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Tumor-derived CD4 + CD25 + regulatory T cells inhibit dendritic cells function by CTLA-4
Accepted Manuscript Title: Tumor-derived CD4+CD25+regulatory T cells inhibit dendritic cells function by CTLA-4 Author: Xin Chen Yong Du Qingqing Hu ZhiMing Huang PII: DOI: Reference:
S0344-0338(16)30236-9 http://dx.doi.org/doi:10.1016/j.prp.2016.12.008 PRP 51695
To appear in: Received date:
6-7-2016
Please cite this article as: Xin Chen, Yong Du, Qingqing Hu, ZhiMing Huang, Tumorderived CD4+CD25+regulatory T cells inhibit dendritic cells function by CTLA-4, Pathology - Research and Practice http://dx.doi.org/10.1016/j.prp.2016.12.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Tumor-derived CD4+CD25+regulatory T cells inhibit dendritic cells function by CTLA-4
Xin Chen1#,Yong Du2#, Qingqing Hu2, ZhiMing Huang1*
1
Department of Gastroenterology and Hepatology, First Affiliated Hospital of Wenzhou Medical
University , Wenzhou, 325000, Zhejiang, China.
2
Department of Pediatrics, First Affiliated Hospital of Wenzhou Medical University, Wenzhou,
325000, ZheJiang, China.
*
Corresponding author: ZhiMing Huang, Department of Gastroenterology and Hepatology, First
Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China; Tel:+577-88069257;
Fax: +577-88069555; E-mail: [email protected]
#
Equal contributors
Tregs: CD4+CD25+ regulatory T cells; DCs: dendritic cells; HCC:hepatocellular carcinoma; CTLA-4: cytotoxic T lymphocyte-associated protein
Highlights
Tumor-derived Tregs down-modulated CD80/CD86 on DCs in a CTLA-4-dependent way. Tregs form stable cluster with DCs. Blockade of CTLA-4 can lead to increase DC-mediated immunity.
Abstract Purpose: CD4+CD25+regulatory T cells (Tregs) play an important role in anti-tumor immune responses. Poor prognosis and declining survival rates have intimate connection with high Treg expression in cancer patients. Cytotoxic T Lymphocyte-associated protein (CTLA-4) is one of the most prominent molecules on Treg. In our previous research, we have demonstrated that HCC-derived Tregs can interfere with Dendritic cells (DCs) function and down-modulate CD80/CD86 on DCs in vitro in a cell-contact dependent way. However the mechanism of how HCC-derived Treg affect DC phenotype are not very clear. Therefore, we investigated the function of CTLA-4 in anti-tumor immune responses. Materials and methods: We established BABL/C mouse with hepatocellular carcinoma model, and tumor-derived Tregs were purified by magnetic cell sorting using mouse CD4+CD25+regulatory T cell isolation kit. Splenic DCs were enriched using CD11c-conjugated microbeads. Then splenic DCs co-cultured with tumor-derived Tregs and antibody-blocking experiments was performed. Results: In our research, we found the down-modulation of CD80/CD86 on DCs was inhibited by blocking CTLA-4. HCC-derived Tregs down-modulated CD80/CD86 on DCs in a CTLA-4-dependent way. Blockade of CTLA-4 can lead to increase DC-mediated immunity. Conclusion: CTLA-4 play a vital role in Treg-mediated immnue inhibition and this discovery can open up new ideas for the development of therapeutic strategies. Keywords: dendritic cell; CD4+CD25+regulatory T cell; CTLA-4.
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide [1] . Chronic viral hepatitis including hepatitis B virus or hepatitis C virus infection is a risk factor for HCC, making HCC prevention a major goal of antiviral therapy[2-4] . Treg-mediated suppression serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and autoinflammatory disorders, allergy, acute and chronic infections, cancer, and metabolic inflammation[5]. Many researches have proved the existence of Treg at tumor sites, indicating that Treg may induce local immune tolerance. The presence of infiltrating Tregs suppresses protective anti-tumor immune responses and their accumulation into the tumor microenvironment correlates with reduced survival[6] . Treg-derived from HCC patients have been proved to be more suppressive than Treg from healthy individuals[7] . Treg-mediated suppression can be important targets for immune intervention. Blockade of Treg-mediated
immune suppression can enhance anti-tumor effects[8-9] . Combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity[10] . Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) expressing on Tregs has been implicated in the function of Tregs. Li et al found cryoablation and anti-CTLA-4 antibody in prostate cancer murine model induced anti-tumor immunological response[11] . There are many mechanisms have been proved for Treg-mediated suppression. In our previous research[12] , increase frequency of Tregs in the peripheral blood and spleen was found in hepatocellular carcinoma mice. HCC-derived Treg could down-regulate the expression of costimulatory molecules CD80/CD86 on DCs by cell-to-cell contact and inhibit TNF-a and IL-12 production from DC. However the mechanism is not very clear. We still need deeper studies on the role of Treg in patients with hepatocellular carcinoma to provide new approaches for tumor immunotherapy. In our study, there is obvious tendency for cell cluster formation between tumor-derived Treg and DC. Treg-DC cluster formation may help Tregs to regulate the phenotype and function of DC. The down-modulation of CD80/CD86 is inhibited by blocking CTLA-4. IL-12 is one of important stimulators produced by DCs. When a high concentration of anti-CTLA-4 antibody is added in the Treg-DC group, the down-modulation of IL-12 is inhibited partially too. Our research illustrates that blockade of CTLA-4 by addition of anti-CTLA-4 mAbs can lead to increased DC-mediated immunity.
