBITC; Bioss, China). After the primary antibodies were incubated overnight at 4 °C, and each corresponding secondary antibody was incubated for 1 h at RT in dark. The slides were washed three times in PBS with 0.05% Tween 20 for 3 min after each incubation. CD8+ and CD4+ showed staining on the membrane, TGF-b+1 and IFN-c+ showed staining in the cytoplasm, and FOXP3+ showed staining in the nucleus and cytoplasm. Five photographs under 400 power fields in each section were taken and the positive cells were counted in cervical cancer tissues. The average number of positive cells in each case was used for statistical comparison. 2.4. Isolation and activation of peripheral blood T cells and co-culture with B7-H4 Fresh heparinized peripheral blood mononuclear cells from cervical cancer patients (n = 10) were collected using the Ficoll-Hypaque gradient. After these cells were passed through a nylon brush column (Kasker Co., German), the lymphocytes were collected. Some 5 105 cells were cultured with phytohaemagglutinin (PHA) 10 lg/ml (Sigma, U.S.) at 37 °C in humidified 5% CO2 for 24 h. Then they were cultured with recombinant human B7H4 2 lg/ml (6576-B7, R&D, U.S.) or equal volumes of PBS (blank control) for 24 and 48 h. 2.5. Flow cytometry The cell subtype, cell cycle, proliferation rate, and apoptosis rate of the T cells were determined using flow cytometry. The antibodies were anti-CD4 FITC, anti-CD8 FITC, anti-CD8a APC, anti Ki67-PE (No. 11-0049, No. 11-0086, No. 17-0086, and No. 561283, eBiosciences, U.S.). A human regulatory T cell staining kit (No. 888999) was also purchased from eBiosciences. After co-cultured with B7-H4 2 lg/ml for 24 or 48 h, the T cells were permeabilized with a saponin buffer and stained with antibodies according to standard protocols. All experiments were performed in triplicate using a Becton Dickinson FACScan.
2.2. Immunohistochemical B7-H4 staining and evaluation
2.6. Enzyme-linked immunosorbent assay
Standard SP immunohistochemical staining (Maxim, China) of B7-H4 antibody (1:150, bs-0673R, Bioss, China) was performed. The immunohistochemical analysis was performed by two independent investigators. Brown or yellow B7-H4 staining of the membrane or cytoplasm was considered positive; and a lack of membrane or cytoplasmic staining was considered negative. Evaluation of the proportion of B7-H4 positive tumor cells was based on a review of the entire section and a final score was assigned. The extent of the staining was semi-quantitatively categorized as follows: 0 (<10% positive cells), 1 (10–25% positive cells), 2 (26– 50% positive cells), 3 (51–75% positive cells), and 4 (>75% positive cells). Staining intensity was scored as negative (0), faint (1+), moderate (2+), or strong (3+). The staining results were determined by the number of positive cells multiplied by the appropriate staining intensity. A combined score of 0–1 was considered negative expression, and P2 was considered positive expression.
The ELISA reagents and components for TGF-b1 (DB100B), IL10(Q1000), and IFN-c (DIF50) were purchased from R&D. The experiment was performed according to the manufacturer’s instructions. The supernatant was collected from the T cells of cervical cancer patients. These were cultured with or without B7-H4 for 24 and 48 h. The experiments were processed as triplicate samples. The analysis was achieved in a Bio-Tek ELX800 microplate reader at 450 nm. 2.7. Statistical analysis The differences in the numbers of tumor-infiltrating T lymphocytes between the groups positive and negative for B7-H4 expression were assessed with the Wilcoxon rank sum test and Fisher’s exact probability method. All statistical calculations were performed with Statistical Product and Service Solutions 17.0 (SPSS
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17.0, IBM) statistical software. Data are here represented as means with standard deviations (SD). Measurement data were treated using an unpaired Student’s t test, or one-way ANOVA analysis for multiple comparisons. Differences were considered significant (P < 0.05).
Table 1 B7-H4 expression in human cervical cancer and cervical epithelium.
a
There was no B7-H4 expression in normal cervical epithelia, but there was weakly positive B7-H4 expression in few neoplastic epithelia of CIN II–III. B7-H4 expression was observed in some cancer cells and mononuclear cells in the tumor matrix. The B7-H4 expression rate of cervical cancer was 46% (31/67). The B7-H4 positive rate in cervical cancer was significantly higher than in normal and neoplastic cervical epithelia (P < 0.01, P < 0.01) (Fig. 1, Table 1 ). 3.2. Association between B7-H4 expression and tumor-infiltrating T cells The tumor-infiltrating T lymphocytes were mainly distributed in the surrounding matrix of cervical squamous cell carcinoma nests, but they were occasionally found in some cancer nests. Few lymphocytes infiltrated in the matrix of cervical adenocarcinoma. The average numbers of CD8+T, CD4+T, and Foxp3+T cells in 5 high-power fields of each cervical carcinoma case was 241.52 ± 126.49, 22.04 ± 13.37, and 106.23 ± 89.15, respectively (Fig. 2). Statistical results showed the average number of infiltrating CD8+T cells in B7-H4-positive cervical carcinoma cases to be significantly lower than in B7-H4-negative cases (P < 0.01). There were fewer infiltrating CD4+T cells in B7-H4-positive cases than in B7-H4-negative cases, but there were more infiltrating Foxp3+ cells in B7-H4-positive cases than in B7-H4-negative cases. However, there were no statistically significant differences in the numbers of infiltrating CD4+T cells and Foxp3+T cells between B7-H4positive and -negative groups (P > 0.05) (Table 2). 3.3. B7-H4 expression and cytokine production of infiltrating T cells in cervical cancers Immunofluorescent double staining showed there to be significantly fewer CD8+T cells producing IFN-c in B7-H4-positive cases than in the B7-H4-negative cases (P < 0.05). There were also slightly fewer CD4+T cells producing TGF-b1 in the B7-H4-positive cervical cancer group than in the B7-H4-negative group, but there were no statistically significant differences between them (P > 0.05) (Figs. 3 and 4, Table 3).
