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Interactions of dendritic cells with cancer cells and modulation of surface molecules affect functional properties of CD8+ T cells
Molecular Immunology 48 (2011) 1744–1752
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Interactions of dendritic cells with cancer cells and modulation of surface molecules affect functional properties of CD8+ T cells Min Ji Seo a , Gi Rak Kim a , Young Min Son a , Deok-Chun Yang b , Hyuk Chu c , Tae Sun Min d , In Duk Jung e , Yeong-Min Park e , Seung Hyun Han f , Cheol-Heui Yun a,g,∗ a
Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea Korean Ginseng Center and Ginseng Genetic Resource Bank, Kyung Hee University, Seocheon-dong, Kiheun-gu, Yongin, Kyunggi-do 449-701, Republic of Korea c Division of Zoonoses, Center for Immunology & Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul, Republic of Korea d National Research Foundation of Korea, Daejeon 305-350, Republic of Korea e Department of Microbiology and Immunology and National Research Laboratory of Dendritic Cell Differentiation and Regulation, Medical Research Institute, Pusan National University, College of Medicine, Busan, Republic of Korea f Department of Oral Microbiology and Immunology, BK21 Program, and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea g Center for Agricultural Biomaterials, Seoul National University, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 9 February 2011 Received in revised form 29 March 2011 Accepted 15 April 2011 Available online 1 June 2011 Keywords: Dendritic cells Cancer cells Major histocompatibility complex CD8+ T cells Antigen processing component
a b s t r a c t To understand the interaction of dendritic cells (DCs) with cancer cells, we investigated molecular changes in DCs following co-culture with cancer cells. DCs co-cultured with Jurkat cancer cells showed remarkable down-regulation of MHC class I molecules, while DCs co-cultured with MCF-7 cancer cells showed minimal changes. Interestingly, down-regulation of MHC class I on DCs was not observed upon treatment with Jurkat cell lysate or culture supernatant, suggesting the importance of direct cell–cell interactions. The expressions of CD40, CD80, CD83, MHC class II, and IL-12p40 on DCs co-cultured with Jurkat cells were only slightly affected. In contrast, DCs co-cultured with MCF-7 cells showed increased expressions of CD80, CD83, CD86, and IL-12p40. Furthermore, DCs co-cultured with Jurkat cells showed a downregulation of low molecular weight polypeptides (LMP) 7, and of transporter associated with antigen processing (TAP) 1 and 2 at the mRNA expression level. LMP7, TAP2 and 2-microglobulin (2M) were also down-regulated at the protein level. We further demonstrated how altered expression of MHC class I on DCs caused by co-culture with cancer cells affected autologous CD8+ T cells, using the model MHC class I-presented HSV antigen. We found that DCs that had been HSV-treated and co-cultured with Jurkat cells showed a reduced potency to activate CD8+ T cells. In contrast, HSV-treated DCs that had been cocultured with MCF-7 cells induced activation of CD8+ T cells, including high expression of CD25, CD69, granzyme B and cytokines, TNF-␣ and IFN-␥. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Dendritic cells (DCs) are heterogeneous professional antigenpresenting cells and are particularly important for the activation
Abbreviations: APM, antigen processing machinery; 2M, 2-microglobulin; CTL, cytotoxic T lymphocyte; DCs, dendritic cells; ERAAP, endoplasmic reticulum aminopeptidase associated with antigen processing; GM-CSF, granulocyte macrophage colony stimulating factor; HSV, herpes simplex virus; LMP, low molecular weight polypeptides; LPS, lipopolysaccharide; M.F.I., mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; TAP, transporter associated with antigen processing. ∗ Corresponding author at: Protein Engineering and Comparative Immunology, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. Tel.: +82 2 880 4802; fax: +82 2 886 4805. E-mail address: [email protected] (C.-H. Yun). 0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2011.04.018