2. Material and Methods 2.1 Tumor model Male BALB/C mice (6-8 weeks old, purchased from the SLACCAS company,China) were bred under specific pathogen-free (SPF) conditions. Animal protocols were approved by the Experimental Animal Management Committee Of Wenzhou Medical University. All surgery was performed under chloralic hydras anesthesia, and all efforts were made to minimize suffering. The H22 hepato-cellular carcinoma cells (China Center for Type Culture Collection, wuhan, CN) were cultured in RPMI 1640 medium and cells were inoculated to abdomen with 1 ×106 cells per mouse. After 8-9 days, cancerous ascites were extracted aseptically. Cells were collected and resuspended to a concentration of 1 ×106 ∕ml for making model. The mice were randomly divided into the tumor group and control group. The mouse was celiac anesthesia. We did a longitudinal incision, exposed the liver, injected 0.01ml cancer cell suspension (104 cells ) to the liver and last closed enterocoelia. About 25 days later, the models were formed. When anatomy, we could see gray and nubbly carcinoma tissues at different sizes from 0.5 to 1.0 cm in diameters in livers. 4-μm thick sections were prepared and stained with haematoxylin and eosin by standard histological procedures (Figure 1).
2.2 Reagents and Materials Mouse CD4+CD25+regulatory T cell isolation kit and mouse CD11c magnetic bead sorting kit (MACS, Miltenyi Biotec, Germany); Fluorescentlylabeled antibody: CD4-FITC, CD25-PE, CD11c-FITC, CD86-PE, CD80-PE and corresponding homotype antibody (eBioscience, California, USA); Mouse IL-12 ELISA kit (IBL, Hamburg, Germany); anti-CTLA4(R&D, Minnesota, USA); Anti-CD3 antibody (BD biosciences, New Jersey, USA ); LPS(Sigma, Santa clara, CA, USA); FCS and RPMI1640 (Gibco, Grand Island, New York, USA); mouse H22
hepatocellular carcinoma cell lines (China Center for Type Culture Collection, wuhan, CN ); Flow cytometry ( BD biosciences, New Jersey, USA ).
2.3 Cell isolation and sorting Splenic single-cell suspension was prepared from normal mice. Then splenic DCs were enriched using CD11c-conjugated microbeads according to the manufacturers’ instructions. The cells were positive for CD11c as assessed by flow cytometry. High purity of DCs were got finally (Figure2).Splenic single-cell suspension was prepared from HCC-bearing mice and then tumor-derived Tregs were purified by magnetic cell sorting using mouse CD4+CD25+regulatory T cell isolation kit according to the manufacturers’ instructions. High purity of tumor-derived Treg or CD4+CD25- Teff cells were gathered respectively (Figure2). Both T cells were stimulated separately with plate-bound anti-CD3 antibody (1µg/ml) and IL-2 (10U/ml) at 5×105 ∕ml in 24-well plates. Preactivated tumor-derived Tregs (4-8×105 ∕ml) or Teff cells (4-8×105 ∕ml) or a mix of two population at a 1:1 ratio were cultured with splenic DCs (DC-T cell ratio of 1:2) and then anti-CTLA-4 mAb (100μg/ml) or control Ab were added. LPS was added（1µg/ml）to stimulate DCs. Last, the co-cultures and the supernatants were collected respectively.
2.4 Flow cytometry and Elisa The co-cultures and the supernatants were collected respectively. The samples were stained with corresponding antibodies at 4℃ for 30 minutes and analyzed by flow cytometry. Isotype-matched antibodies were used as controls. IL-12 was assessed by ELISA using corresponding enzyme-linked immunosorbent assay kits according to the manufacturer’ s instruction.
2.5 Statistical Analysis Data are expressed as mean±sd. Statistical analysis was performed using Student’s t test with SPSS18.0 program (SPSS, Chicago). All P values ＜0.05 were considered statistically significant.