A
B
Cases
Cervical cancer CIN II–III Normal cervical epithelium
67 30 30
B7-H4
X2
P
20.400 17.280 1.017
0.000a 0.000* 0.500**
+
3. Results 3.1. Expression of B7-H4 in human cervical cancer, neoplastic and normal cervical epithelia
Groups
* **
31 (46.24) 1 (10.00) 0 (0.00)
36 29 30
Normal cervical epithelium and cervical cancer. Normal cervical epithelium and CIN II–III. CIN II–III and cervical cancer.
3.4. Recombinant B7-H4 and the proliferation of T cells After co-culture with recombinant B7-H4 for 48 h, the relative numbers of G1, G2, and S phase T cells were 90.59%, 8.55%, and 0.87%, respectively of peripheral blood of cervical cancer patients but 92.83%, 6.09%, and 1.13%, respectively, in the blank control group (Fig. 5). There were significantly fewer cells in S phase in the B7-H4 group than in the blank control group. In addition, the Ki67 positive rates of CD4+T and CD8+T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, respectively, which were lower than those of the blank control group, which were 2.74 ± 0.98% and 1.71 ± 1.32% (Fig. 6).
3.5. B7-H4 and apoptosis in T cells After 48 h of co-culture with B7-H4, the apoptotic rate of peripheral blood T cells of cervical cancer patients was 27.4 ± 1.42% but 23.8 ± 3.14% in the blank control group. There was no significant difference between the two groups.
3.6. B7-H4 and the subtypes of T cells in cultured peripheral blood After 48 h of co-culture with B7-H4, the relative numbers of CD4+T and CD8+T cells among the T cells were 40.02 ± 2.70% and 17.93 ± 1.90%, respectively. There were 46.75 ± 7.30% and 25.98 ± 3.42% in the blank control group. The ratio of CD4+T/ CD8+T was 2.23 in the B7-H4 group, which was higher than the 1.79 in the blank control group. Among CD4+T cells, the relative number of CD25+FOXP3+T cells in the B7-H4 group was 15.9%, which was higher than the 12.8% in the blank group (Fig. 7).
3.7. B7-H4 and cytokine secretion by T cells ELISA tests showed that, after co-culture with B7-H4, the concentration of IFN-c decreased slightly but, at 24 and 48 h, the concentrations of IL-10 and TGF-b1 were significantly higher than in the blank control groups (P < 0.05, P < 0.05, Fig. 8).
C
Fig. 1. Immunohistochemical analysis of B7-H4 in cervical cancer and cervical epithelium. Representative results are shown. (A) Positive B7-H4 expression in cervical squamous cell carcinoma. (B) Weakly positive B7-H4 expression in cervical neoplastic epithelium. (C) Negative B7-H4 expression in normal cervical epithelium.
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A
CD8 + T
B
HE
C
CD4 + T
FOXP3+ T
D
Fig. 2. Representative figures of infiltrating T cells in cervical squamous cell carcinoma.