of naïve T cells (Jang et al., 2009; Son et al., 2008). DCs play a key role in the induction of tumor-specific immune responses by enabling cross-priming where tumor antigens are presented by MHC class I to CD8+ cytotoxic T lymphocytes (CTLs) (Armstrong et al., 1998; Huang et al., 1994). However, tumor cells utilize a variety of mechanisms to escape immune recognition and attack during development and progression (Marincola et al., 2000). For example, tumor cells are necessary and sufficient to convert DCs into tolerogenic TGF--secreting cells that induce CD4+ CD25+ regulatory T cells (Ghiringhelli et al., 2005; Ha, 2009). A previous study showed that functional changes, in particular alterations in the ability to process and present cancer-specific antigens to T cells, are associated with DC maturation (Banchereau and Steinman, 1998; Steinman, 1996) and with exposure of DCs to tumor cells (Esche et al., 1999; Katsenelson et al., 2001; Pirtskhalaishvili et al., 2000). However, the molecular mechanisms of the modulation of MHC class I expression and association with the antigen processing
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machinery (APM) have not been well defined in DCs that interact with cancer cells. The APM is composed of multiple molecular species organized to efficiently process antigens entering the pathway (Campoli et al., 2002; Seliger et al., 2000). MHC class I heavy chains are initially assembled with 2-microglobulin (2M), followed by recruitment into the peptide-loading complex in the endoplasmic reticulum. Endogenous peptides, generated in the cytoplasm through the action of proteasomes and other peptidases, are transported into the endoplasmic reticulum by transporter associated with antigen processing (TAP). Endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) mediates the final amino-terminal trimming of peptides, either before or after their initial binding to MHC class I molecules. Components of the peptide loading complex promote peptide loading and exchange, providing a ‘quality-control’ mechanism for the preferential export of kinetically stable peptide-MHC class I complexes to the cell surface (Hammer and Shastri, 2007; Jensen, 2007; Paulsson, 2004; Peaper and Cresswell, 2008; Zhang and Williams, 2006). Previous studies on the involvement of APM in DCs demonstrated that tumor cells down-regulate the expression of several APM components in immature DCs (iDCs) in a transwell system, indicating the importance of cancer cell-derived secretory molecules (Whiteside et al., 2004). However, it is unclear whether a direct interaction between cancer cells and DCs is required to modulate the molecular changes of DCs and CD8+ T cells. Here we analyzed functional properties of CD8+ T cells in response to the regulation of surface markers and the antigen presenting capacity of DCs co-cultured with MCF-7 or Jurkat cells. 2. Materials and methods 2.1. Cell lines, reagents and antibodies A549 (lung cancer), SW480 (colorectal cancer), HeLa (cervical cancer), MCF-7 (breast cancer), Jurkat (T cell lymphoma), and Raji (B cell lymphoma) cell-lines were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS), and 1% antibiotics (all from Invitrogen, NY, USA). For flow cytometiric analysis, fluorescence-labeled monoclonal antibodies to cell surface and intracellular molecules of lymphocytes (CD8-FITC, CD25-APC, CD69-APC, IFN-␥-APC, and granzyme BAlexa 647) and DCs (CD40-FITC, CD80-PE, CD83-FITC, CD86-APC, MHC class I-FITC, MHC class II-APC, and CD11c-APC) were purchased from BD Biosciences (CA, USA). Western blot analysis was performed with primary antibodies specific for TAP2, 2M, actin (Santa Cruz Biotechnology, CA, USA) and LMP7 (R&D Systems, MN, USA), and secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) or HRP-conjugated mouse anti-rabbit IgG (Chemicon, IL, USA). 2.2. DC generation Peripheral blood mononuclear cells (PBMCs) were obtained from human blood by density gradient centrifugation using FicollPaque PlusTM (Amersham Healthcare, Aylesbury, UK). Monocytes were isolated from the PBMCs using a magnetic bead-based positive selection kit (IMagTM ) and anti-human CD14 antibody (BD Biosciences). This procedure routinely yielded over 90% pure CD14positive cells as verified by flow cytometry (data not shown). To generate immature DCs (iDCs), CD14-positive cells were treated with 500 U/ml human recombinant Interleukin-4 (hrIL-4; R&D Systems, Minneapolis, MN, USA) and 800 U/ml human recombinant granulocyte-macrophage colony stimulating factor (hrGM-CSF;