3. Results 3.1 Tregs form stable cluster with DCs Pre-activated tumor-derived Tregs were co-cultured with splenic DCs in the presence of LPS, and typical cluster formation was observed (Figure3). Onishi et al confirmed that Tregs form aggregates around Splenic DCs using immunofluorescence(13). In our previous research(12), we have confirmed Tregs down-regulated the expression of costimulatory molecules CD80/CD86 on DCs by cell-to-cell contact. This indicated that Treg-DC cluster formation might enable Tregs to modulate phenotypic and functional characteristics of DCs. Tregs might diminish the up-regulation of costimulatory molecule expression on DC by formation stable cell cluster. Treg-DC cluster might constitute a physical barrier and interfere with the function of DC.
3.2 HCC-derived Tregs down-regulate CD80/86 expression on splenic DCs depending on CTLA-4 Tregs could down-regulate CD80/86 expression on splenic DCs in our previous research, but
by what mechanism for Treg mediating CD80/86 down-regulation was unkonwn. To further address the suppression mechanism induced by Tregs, we then analyzed the possible contribution of CTLA-4 and performed antibody-blocking experiments (Figure4). HCC-derived Treg either when co-cultured with DC separately or when co-cultured with DC and Teff simultaneously, reduced the expression of CD80/CD86. But when splenic DCs were co-cultured with HCC-derived Tregs in the presence of anti-CTLA-4 mAb, the CD80/86 expression was found not to down-regulate. To avoid cross-linking by anti-CTLA-4, control antibody was added to the cultures. The findings indicated that there exsited CTLA-4/B7 interaction. HCC-derived Tregs down-regulated CD80/86 expression in a CTLA-4 dependent manner and blockade of CTLA-4 with anti-CTLA-4 mAb could abrogate the Treg-mediated suppression. 3.3 CTLA-4 expression strengthen Treg-mediated suppression The production of pro-inflammatory and immunoregulatory cytokines by DCs is important for the generation of adaptive immune responses. IL-12 is one of important stimulator produced by DCs. LPS-induced mature DCs can secrete high level of IL-12 while Tregs down-regulate the production of IL-12 from DCs. But how CTLA-4 affect the cytokine secretion by DCs. To figure out this problem, we did antibody-blocking experiments. We added a high concentration of anti-CTLA-4 antibody into different groups. The down-modulation of IL-12 was inhibited partially by blocking CTLA-4. Treg suppression of DCs function depends on CTLA-4. (Figure5)
4. Discussion Treg as a functional subset of inhibitory T cells, plays a crucial role in the maintenance of immune tolerance. Treg can secret immunosuppressive cytokines such as IL-10, TGF-β, IL-35, which can directly inhibit the function of responder T cells and myeloid cells[14-15] . Treg not only involves in autoimmune disease, infection and transplantation tolerance, but also plays a pivotal role in the suppression of anti-tumor immunity during development[16] . It has the features of hyporesponsive and anergic to antigen stimulation. Depletion of Treg can augment anti-tumor immune responses and could be a new approach for tumor therapy[17] . Administration of Treg-depleting anti-CD25 monoclonal antibody enhances both the anti-tumor response and the effectiveness of cancer vaccines[10] . The high level expression of Treg in tumor tissues and peripheral blood of cancer patients was reported in a lot of literature[18-20] . A high expression of Treg may inhibit the anti-tumor effect of T cells or induce immunological resistance to tumor antigens among cytotoxic T lymphocytes without producing acute or memory cytotoxic T lymphocytes responses. Strauss et al reported that Treg produced interleukin-10 and transforming growth factor-beta1 to mediate suppression in the tumor microenvironment[21] . Many researches found the prevalence of Treg cells was obviously higher in hepatocellular carcinoma than in the nontumorous liver and the patient with high expression of Treg cells showed a lower survival rate[22-24] . Among the cell-surface markers associated with Treg phenotype and function, CTLA-4 is one of the most prominent molecules. CTLA-4 which is an essential receptor involved in the negative regulation of T cell activation is a negative immunomodulator expressed on activated T cells and delivers an inhibitory signal during immune responses[25] . Blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases[26] . CTLA-4 deficiency in Tregs not only breaks self-tolerance but also impairs the suppressive function of Tregs in tumor immunity. Researches