There were more infiltrating CD8+T and FOXP3+T cells than infiltrating CD4+T cells, but there was no significant difference in the number of infiltrating CD4+T and FOXP3+T cells between B7-H4positive and -negative cases. However, the average number of infiltrating CD8+T cells and their IFN-c production was significantly lower in B7-H4-positive cases than in -negative cases. This was consistent with a report by Bedoya [12]. Another study of nonsmall cell lung cancer also showed there to be fewer infiltrating T lymphocytes in B7-H4-positive cases than in B7-H4-negative cases, but the subtypes of the T lymphocytes were not analyzed [13]. The current study demonstrated that there were slightly fewer infiltrating CD4+T cells producing TGF-b1 in B7-H4-positive than in B7-H4-negative cases but the difference was not significant. This suggested that B7-H4 probably inhibited the infiltration and immune effect of CD8+T cells but had no significant role in the infiltration of CD4+T cells and Foxp3+Tregs. The transmembrane B7-H4 on cancer cells is inducible and unstable, but soluble B7-H4 has been detected in blood of cancer patients and is associated with a poor progress [14]. For this reason, recombinant human B7-H4 protein was co-cultured with active peripheral blood T cells of cervical cancer patients in vitro. Current flow cytometry data showed that T cells were arrested in the G1/G2 phase, and the number of S phase cells decreased after co-culture with human B7-H4 for 48 h. Sica GL also reported that B7-H4-positive cells arrested the proliferation of early activate T cells and inhibited CD8+T cells mature and IFN-c production by down-regulating protein kinase AKT pathway and the expression
Table 2 B7-H4 expression and the infiltrating T lymphocytesin cervical cancer. T lymphocytes
± S) SUM (X
CD8+ FOXP3+ CD4+
241.52 ± 126.49 106.23 ± 89.15 22.04 ± 13.37
B7-H4
P
+ 115.00 ± 49.045 111.83 ± 107.85 19.63 ± 12.76
267.00 ± 142.74 102.63 ± 89.20 23.19 ± 13.87
0.001 0.664 0.628
4. Discussion The suppression of antitumor immune response can allow tumor cells to escape immune surveillance. Members of the B7 family are involved in this process. Aberrant B7-H1 and B7-DC expression has been observed in cervical cancer tissues before (data not shown). In this study, B7-H4 positive expression was observed in 46% (31/67) of tumor cells and some matrix cells of cervical cancer cases, and weakly positive B7-H4 expression was observed in few high-grade neoplastic cervical epithelia, but no B7-H4 expression was observed in normal cervical epithelia. Aberrant B7-H4 expression was found to be associated with poor prognosis for cervical cancer. This indicated that B7-H4 suppressed anti-tumor immune response in the cervical microenvironment, worsening prognosis. The results of immunofluorescent staining showed CD4+T, CD8+T, and FOXP3+T cell infiltration in cervical cancer tissues.
CD8+ T
HE
IFN-γ+
CD8+ T /IFN-γ+
Fig. 3. Representative infiltrating CD8+ T and IFN-c producing cells in cervical carcinoma.
A
B7-H4
B
CD8+T
C
B7-H4
D
CD8+T
Fig. 4. B7-H4 expression and infiltrating CD8+T cells in cervical carcinomas (A) Absence of B7-H4 expression in cervical carcinoma cells. (C) B7-H4 expression in cervical carcinoma cells. (B) and (D) Tumor-infiltrating CD8+T cells.
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± S) SUM (X
IFN-c/CD8+ TGF-b1/CD4+
78.78 ± 46.74 16.18 ± 11.69
B7-H4
P
+ 58.25 ± 24.73 12.53 ± 9.09
80.23 ± 55.99 17.88 ± 12.61
0.035 0.344
of CDK4/6 and cyclin E/D. They also up-regulate the level of P21 [15]. Xiaojie Wang demonstrated that B7-H4 protein inhibited proliferation of mouse CD4+T and CD8+T cells by interfering with the
Blank group
activation of protein kinase ERK, p38, JNK, and the AKT pathway of T cells [16]. However, Kristensen did not find B7-H4 to have any inhibitive effect on the proliferation of T cells collected from the mesenteric lymph nodes of normal mice [17]. These different results may have been caused by the collection of B7-H4 protein and T cells from different sources. The current experiment also showed that there were fewer Ki67 of CD4+T and CD8+T cells, but the ratio of CD4+T/CD8+T was higher after co-culture with B7-H4 protein. This indicated that B7-H4 protein had a stronger depressive effect on CD8+T cells than on CD4+T cells, which is consistent with the results of immunohistochemical staining of cervical cancer tissues.
B7-H4 group
Fig. 5. Representative cell cycle of the cultured peripheral T cells.
Fig. 6. Representative Ki67 positive rates of the cultured T cells.
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Fig. 7. Representative flow cytometry profiles of T cell subtypes in the cultured peripheral blood.
Foxp3+Tregs are important negative immune regulators. They make direct contact with the target cells and also secrete TGF-b1 and IL-10. The role of B7-H4 protein on Foxp3+T cells was here analyzed in vitro by flow cytometry, and the proportion of CD25+Foxp3+T cells in CD4+T cells was found to be higher after 48 h of co-culture with B7-H4 protein. The results suggested that B7-H4 may promote the proliferation of Tregs. Because cytokine secretion reflects T cells’ immune functional status, the concentration of IFN-c, TGF-b1, and IL-10 was assessed in the supernatant of B7-H4 co-cultured T cells. These results showed that the concentration of IFN-c was lower, but the
concentration of TGF-b1 and IL-10 was higher. IFN-c is mainly secreted by active CD8+T cells. Low levels of IFN-c secretion suggested that B7-H4 depressed the function of CD8+T cells. IL-10 and TGF-b1 are depressive cytokines. They are mainly produced by Tregs. TGF-b1 may suppress the immune response in the tumor microenvironment by reducing the number of infiltrating CD8+T, CD4+T, and dendritic cells and promoting tumor angiogenesis, thereby promoting tumor progression [18]. In this experiment, both the higher concentration of IL-10 and TGF-b1 in the supernatant and the greater number of CD4+CD25+Foxp3+T cells in co-cultured T cells suggested that B7-H4 may promote
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Ethics statement This study protocol was approved by the institutional Ethics Committee of Chongqing Medical University. Written informed consent was obtained from all individuals prior to their inclusion in the study. Acknowledgments We would like to thank Mr. Ming Li for his support in flow cytometry. This research was supported by the Natural Science Foundation of Chongqing, China (CSTC, 2009BB5268). References
Fig. 8. Representative cytokine concentrations in supernatants of the cultured T cells.