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R&D Systems) at 1 × 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with heat-inactivated 10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin (all from Invitrogen) and 0.05 mM 2-mercaptoethanol (Sigma–Aldrich, MO, USA) in 100 mm tissue culture plates (Costar Corp., MA, USA) for 5 days at 37 ◦ C under 5% CO2 with a media change after 3 days. All experiments involving human blood were approved by the Institutional Review Board of Seoul National University (IRB no. 0705/001-002). 2.3. Co-culture of DCs with cancer cells To examine cancer cell number-dependent modulation of DCs, human DCs generated from PBMCs were co-cultured with each cancer cell-line for 24 h at 37 ◦ C, in a 12-well plate for a 1:1 (DCs:cancer cells) ratio, 6-well plate for 1:2 ratio and 60 mm culture dish for 1:4 ratio. Cancer cells were seeded and incubated in a 37 ◦ C CO2 incubator for 8 h to allow attachment before the addition of DCs. DCs cultured in the absence of cancer cells were used as a control. 2.4. Isolation of DCs after co-culture with cancer cells For the isolation of DCs, co-cultured cells were stained with antihuman CD11c-APC monoclonal antibody (mAb) for 10 min at 4 ◦ C. The cells were washed twice with phosphate buffered saline (PBS) and incubated with anti-APC microbeads for 15 min at 4 ◦ C. After washing with buffer (PBS pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA), the cell suspension was loaded onto a MACS column, and placed in the magnetic field of a MACS separator (Miltenyi Biotec, CA, USA). The magnetically labeled cells were eluted and confirmed as DCs (data not shown). 2.5. RT-PCR analysis DCs at 1 × 106 cells were disrupted in 1 ml TRI Reagent (Invitrogen) and RNA was extracted using chloroform and isopropyl alcohol. Samples were kept frozen at −80 ◦ C until use. Total RNA was quantitated by A260 measurements (NanoDrop® ND-1000 Spectrophotometer, Amersham Bioscience). RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase and oligo(dT) (Invitrogen), and 2 g of each reaction mixture was then used for PCR. Sense and antisense oligonucleotide primers were designed for RT-PCR using DNA sequence information obtained from the Genome Database (NCBI). Primers were synthesized by Bioneer (Korea), with the following sequences: 5 -gatcacactgacctggcagc-3 , 5 -ggtgcctttgcagaaacaaa-3 for HLA-A; 5 -accaccccatctctgaccat-3 , 5 -tctcggtccctcacaagaca-3 for HLA-B; 5 -ggaccgggagacacagaagt-3 , 5 -gtcagtgtgatctccgcagg-3 for HLA-C; 5 -ctcgcgctactctctctttctgg3 , 5 -gcttacatgtctcgatcccacttaa-3 for beta2-microglobulin; 5 cagaatctgtaccagccc-3 , 5 -ctggctgtttgcatccagg-3 for TAP1; 5 tacctgctcgtaaggagggtgc-3 , 5 -attgggatatgcaaaggagacg -3 for TAP2; 5 -ttgtgatgggttctgattcccg-3 , 5 -cagagcaatagcgtctgtgc-3 for LMP2; 5 -tcgccttcaagttccagcatgc-3 , 5 -ccaaccatcttccttcatgtgg-3 for LMP7; and 5 -caaagtgtccctgatgccagc-3 , 5 -ggtgaattcgacaggcatagcg-3 for tapasin. PCR was performed in a thermal cycler (Bio-Rad Laboratories, CA, USA) under the following conditions: pre-denaturation at 95 ◦ C for 4 min, followed by 30 cycles of denaturation at 95 ◦ C for 30 s, annealing at 55 ◦ C for 30 s and extension at 72 ◦ C for 1 min, and finally an additional elongation step for 5 min at 72 ◦ C. The PCR products were separated on a 1.5% agarose gel containing ethidium bromide. 2.6. Isolation of T cells CD8+ T cells were isolated from PBMCs using a magnetic bead-based positive selection kit (IMagTM , BD Biosciences) and
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Fig. 1. Expression of MHC class I on dendritic cells co-cultured with different cancer cell-lines. DCs were co-cultured with each cancer cell-line. (A) Expression levels of MHC class I on the surface of DCs alone (shaded area) and DCs with cancer cells (ratio of 1:1 for dashed line, 1:2 for dotted line, 1:4 for bold line) were assessed by flow cytometry using APC-conjugated anti-CD11c and FITC-conjugated anti-MHC class I monoclonal antibodies. (B) The data are shown as the mean of five independent experiments ± SD (P < 0.05).