have reported that CTLA-4 blockade results in an accumulation of Tregs and CD8+T cells in tumor tissue[27-28] . DCs play a crucial role in the induction of primary T cell responses. Now various DC vaccines are considered as effective treatment for cancers. A high frequency of immature DCs in the cancerous epithelium and a low density of mature DCs in the tumor stroma was found in esophageal squamous cell carcinoma[29] . Activated DCs secrete high level of IL-12 which plays a crucial role in the stimulation of T cells. Treg can affect the maturation of DC and restrain DC presenting antigen to T cells. In our previous study we have demonstrated the suppressive function of Tregs was mediated by cell-to-cell contact. Tregs can hamper the up-regulation of CD80/CD86 on DCs. However the mechanism of how HCC-derived Treg affect DC phenotype is not very clear. To figure out this problem, we did antibody-blocking experiments. Tregs co-cultured with DCs, or co-cultured with Teff and DC, could decrease the expression of CD80/CD86 on DCs. But when we added anti-CTLA-4 antibody into co-cultured cells, there was no reduction of CD80/CD86 on DCs. The down-modulation of CD80/CD86 could be interdicted by blocking CTLA-4 on Tregs. CTLA-4 interacted with two ligands (CD80/CD86) was found on antigen-presenting cells and delivered a cell-intrinsic negative signal. CTLA-4 on the surface of Treg down-regulated or prevented the up-regulation of CD80/CD86. The down-modulation was likely caused by a direct effect on the co-cultured DCs. IL-12 production could be strongly inhibited by the presence of Treg. But the down-modulation of IL-12 was inhibited partially by anti-CTLA-4 antibody. In conclusion, the effector function of Tregs is cell-contact dependent. CTLA-4 plays an important role in Treg-mediated suppression. In our research we have clearly proved that the down-modulation of CD80/86 by Tregs is CTLA-4-dependent. Tregs can modulate the phenotype and function of DCs. Disclosures The authors have no financial commercial conflicts of interest.
Acknowledgements This work was financially supported by Natural Science Fund of Zhejiang province (Y2090660).
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Figure 1. Histopathological examination (A) Histopathological examination of carcinoma tissue. The cancer cells were arranging disorderly, pleomorphism obviously and lost original liver normal structure. Necrosis region could be observed visibly. (magnification×200) (B) Histopathological examination of normal liver tissue. We could observe normal hepatic lobule structure. (magnification×200)
Figure2. Representative figures of flow cytometry examination. The proportion of CD4+CD25+ Treg/CD4+ T cells in HCC-mice is low before cell isolation. CD4+T cells from HCC-mice spleen were sorted into CD25- and CD25+ cells by immunomagnetic beads. The purities of Tregs or effector T cells (Teffs ) were ＞90% respectively. DCs from BALB/C mice spleen were isolated by CD11C+ immunomagnetic beads and the purity was ＞90%.
Figure3. Cell cluster formation in co-cultures of DC with Treg. Representative photomicrographs of culture cells. Different groups of cells were observed by conventional bright field microscopy (Olympus, Japan). (A) Cultures of DC. DCs from BALB/C mice spleen were isolated by CD11C+immunomagnetic beads and cultured alone in medium in the presence of LPS. They were colony growth and distribution of clusters(magnification×10). (B)DCs under high magnification (magnification×40). (C) Cultures of tumor-derived Treg. Tregs from HCC-mice spleen were sorted by immunomagnetic beads and then cultured alone in medium. They were sporadic growth and distribution decentralized. (magnification×40). (D) Tregs co-cultured with DCs. A strong tendency for cell cluster formation between Treg and DC was observed. Tregs formed stable cluster around DCs(magnification×10).
Figure4. HCC-derived Tregs down-regulate CD80/86 expression on splenic DCs depending on CTLA-4. Splenic DCs cultured in medium only or co-cultured with HCC-derived Tregs in the presence of LPS or with a mix of HCC-derived Tregs and Teff cells in the presence of LPS and then anti-CTLA-4 mAb (100μg/ml) or control Ab were added. CD80/86 expression on CD11c-positive cells was analyzed. The data show mean fluorescence intensity (MFI) of gated CD11c positive cells. Results were the mean ± SD of triplicate wells. * is a significant difference. When the DC+Treg+anti-CTLA4 group compared to DC+Treg group, the CD80/86 expression was found not to down-regulate. When the DC+Treg+Teff+anti-CTLA4 group compared to DC+Treg+Teff group, the CD80/86 expression was found not to down-regulate.
Fig.5 Splenic DCs were co-cultured with pre-activated tumor-derived Tregs with or without LPS, anti-CTLA-4 Ab, or isotype control antibody. IL-12 production was determined by ELISA in the culture supernatants. Results were the mean ± SD of triplicate wells. * is a significant difference when compared to DC+Lps+Treg group (P<0.05). When the DC+Lps+Treg+anti-CTLA4 group compared to DC+Lps+Treg group, the down-modulation of IL-12 was inhibited partially by blocking CTLA-4.