the proliferation of Tregs and their immunosuppressive function, although the mechanism remains to be elucidated. Podojil demonstrated B7-H4Ig inhibits mouse and human T-cell function via IL-10/Treg-dependent mechanisms [19]. The current data showed B7-H4 had no significant effect on the apoptosis of the co-cultured T cells of cervical patients’ peripheral blood. In summary, B7-H4 is overexpressed in human cervical cancers, and it is associated with low numbers of infiltrating CD8+T lymphocytes and correspondingly low IFN-c production. In vitro, B7-H4 inhibits the proliferation of CD4+T and CD8+T but promotes the proliferation, and IL-10 and TGF-b1 production of Tregs. B7-H4 plays an important role in suppressing anti-tumor immune in microenvironment of cervical cancer and it may be a suitable immunotherapy target of the cancer. Author contributions X. Wang and T.T. Wang made equal contributions to the experiment and the publication process. W.L. Huang, Y. Zhang, and W.P. Geng performed the experiments. L. Xiao and Y. Luo supplied the clinical archive. Dr. M. Xu critically discussed and reviewed the paper and supervised the project.
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B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas Xin Wang a, Tingting Wang a, Man Xu a,⇑, Ling Xiao b, Yi Luo b, Wenlian Huang a, Yan Zhang a, Weipu Geng a a b
Department of Pathology, Molecular Medicine and Tumor Research Center of Chongqing Medical University, Chongqing, China Department of Obstetrics and Gynecology, The First Affiliate Hospital of Chongqing Medical University, Chongqing, China
a r t i c l e
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Article history: Received 14 February 2014 Accepted 6 October 2014 Available online 13 October 2014 Keywords: B7-H4 T lymphocyte Cytokine Cell proliferation Cervical carcinoma
a b s t r a c t Objective: To investigate B7-H4 expression and its correlation with the number of infiltrating T lymphocytes and cytokine production by those lymphocytes in human cervical cancer and to determine the effect of recombinant B7-H4 on the active peripheral blood T cells of the patients in vitro. Methods: B7-H4 expression was detected in 67 cases of cervical cancer using immunohistochemical staining. Tumor-infiltrating CD8+T, CD4+T, and FOXP3+ (Forkhead Box P3) T lymphocytes and their levels of IFN-c and TGF-b1 production were determined by immunofluorescent double-staining. After the peripheral blood T lymphocytes of patients were co-cultured with B7-H4, proliferation, apoptosis, and cell subtypes were analyzed using flow cytometry. Cytokines in the supernatant were detected by ELISA. Results: B7-H4 was expressed in 46% (31/67) of the cases of cervical cancer. The number of infiltrating CD8+T lymphocytes and their IFN-c production in positive B7-H4 expression cervical cancers was significantly lower than in negative B7-H4 cases (P < 0.01, P < 0.05), but there was no significant difference between cases positive and negative for B7-H4 with respect to infiltrating FOXP3+T and CD4+T cells or TGF-b1 production. After co-culture with B7-H4 for 48 h, the patients’ activated T lymphocytes were arrested at G1/G2 phase. The Ki67 positive rates of CD4+T and CD8+T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, and they were lower than in the blank group. The proportion of CD4+T and CD8+T cells decreased, but CD4+T/CD8+T and the proportion of CD4+CD25+Foxp3+T cells increased. In addition, concentrations of IL-10 and TGF-b1 in the supernatant of co-cultured T cells increased significantly (P < 0.05, P < 0.05), but that of IFN-c decreased. B7-H4 had no significant effect on apoptosis of the T cells. Conclusion: B7-H4 is overexpressed in human cervical cancers, and it is associated with lower numbers of tumor-infiltrating CD8+T lymphocytes and therefore less IFN-c production. In vitro, B7-H4 inhibits the proliferation of CD4+T and CD8+T but promotes the proliferation of Tregs and the secretion of IL-10 and TGF-b1. B7-H4 plays an important role in depressing the anti-tumor immunity of CD8+T cell in microenvironments of cervical cancer. Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
1. Introduction Cervical cancer is a malignant tumor common in Chinese women; the morbidity, the proportion of young patients, and the number of early stage cancer cases are increasing recently [1,2]. Persistent infection of high-risk human papilloma-virus (HPV) is a key inducing factor of this cancer. In most cases, HPV infection is self-limiting and can be eradicated by immune response in cervical microenvironment. The decrease in immune surveillance and clearance plays an important role in cervical cancer carcinogenesis ⇑ Corresponding author.
and development [3]. Recent studies have shown immunosuppression of tumor microenvironment to be associated with certain factors, including expression of inhibitory ligand B7s, recruitment of regulatory T cells and tumor-induced myeloid-derived suppressor cells, and up-regulation of transforming growth factor-beta (TGFb), galectin-1, indoleamine 2,3-dioxygenase, and arginase [4]. B7 family molecules, including B7-H1 (PD-L1), B7-DC (PD-L2), and B7-H4 (B7S1), combine with corresponding receptors of activated T lymphocytes and negatively regulate T cell immunity. This plays an important role in down-regulating anti-tumor immunity in cancer microenvironments [5]. Overexpression of PD-L1 and B7-DC has been observed in cervical cancer cells, and these were
http://dx.doi.org/10.1016/j.humimm.2014.10.002 0198-8859/Ó 2014 Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics.