anti-human CD8 antibodies. CD8+ T cells were cultured in RPMI 1640, supplemented with heat-inactivated 10% FBS and 1% antibiotics. It should be noted that the cells were treated with 2.5 ng/ml of hrIL-2 (R&D Systems), which is the minimal dose for maintaining the survival of T cells (data not shown).
2.7. Western blot analysis DCs treated with different stimuli were washed three times with cold PBS and lysed in cold RIPA lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 7.4, a protease inhibitor cocktail (Roche, Mannheim, Germany), 2 mM NaF, 0.1 mM sodium orthovanadate and 2 mM glycerol phosphate. After centrifugation at 28,000 × g for 7 min at 4 ◦ C, the insoluble material was removed and the protein concentration was measured by Bradford Assay (Bio-Rad Laboratories) with BSA as a standard. Twenty micrograms of lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride microporous membrane (Amersham Bioscience). Membranes were blocked with 3% BSA in TBST (0.1 M Tris, 0.9% NaCl, and 0.1% Tween 20) for 60 min at room temperature before being probed with primary antibodies. Primary antibodies were detected using HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) or HRP-conjugated mouse anti-rabbit IgG (Chemicon) and visualized by the enhanced chemiluminescence system (GE Healthcare, Chalfont, UK).
2.8. ELISA to detect IL-12p40, TNF-˛, and IFN- DCs were co-cultured with MCF-7 or Jurkat cells at a ratio of 1:4, or stimulated with lipopolysaccharide (LPS) as a positive control for 24 h. The culture supernatant was then harvested to detect the level of IL-12p40 secretion. After co-culture with cancer cells, DCs were isolated using APC-conjugated anti-CD11c mAb and anti-APC microbeads. DCs were then treated with herpes simplex virus (HSV) at a multiplicity of infection (MOI) of 10 for 2 h at 37 ◦ C, and subsequently co-cultured with autologus CD8+ T cells. After 3 days, the production of TNF-␣ and IFN-␥ was measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D system) according to the manufacturer’s instructions.