S0344-0338(16)30236-9 http://dx.doi.org/doi:10.1016/j.prp.2016.12.008 PRP 51695
To appear in: Received date:
6-7-2016
Please cite this article as: Xin Chen, Yong Du, Qingqing Hu, ZhiMing Huang, Tumorderived CD4+CD25+regulatory T cells inhibit dendritic cells function by CTLA-4, Pathology - Research and Practice http://dx.doi.org/10.1016/j.prp.2016.12.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Tumor-derived CD4+CD25+regulatory T cells inhibit dendritic cells function by CTLA-4
Xin Chen1#,Yong Du2#, Qingqing Hu2, ZhiMing Huang1*
1
Department of Gastroenterology and Hepatology, First Affiliated Hospital of Wenzhou Medical
University , Wenzhou, 325000, Zhejiang, China.
2
Department of Pediatrics, First Affiliated Hospital of Wenzhou Medical University, Wenzhou,
325000, ZheJiang, China.
*
Corresponding author: ZhiMing Huang, Department of Gastroenterology and Hepatology, First
Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China; Tel:+577-88069257;
Fax: +577-88069555; E-mail: [email protected]
#
Equal contributors
Tregs: CD4+CD25+ regulatory T cells; DCs: dendritic cells; HCC:hepatocellular carcinoma; CTLA-4: cytotoxic T lymphocyte-associated protein
Highlights
Tumor-derived Tregs down-modulated CD80/CD86 on DCs in a CTLA-4-dependent way. Tregs form stable cluster with DCs. Blockade of CTLA-4 can lead to increase DC-mediated immunity.
Abstract Purpose: CD4+CD25+regulatory T cells (Tregs) play an important role in anti-tumor immune responses. Poor prognosis and declining survival rates have intimate connection with high Treg expression in cancer patients. Cytotoxic T Lymphocyte-associated protein (CTLA-4) is one of the most prominent molecules on Treg. In our previous research, we have demonstrated that HCC-derived Tregs can interfere with Dendritic cells (DCs) function and down-modulate CD80/CD86 on DCs in vitro in a cell-contact dependent way. However the mechanism of how HCC-derived Treg affect DC phenotype are not very clear. Therefore, we investigated the function of CTLA-4 in anti-tumor immune responses. Materials and methods: We established BABL/C mouse with hepatocellular carcinoma model, and tumor-derived Tregs were purified by magnetic cell sorting using mouse CD4+CD25+regulatory T cell isolation kit. Splenic DCs were enriched using CD11c-conjugated microbeads. Then splenic DCs co-cultured with tumor-derived Tregs and antibody-blocking experiments was performed. Results: In our research, we found the down-modulation of CD80/CD86 on DCs was inhibited by blocking CTLA-4. HCC-derived Tregs down-modulated CD80/CD86 on DCs in a CTLA-4-dependent way. Blockade of CTLA-4 can lead to increase DC-mediated immunity. Conclusion: CTLA-4 play a vital role in Treg-mediated immnue inhibition and this discovery can open up new ideas for the development of therapeutic strategies. Keywords: dendritic cell; CD4+CD25+regulatory T cell; CTLA-4.
1. Introduction Hepatocellular carcinoma (HCC) is one of the most common cancer worldwide [1] . Chronic viral hepatitis including hepatitis B virus or hepatitis C virus infection is a risk factor for HCC, making HCC prevention a major goal of antiviral therapy[2-4] . Treg-mediated suppression serves as a vital mechanism of negative regulation of immune-mediated inflammation and features prominently in autoimmune and autoinflammatory disorders, allergy, acute and chronic infections, cancer, and metabolic inflammation[5]. Many researches have proved the existence of Treg at tumor sites, indicating that Treg may induce local immune tolerance. The presence of infiltrating Tregs suppresses protective anti-tumor immune responses and their accumulation into the tumor microenvironment correlates with reduced survival[6] . Treg-derived from HCC patients have been proved to be more suppressive than Treg from healthy individuals[7] . Treg-mediated suppression can be important targets for immune intervention. Blockade of Treg-mediated
immune suppression can enhance anti-tumor effects[8-9] . Combinations of monoclonal antibodies capable of modulating Treg functions synergistically enhance antitumor activity[10] . Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) expressing on Tregs has been implicated in the function of Tregs. Li et al found cryoablation and anti-CTLA-4 antibody in prostate cancer murine model induced anti-tumor immunological response[11] . There are many mechanisms have been proved for Treg-mediated suppression. In our previous research[12] , increase frequency of Tregs in the peripheral blood and spleen was found in hepatocellular carcinoma mice. HCC-derived Treg could down-regulate the expression of costimulatory molecules CD80/CD86 on DCs by cell-to-cell contact and inhibit TNF-a and IL-12 production from DC. However the mechanism is not very clear. We still need deeper studies on the role of Treg in patients with hepatocellular carcinoma to provide new approaches for tumor immunotherapy. In our study, there is obvious tendency for cell cluster formation between tumor-derived Treg and DC. Treg-DC cluster formation may help Tregs to regulate the phenotype and function of DC. The down-modulation of CD80/CD86 is inhibited by blocking CTLA-4. IL-12 is one of important stimulators produced by DCs. When a high concentration of anti-CTLA-4 antibody is added in the Treg-DC group, the down-modulation of IL-12 is inhibited partially too. Our research illustrates that blockade of CTLA-4 by addition of anti-CTLA-4 mAbs can lead to increased DC-mediated immunity.