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associated with decreasing CD8+T cell infiltration. B7-H4 is a transmembrane protein. It is expressed in human tissues in the cancers of the lung, esophagus, stomach, liver, pancreas, breast, ovary, brain, prostate, and kidney [6,7]. It combines with surface receptors of the activated T cell, thus regulating T cells’ growth, cell cycle, and immune function. It is associated with cancer progression and poorer patient outcomes [8]. The soluble form of B7-H4 has also been detected in blood samples from cancer patients. Recently, B7-H4 was found in cervical cancer cells and infiltrating cells in the matrix. It was associated with distant spread, lymph node metastasis, and poor prognosis for cervical cancer patients [9,10]. However, the relationship between B7-H4 expression and the immune status of cervical environment has not yet been explored. In cervical cancer tissue, the infiltrating T lymphocytes include CD4+T cells, CD8+T cells, and FOXP3+ regulatory T cells (Treg), and the CD8+/FOXP3+T cell ratio is an independent and prognostic factor of cervical cancer patients [11]. Data have shown B7-H4 overexpression to be associated with immunosuppression in the tumor microenvironment and with poor prognosis in patients [7,8,10]. In order to assess B7-H4 expression in cervical cancer and its effect on T-cell immunity in the cervical microenvironment, immunohistochemical and immunofluorescent staining were here used to detect B7-H4 expression in 67 cases of human cervical cancers and analyze its association with the infiltration of FOXP3+T, CD4+T, and CD8+T cells and their TGF-b1 and IFN-c production in tumors. Flow cytometry and ELISA were used to assess the effects of recombinant human B7-H4 on peripheral blood T cells’ proliferation, apoptosis, and cytokine production in cervical cancer patients. 2. Materials and methods 2.1. Specimens Here, 67 cases of cervical carcinoma (60 cases of squamous cell carcinoma and 7 cases of adenocarcinoma) were collected from pathology department of the first affiliate Hospital of Chongqing Medical University from 2010 to 2012. The average patient age was 43.12 y (median 43 y; range, 32–65 y). Patients who underwent pre-operative treatment, such as chemotherapy or radiotherapy, were excluded. Then, 30 samples of high grade cervical intraepithelial neoplasia (CIN II-III) and 30 samples of normal cervix were obtained from biopsy or uterine hysterectomy due to leiomyoma, and 4 lm sections of paraffin-embedded tissues were collected for immunohistochemical staining and immunofluorescent staining.
2.3. Immunofluorescent double-staining Immunofluorescent double-staining was performed to detect FOXP3+T, CD4+T, and CD8+T cells and TGF-b1- and IFN-c-producing cells in cervical tissues. Primary antibodies were mouse anti human FOXP3 (1:100, 14-4777-82, eBiosciences, U.S.), mouse anti human CD4, rabbit anti-human CD8, and rabbit anti-human TGF-b1 (MAB-0251, RMA-0514, RAB-0238, Maxim, China) and mouse antihuman IFN-c (1:200, ab82447, Abcam, U.K.). The secondary antibodies were goat anti mouse IgG-FTTC and goat anti-rabbit IgGRBITC (1:1200, bs-0296G-FTTC, bs-0295G-RBITC; Bioss, China). After the primary antibodies were incubated overnight at 4 °C, and each corresponding secondary antibody was incubated for 1 h at RT in dark. The slides were washed three times in PBS with 0.05% Tween 20 for 3 min after each incubation. CD8+ and CD4+ showed staining on the membrane, TGF-b+1 and IFN-c+ showed staining in the cytoplasm, and FOXP3+ showed staining in the nucleus and cytoplasm. Five photographs under 400 power fields in each section were taken and the positive cells were counted in cervical cancer tissues. The average number of positive cells in each case was used for statistical comparison. 2.4. Isolation and activation of peripheral blood T cells and co-culture with B7-H4 Fresh heparinized peripheral blood mononuclear cells from cervical cancer patients (n = 10) were collected using the Ficoll-Hypaque gradient. After these cells were passed through a nylon brush column (Kasker Co., German), the lymphocytes were collected. Some 5 105 cells were cultured with phytohaemagglutinin (PHA) 10 lg/ml (Sigma, U.S.) at 37 °C in humidified 5% CO2 for 24 h. Then they were cultured with recombinant human B7H4 2 lg/ml (6576-B7, R&D, U.S.) or equal volumes of PBS (blank control) for 24 and 48 h. 2.5. Flow cytometry The cell subtype, cell cycle, proliferation rate, and apoptosis rate of the T cells were determined using flow cytometry. The antibodies were anti-CD4 FITC, anti-CD8 FITC, anti-CD8a APC, anti Ki67-PE (No. 11-0049, No. 11-0086, No. 17-0086, and No. 561283, eBiosciences, U.S.). A human regulatory T cell staining kit (No. 888999) was also purchased from eBiosciences. After co-cultured with B7-H4 2 lg/ml for 24 or 48 h, the T cells were permeabilized with a saponin buffer and stained with antibodies according to standard protocols. All experiments were performed in triplicate using a Becton Dickinson FACScan.
2.2. Immunohistochemical B7-H4 staining and evaluation
2.6. Enzyme-linked immunosorbent assay
Standard SP immunohistochemical staining (Maxim, China) of B7-H4 antibody (1:150, bs-0673R, Bioss, China) was performed. The immunohistochemical analysis was performed by two independent investigators. Brown or yellow B7-H4 staining of the membrane or cytoplasm was considered positive; and a lack of membrane or cytoplasmic staining was considered negative. Evaluation of the proportion of B7-H4 positive tumor cells was based on a review of the entire section and a final score was assigned. The extent of the staining was semi-quantitatively categorized as follows: 0 (<10% positive cells), 1 (10–25% positive cells), 2 (26– 50% positive cells), 3 (51–75% positive cells), and 4 (>75% positive cells). Staining intensity was scored as negative (0), faint (1+), moderate (2+), or strong (3+). The staining results were determined by the number of positive cells multiplied by the appropriate staining intensity. A combined score of 0–1 was considered negative expression, and P2 was considered positive expression.
The ELISA reagents and components for TGF-b1 (DB100B), IL10(Q1000), and IFN-c (DIF50) were purchased from R&D. The experiment was performed according to the manufacturer’s instructions. The supernatant was collected from the T cells of cervical cancer patients. These were cultured with or without B7-H4 for 24 and 48 h. The experiments were processed as triplicate samples. The analysis was achieved in a Bio-Tek ELX800 microplate reader at 450 nm. 2.7. Statistical analysis The differences in the numbers of tumor-infiltrating T lymphocytes between the groups positive and negative for B7-H4 expression were assessed with the Wilcoxon rank sum test and Fisher’s exact probability method. All statistical calculations were performed with Statistical Product and Service Solutions 17.0 (SPSS
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17.0, IBM) statistical software. Data are here represented as means with standard deviations (SD). Measurement data were treated using an unpaired Student’s t test, or one-way ANOVA analysis for multiple comparisons. Differences were considered significant (P < 0.05).
Table 1 B7-H4 expression in human cervical cancer and cervical epithelium.
a
There was no B7-H4 expression in normal cervical epithelia, but there was weakly positive B7-H4 expression in few neoplastic epithelia of CIN II–III. B7-H4 expression was observed in some cancer cells and mononuclear cells in the tumor matrix. The B7-H4 expression rate of cervical cancer was 46% (31/67). The B7-H4 positive rate in cervical cancer was significantly higher than in normal and neoplastic cervical epithelia (P < 0.01, P < 0.01) (Fig. 1, Table 1 ). 3.2. Association between B7-H4 expression and tumor-infiltrating T cells The tumor-infiltrating T lymphocytes were mainly distributed in the surrounding matrix of cervical squamous cell carcinoma nests, but they were occasionally found in some cancer nests. Few lymphocytes infiltrated in the matrix of cervical adenocarcinoma. The average numbers of CD8+T, CD4+T, and Foxp3+T cells in 5 high-power fields of each cervical carcinoma case was 241.52 ± 126.49, 22.04 ± 13.37, and 106.23 ± 89.15, respectively (Fig. 2). Statistical results showed the average number of infiltrating CD8+T cells in B7-H4-positive cervical carcinoma cases to be significantly lower than in B7-H4-negative cases (P < 0.01). There were fewer infiltrating CD4+T cells in B7-H4-positive cases than in B7-H4-negative cases, but there were more infiltrating Foxp3+ cells in B7-H4-positive cases than in B7-H4-negative cases. However, there were no statistically significant differences in the numbers of infiltrating CD4+T cells and Foxp3+T cells between B7-H4positive and -negative groups (P > 0.05) (Table 2). 3.3. B7-H4 expression and cytokine production of infiltrating T cells in cervical cancers Immunofluorescent double staining showed there to be significantly fewer CD8+T cells producing IFN-c in B7-H4-positive cases than in the B7-H4-negative cases (P < 0.05). There were also slightly fewer CD4+T cells producing TGF-b1 in the B7-H4-positive cervical cancer group than in the B7-H4-negative group, but there were no statistically significant differences between them (P > 0.05) (Figs. 3 and 4, Table 3).
A
B
Cases
Cervical cancer CIN II–III Normal cervical epithelium
67 30 30
B7-H4
X2
P
20.400 17.280 1.017
0.000a 0.000* 0.500**
+
3. Results 3.1. Expression of B7-H4 in human cervical cancer, neoplastic and normal cervical epithelia
Groups
* **
31 (46.24) 1 (10.00) 0 (0.00)
36 29 30
Normal cervical epithelium and cervical cancer. Normal cervical epithelium and CIN II–III. CIN II–III and cervical cancer.