2. Material and Methods 2.1 Tumor model Male BALB/C mice (6-8 weeks old, purchased from the SLACCAS company,China) were bred under specific pathogen-free (SPF) conditions. Animal protocols were approved by the Experimental Animal Management Committee Of Wenzhou Medical University. All surgery was performed under chloralic hydras anesthesia, and all efforts were made to minimize suffering. The H22 hepato-cellular carcinoma cells (China Center for Type Culture Collection, wuhan, CN) were cultured in RPMI 1640 medium and cells were inoculated to abdomen with 1 ×106 cells per mouse. After 8-9 days, cancerous ascites were extracted aseptically. Cells were collected and resuspended to a concentration of 1 ×106 ∕ml for making model. The mice were randomly divided into the tumor group and control group. The mouse was celiac anesthesia. We did a longitudinal incision, exposed the liver, injected 0.01ml cancer cell suspension (104 cells ) to the liver and last closed enterocoelia. About 25 days later, the models were formed. When anatomy, we could see gray and nubbly carcinoma tissues at different sizes from 0.5 to 1.0 cm in diameters in livers. 4-μm thick sections were prepared and stained with haematoxylin and eosin by standard histological procedures (Figure 1).
2.2 Reagents and Materials Mouse CD4+CD25+regulatory T cell isolation kit and mouse CD11c magnetic bead sorting kit (MACS, Miltenyi Biotec, Germany); Fluorescentlylabeled antibody: CD4-FITC, CD25-PE, CD11c-FITC, CD86-PE, CD80-PE and corresponding homotype antibody (eBioscience, California, USA); Mouse IL-12 ELISA kit (IBL, Hamburg, Germany); anti-CTLA4(R&D, Minnesota, USA); Anti-CD3 antibody (BD biosciences, New Jersey, USA ); LPS(Sigma, Santa clara, CA, USA); FCS and RPMI1640 (Gibco, Grand Island, New York, USA); mouse H22
hepatocellular carcinoma cell lines (China Center for Type Culture Collection, wuhan, CN ); Flow cytometry ( BD biosciences, New Jersey, USA ).
2.3 Cell isolation and sorting Splenic single-cell suspension was prepared from normal mice. Then splenic DCs were enriched using CD11c-conjugated microbeads according to the manufacturers’ instructions. The cells were positive for CD11c as assessed by flow cytometry. High purity of DCs were got finally (Figure2).Splenic single-cell suspension was prepared from HCC-bearing mice and then tumor-derived Tregs were purified by magnetic cell sorting using mouse CD4+CD25+regulatory T cell isolation kit according to the manufacturers’ instructions. High purity of tumor-derived Treg or CD4+CD25- Teff cells were gathered respectively (Figure2). Both T cells were stimulated separately with plate-bound anti-CD3 antibody (1µg/ml) and IL-2 (10U/ml) at 5×105 ∕ml in 24-well plates. Preactivated tumor-derived Tregs (4-8×105 ∕ml) or Teff cells (4-8×105 ∕ml) or a mix of two population at a 1:1 ratio were cultured with splenic DCs (DC-T cell ratio of 1:2) and then anti-CTLA-4 mAb (100μg/ml) or control Ab were added. LPS was added（1µg/ml）to stimulate DCs. Last, the co-cultures and the supernatants were collected respectively.
2.4 Flow cytometry and Elisa The co-cultures and the supernatants were collected respectively. The samples were stained with corresponding antibodies at 4℃ for 30 minutes and analyzed by flow cytometry. Isotype-matched antibodies were used as controls. IL-12 was assessed by ELISA using corresponding enzyme-linked immunosorbent assay kits according to the manufacturer’ s instruction.
2.5 Statistical Analysis Data are expressed as mean±sd. Statistical analysis was performed using Student’s t test with SPSS18.0 program (SPSS, Chicago). All P values ＜0.05 were considered statistically significant.