3.4. Recombinant B7-H4 and the proliferation of T cells After co-culture with recombinant B7-H4 for 48 h, the relative numbers of G1, G2, and S phase T cells were 90.59%, 8.55%, and 0.87%, respectively of peripheral blood of cervical cancer patients but 92.83%, 6.09%, and 1.13%, respectively, in the blank control group (Fig. 5). There were significantly fewer cells in S phase in the B7-H4 group than in the blank control group. In addition, the Ki67 positive rates of CD4+T and CD8+T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, respectively, which were lower than those of the blank control group, which were 2.74 ± 0.98% and 1.71 ± 1.32% (Fig. 6).
3.5. B7-H4 and apoptosis in T cells After 48 h of co-culture with B7-H4, the apoptotic rate of peripheral blood T cells of cervical cancer patients was 27.4 ± 1.42% but 23.8 ± 3.14% in the blank control group. There was no significant difference between the two groups.
3.6. B7-H4 and the subtypes of T cells in cultured peripheral blood After 48 h of co-culture with B7-H4, the relative numbers of CD4+T and CD8+T cells among the T cells were 40.02 ± 2.70% and 17.93 ± 1.90%, respectively. There were 46.75 ± 7.30% and 25.98 ± 3.42% in the blank control group. The ratio of CD4+T/ CD8+T was 2.23 in the B7-H4 group, which was higher than the 1.79 in the blank control group. Among CD4+T cells, the relative number of CD25+FOXP3+T cells in the B7-H4 group was 15.9%, which was higher than the 12.8% in the blank group (Fig. 7).
3.7. B7-H4 and cytokine secretion by T cells ELISA tests showed that, after co-culture with B7-H4, the concentration of IFN-c decreased slightly but, at 24 and 48 h, the concentrations of IL-10 and TGF-b1 were significantly higher than in the blank control groups (P < 0.05, P < 0.05, Fig. 8).
C
Fig. 1. Immunohistochemical analysis of B7-H4 in cervical cancer and cervical epithelium. Representative results are shown. (A) Positive B7-H4 expression in cervical squamous cell carcinoma. (B) Weakly positive B7-H4 expression in cervical neoplastic epithelium. (C) Negative B7-H4 expression in normal cervical epithelium.
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A
CD8 + T
B
HE
C
CD4 + T
FOXP3+ T
D
Fig. 2. Representative figures of infiltrating T cells in cervical squamous cell carcinoma.
There were more infiltrating CD8+T and FOXP3+T cells than infiltrating CD4+T cells, but there was no significant difference in the number of infiltrating CD4+T and FOXP3+T cells between B7-H4positive and -negative cases. However, the average number of infiltrating CD8+T cells and their IFN-c production was significantly lower in B7-H4-positive cases than in -negative cases. This was consistent with a report by Bedoya [12]. Another study of nonsmall cell lung cancer also showed there to be fewer infiltrating T lymphocytes in B7-H4-positive cases than in B7-H4-negative cases, but the subtypes of the T lymphocytes were not analyzed [13]. The current study demonstrated that there were slightly fewer infiltrating CD4+T cells producing TGF-b1 in B7-H4-positive than in B7-H4-negative cases but the difference was not significant. This suggested that B7-H4 probably inhibited the infiltration and immune effect of CD8+T cells but had no significant role in the infiltration of CD4+T cells and Foxp3+Tregs. The transmembrane B7-H4 on cancer cells is inducible and unstable, but soluble B7-H4 has been detected in blood of cancer patients and is associated with a poor progress [14]. For this reason, recombinant human B7-H4 protein was co-cultured with active peripheral blood T cells of cervical cancer patients in vitro. Current flow cytometry data showed that T cells were arrested in the G1/G2 phase, and the number of S phase cells decreased after co-culture with human B7-H4 for 48 h. Sica GL also reported that B7-H4-positive cells arrested the proliferation of early activate T cells and inhibited CD8+T cells mature and IFN-c production by down-regulating protein kinase AKT pathway and the expression
Table 2 B7-H4 expression and the infiltrating T lymphocytesin cervical cancer. T lymphocytes
± S) SUM (X
CD8+ FOXP3+ CD4+
241.52 ± 126.49 106.23 ± 89.15 22.04 ± 13.37
B7-H4
P
+ 115.00 ± 49.045 111.83 ± 107.85 19.63 ± 12.76
267.00 ± 142.74 102.63 ± 89.20 23.19 ± 13.87
0.001 0.664 0.628
4. Discussion The suppression of antitumor immune response can allow tumor cells to escape immune surveillance. Members of the B7 family are involved in this process. Aberrant B7-H1 and B7-DC expression has been observed in cervical cancer tissues before (data not shown). In this study, B7-H4 positive expression was observed in 46% (31/67) of tumor cells and some matrix cells of cervical cancer cases, and weakly positive B7-H4 expression was observed in few high-grade neoplastic cervical epithelia, but no B7-H4 expression was observed in normal cervical epithelia. Aberrant B7-H4 expression was found to be associated with poor prognosis for cervical cancer. This indicated that B7-H4 suppressed anti-tumor immune response in the cervical microenvironment, worsening prognosis. The results of immunofluorescent staining showed CD4+T, CD8+T, and FOXP3+T cell infiltration in cervical cancer tissues.
CD8+ T
HE
IFN-γ+
CD8+ T /IFN-γ+
Fig. 3. Representative infiltrating CD8+ T and IFN-c producing cells in cervical carcinoma.
A
B7-H4
B
CD8+T
C
B7-H4
D
CD8+T
Fig. 4. B7-H4 expression and infiltrating CD8+T cells in cervical carcinomas (A) Absence of B7-H4 expression in cervical carcinoma cells. (C) B7-H4 expression in cervical carcinoma cells. (B) and (D) Tumor-infiltrating CD8+T cells.
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± S) SUM (X
IFN-c/CD8+ TGF-b1/CD4+
78.78 ± 46.74 16.18 ± 11.69
B7-H4
P
+ 58.25 ± 24.73 12.53 ± 9.09
80.23 ± 55.99 17.88 ± 12.61
0.035 0.344
of CDK4/6 and cyclin E/D. They also up-regulate the level of P21 [15]. Xiaojie Wang demonstrated that B7-H4 protein inhibited proliferation of mouse CD4+T and CD8+T cells by interfering with the
Blank group
activation of protein kinase ERK, p38, JNK, and the AKT pathway of T cells [16]. However, Kristensen did not find B7-H4 to have any inhibitive effect on the proliferation of T cells collected from the mesenteric lymph nodes of normal mice [17]. These different results may have been caused by the collection of B7-H4 protein and T cells from different sources. The current experiment also showed that there were fewer Ki67 of CD4+T and CD8+T cells, but the ratio of CD4+T/CD8+T was higher after co-culture with B7-H4 protein. This indicated that B7-H4 protein had a stronger depressive effect on CD8+T cells than on CD4+T cells, which is consistent with the results of immunohistochemical staining of cervical cancer tissues.
B7-H4 group
Fig. 5. Representative cell cycle of the cultured peripheral T cells.
Fig. 6. Representative Ki67 positive rates of the cultured T cells.
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Fig. 7. Representative flow cytometry profiles of T cell subtypes in the cultured peripheral blood.
Foxp3+Tregs are important negative immune regulators. They make direct contact with the target cells and also secrete TGF-b1 and IL-10. The role of B7-H4 protein on Foxp3+T cells was here analyzed in vitro by flow cytometry, and the proportion of CD25+Foxp3+T cells in CD4+T cells was found to be higher after 48 h of co-culture with B7-H4 protein. The results suggested that B7-H4 may promote the proliferation of Tregs. Because cytokine secretion reflects T cells’ immune functional status, the concentration of IFN-c, TGF-b1, and IL-10 was assessed in the supernatant of B7-H4 co-cultured T cells. These results showed that the concentration of IFN-c was lower, but the
concentration of TGF-b1 and IL-10 was higher. IFN-c is mainly secreted by active CD8+T cells. Low levels of IFN-c secretion suggested that B7-H4 depressed the function of CD8+T cells. IL-10 and TGF-b1 are depressive cytokines. They are mainly produced by Tregs. TGF-b1 may suppress the immune response in the tumor microenvironment by reducing the number of infiltrating CD8+T, CD4+T, and dendritic cells and promoting tumor angiogenesis, thereby promoting tumor progression [18]. In this experiment, both the higher concentration of IL-10 and TGF-b1 in the supernatant and the greater number of CD4+CD25+Foxp3+T cells in co-cultured T cells suggested that B7-H4 may promote
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Ethics statement This study protocol was approved by the institutional Ethics Committee of Chongqing Medical University. Written informed consent was obtained from all individuals prior to their inclusion in the study. Acknowledgments We would like to thank Mr. Ming Li for his support in flow cytometry. This research was supported by the Natural Science Foundation of Chongqing, China (CSTC, 2009BB5268). References
Fig. 8. Representative cytokine concentrations in supernatants of the cultured T cells.
the proliferation of Tregs and their immunosuppressive function, although the mechanism remains to be elucidated. Podojil demonstrated B7-H4Ig inhibits mouse and human T-cell function via IL-10/Treg-dependent mechanisms [19]. The current data showed B7-H4 had no significant effect on the apoptosis of the co-cultured T cells of cervical patients’ peripheral blood. In summary, B7-H4 is overexpressed in human cervical cancers, and it is associated with low numbers of infiltrating CD8+T lymphocytes and correspondingly low IFN-c production. In vitro, B7-H4 inhibits the proliferation of CD4+T and CD8+T but promotes the proliferation, and IL-10 and TGF-b1 production of Tregs. B7-H4 plays an important role in suppressing anti-tumor immune in microenvironment of cervical cancer and it may be a suitable immunotherapy target of the cancer. Author contributions X. Wang and T.T. Wang made equal contributions to the experiment and the publication process. W.L. Huang, Y. Zhang, and W.P. Geng performed the experiments. L. Xiao and Y. Luo supplied the clinical archive. Dr. M. Xu critically discussed and reviewed the paper and supervised the project.
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