3. Results 3.1 Tregs form stable cluster with DCs Pre-activated tumor-derived Tregs were co-cultured with splenic DCs in the presence of LPS, and typical cluster formation was observed (Figure3). Onishi et al confirmed that Tregs form aggregates around Splenic DCs using immunofluorescence(13). In our previous research(12), we have confirmed Tregs down-regulated the expression of costimulatory molecules CD80/CD86 on DCs by cell-to-cell contact. This indicated that Treg-DC cluster formation might enable Tregs to modulate phenotypic and functional characteristics of DCs. Tregs might diminish the up-regulation of costimulatory molecule expression on DC by formation stable cell cluster. Treg-DC cluster might constitute a physical barrier and interfere with the function of DC.
3.2 HCC-derived Tregs down-regulate CD80/86 expression on splenic DCs depending on CTLA-4 Tregs could down-regulate CD80/86 expression on splenic DCs in our previous research, but
by what mechanism for Treg mediating CD80/86 down-regulation was unkonwn. To further address the suppression mechanism induced by Tregs, we then analyzed the possible contribution of CTLA-4 and performed antibody-blocking experiments (Figure4). HCC-derived Treg either when co-cultured with DC separately or when co-cultured with DC and Teff simultaneously, reduced the expression of CD80/CD86. But when splenic DCs were co-cultured with HCC-derived Tregs in the presence of anti-CTLA-4 mAb, the CD80/86 expression was found not to down-regulate. To avoid cross-linking by anti-CTLA-4, control antibody was added to the cultures. The findings indicated that there exsited CTLA-4/B7 interaction. HCC-derived Tregs down-regulated CD80/86 expression in a CTLA-4 dependent manner and blockade of CTLA-4 with anti-CTLA-4 mAb could abrogate the Treg-mediated suppression. 3.3 CTLA-4 expression strengthen Treg-mediated suppression The production of pro-inflammatory and immunoregulatory cytokines by DCs is important for the generation of adaptive immune responses. IL-12 is one of important stimulator produced by DCs. LPS-induced mature DCs can secrete high level of IL-12 while Tregs down-regulate the production of IL-12 from DCs. But how CTLA-4 affect the cytokine secretion by DCs. To figure out this problem, we did antibody-blocking experiments. We added a high concentration of anti-CTLA-4 antibody into different groups. The down-modulation of IL-12 was inhibited partially by blocking CTLA-4. Treg suppression of DCs function depends on CTLA-4. (Figure5)
4. Discussion Treg as a functional subset of inhibitory T cells, plays a crucial role in the maintenance of immune tolerance. Treg can secret immunosuppressive cytokines such as IL-10, TGF-β, IL-35, which can directly inhibit the function of responder T cells and myeloid cells[14-15] . Treg not only involves in autoimmune disease, infection and transplantation tolerance, but also plays a pivotal role in the suppression of anti-tumor immunity during development[16] . It has the features of hyporesponsive and anergic to antigen stimulation. Depletion of Treg can augment anti-tumor immune responses and could be a new approach for tumor therapy[17] . Administration of Treg-depleting anti-CD25 monoclonal antibody enhances both the anti-tumor response and the effectiveness of cancer vaccines[10] . The high level expression of Treg in tumor tissues and peripheral blood of cancer patients was reported in a lot of literature[18-20] . A high expression of Treg may inhibit the anti-tumor effect of T cells or induce immunological resistance to tumor antigens among cytotoxic T lymphocytes without producing acute or memory cytotoxic T lymphocytes responses. Strauss et al reported that Treg produced interleukin-10 and transforming growth factor-beta1 to mediate suppression in the tumor microenvironment[21] . Many researches found the prevalence of Treg cells was obviously higher in hepatocellular carcinoma than in the nontumorous liver and the patient with high expression of Treg cells showed a lower survival rate[22-24] . Among the cell-surface markers associated with Treg phenotype and function, CTLA-4 is one of the most prominent molecules. CTLA-4 which is an essential receptor involved in the negative regulation of T cell activation is a negative immunomodulator expressed on activated T cells and delivers an inhibitory signal during immune responses[25] . Blockade of CTLA-4 for a limited period in normal mice leads to spontaneous development of chronic organ-specific autoimmune diseases[26] . CTLA-4 deficiency in Tregs not only breaks self-tolerance but also impairs the suppressive function of Tregs in tumor immunity. Researches
have reported that CTLA-4 blockade results in an accumulation of Tregs and CD8+T cells in tumor tissue[27-28] . DCs play a crucial role in the induction of primary T cell responses. Now various DC vaccines are considered as effective treatment for cancers. A high frequency of immature DCs in the cancerous epithelium and a low density of mature DCs in the tumor stroma was found in esophageal squamous cell carcinoma[29] . Activated DCs secrete high level of IL-12 which plays a crucial role in the stimulation of T cells. Treg can affect the maturation of DC and restrain DC presenting antigen to T cells. In our previous study we have demonstrated the suppressive function of Tregs was mediated by cell-to-cell contact. Tregs can hamper the up-regulation of CD80/CD86 on DCs. However the mechanism of how HCC-derived Treg affect DC phenotype is not very clear. To figure out this problem, we did antibody-blocking experiments. Tregs co-cultured with DCs, or co-cultured with Teff and DC, could decrease the expression of CD80/CD86 on DCs. But when we added anti-CTLA-4 antibody into co-cultured cells, there was no reduction of CD80/CD86 on DCs. The down-modulation of CD80/CD86 could be interdicted by blocking CTLA-4 on Tregs. CTLA-4 interacted with two ligands (CD80/CD86) was found on antigen-presenting cells and delivered a cell-intrinsic negative signal. CTLA-4 on the surface of Treg down-regulated or prevented the up-regulation of CD80/CD86. The down-modulation was likely caused by a direct effect on the co-cultured DCs. IL-12 production could be strongly inhibited by the presence of Treg. But the down-modulation of IL-12 was inhibited partially by anti-CTLA-4 antibody. In conclusion, the effector function of Tregs is cell-contact dependent. CTLA-4 plays an important role in Treg-mediated suppression. In our research we have clearly proved that the down-modulation of CD80/86 by Tregs is CTLA-4-dependent. Tregs can modulate the phenotype and function of DCs. Disclosures The authors have no financial commercial conflicts of interest.
Acknowledgements This work was financially supported by Natural Science Fund of Zhejiang province (Y2090660).
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Figure 1. Histopathological examination (A) Histopathological examination of carcinoma tissue. The cancer cells were arranging disorderly, pleomorphism obviously and lost original liver normal structure. Necrosis region could be observed visibly. (magnification×200) (B) Histopathological examination of normal liver tissue. We could observe normal hepatic lobule structure. (magnification×200)
Figure2. Representative figures of flow cytometry examination. The proportion of CD4+CD25+ Treg/CD4+ T cells in HCC-mice is low before cell isolation. CD4+T cells from HCC-mice spleen were sorted into CD25- and CD25+ cells by immunomagnetic beads. The purities of Tregs or effector T cells (Teffs ) were ＞90% respectively. DCs from BALB/C mice spleen were isolated by CD11C+ immunomagnetic beads and the purity was ＞90%.
Figure3. Cell cluster formation in co-cultures of DC with Treg. Representative photomicrographs of culture cells. Different groups of cells were observed by conventional bright field microscopy (Olympus, Japan). (A) Cultures of DC. DCs from BALB/C mice spleen were isolated by CD11C+immunomagnetic beads and cultured alone in medium in the presence of LPS. They were colony growth and distribution of clusters(magnification×10). (B)DCs under high magnification (magnification×40). (C) Cultures of tumor-derived Treg. Tregs from HCC-mice spleen were sorted by immunomagnetic beads and then cultured alone in medium. They were sporadic growth and distribution decentralized. (magnification×40). (D) Tregs co-cultured with DCs. A strong tendency for cell cluster formation between Treg and DC was observed. Tregs formed stable cluster around DCs(magnification×10).
Figure4. HCC-derived Tregs down-regulate CD80/86 expression on splenic DCs depending on CTLA-4. Splenic DCs cultured in medium only or co-cultured with HCC-derived Tregs in the presence of LPS or with a mix of HCC-derived Tregs and Teff cells in the presence of LPS and then anti-CTLA-4 mAb (100μg/ml) or control Ab were added. CD80/86 expression on CD11c-positive cells was analyzed. The data show mean fluorescence intensity (MFI) of gated CD11c positive cells. Results were the mean ± SD of triplicate wells. * is a significant difference. When the DC+Treg+anti-CTLA4 group compared to DC+Treg group, the CD80/86 expression was found not to down-regulate. When the DC+Treg+Teff+anti-CTLA4 group compared to DC+Treg+Teff group, the CD80/86 expression was found not to down-regulate.
Fig.5 Splenic DCs were co-cultured with pre-activated tumor-derived Tregs with or without LPS, anti-CTLA-4 Ab, or isotype control antibody. IL-12 production was determined by ELISA in the culture supernatants. Results were the mean ± SD of triplicate wells. * is a significant difference when compared to DC+Lps+Treg group (P<0.05). When the DC+Lps+Treg+anti-CTLA4 group compared to DC+Lps+Treg group, the down-modulation of IL-12 was inhibited partially by blocking CTLA-4.