2.9. Statistical analysis The mean value with standard deviation was determined for each treatment group in a given experiment, and all experiments were performed at least three times. Treatment groups were compared with the appropriate control group, and statistical significance was measured using a two-tailed paired t-test. Differences were considered significant when P < 0.05. 3. Results 3.1. Expression of MHC class I on dendritic cells co-cultured with various cancer cell-lines We evaluated the expression of MHC class I on DCs co-cultured with A549, SW480, HeLa, MCF-7, Jurkat, or Raji cells at a ratio of 1:1, 1:2 or 1:4 for 24 h at 37 ◦ C. DCs co-cultured with cancer cells were stained with APC-conjugated CD11c and FITC-conjugated MHC I antibodies. Only CD11c-positive cells were then gated and assessed for the expression of MHC class I. The surface levels of MHC class I on DCs were down-regulated when co-cultured with A549, SW480, Jurkat, or Raji, but not with HeLa and MCF-7 cells (Fig. 1A). These results suggested that expression of MHC class I on DCs depended on the type of cancer cells that they were co-cultured with. The expression of MHC class I on DCs co-cultured with Jurkat cells was decreased the most, showing more than 50% down-regulation for a 1:4 ratio when compared to DCs without cancer cells. These results indicated that down-regulation of the expression level of MHC class I on DCs upon exposure to Jurkat cells was cell number-dependent. We therefore fixed the co-culture setting at a ratio of 1:4 in all further experiments. For these findings to be relevant, we presented the data on several donors and examined the variability from donor to donor. As shown in Fig. 1B, the expression of MHC class I on DCs was decreased in similar pattern as observed in Fig. 1A. 3.2. Expressions of CD80, CD83, CD86 and IL-12p40 are increased in DCs co-cultured with MCF-7 cells, but not with Jurkat cells We also investigated the expressions of other DC surface molecules including CD40, CD80, CD83, CD86, and MHC class II. For this, we selected two cancer cell lines: Jurkat cells, which downregulated the expression of MHC class I molecules on DCs the most; and MCF-7 cells, which hardly affected MHC class I expression. DCs
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Fig. 2. Expression of surface markers on dendritic cells co-cultured with MCF-7 or Jurkat cells. Dendritic cells were co-cultured with MCF-7 or Jurkat cells at a 1:4 ratio. (A) Expression of CD40, CD80, CD83, CD86, and MHC class II (shaded area, DCs alone; dashed line, DCs co-cultured with MCF-7 cells; dotted line, DCs co-cultured with Jurkat cells) was assessed by flow cytometry. Data represent one of the three independent experiments. (B) Culture supernatants were harvested at 72 h after incubation and used for the detection of IL-12p40 in DCs alone, DCs co-cultured with MCF-7 cells, DCs co-cultured with Jurkat cells, and DCs treated with 100 ng/ml LPS (as a positive control), by ELISA. The data are representative of three independent experiments (P < 0.05).
were co-cultured with either MCF-7 or Jurkat cells for 24 h at a ratio of 1:4, followed by assessment of the expression of CD40, CD80, CD83, CD86, and MHC class II in CD11c-positive cells. Expression levels of CD80, CD83 and CD86 changed minimally on DCs after coculture with Jurkat cells, but were increased on DCs after co-culture with MCF-7 cells (Fig. 2A). On the other hand, expression levels of CD40 and MHC class II on DCs were not affected by co-culture with either MCF-7 or Jurkat cells (Fig. 2A). To ensure this, we conducted three independent experiments for investigating the expression of CD40, CD80, CD83, CD86 and MHC class II (Supplementary Fig. 1). IL-12 is mainly produced by DCs and is one of the key mediators for inducing IFN-␥ and TNF-␣ production from T cells. IL-12 also mediates enhancement of the cytotoxic activity of CD8+ T lymphocytes (Shurin et al., 2009). IL-12 is heterodimer form of IL-12p40 and IL-12p35, which is IL-12p70 active form. It has been shown that IL-12p40 expression could be a criterion for the activation of dendritic cells (Jelinek et al., 2011; Maroof et al., 2009). The functional changes in DCs co-cultured with cancer cells were examined by testing IL-12p40 secretion. As expected, the highest level of IL12p40 secretion was detected in DCs treated with 100 ng/ml LPS, and used as a positive control (Fig. 2B). Untreated DCs, and DCs cocultured with Jurkat cells hardly secreted any IL-12p40. However, DCs co-cultured with MCF-7 cells showed a greater than 100-fold increase in IL-12p40, indicating that MCF-7 cells could strongly activate DCs (Fig. 2B). 3.3. Direct contacts between DCs and Jurkat cells mediate down-regulation of MHC class I molecules on DCs To elucidate the cause of down-regulation of MHC class I on DCs co-cultured with Jurkat cells, we treated DCs with either lysates or culture media from these cells. MHC class I expression on DCs
was not affected by treatment with Jurkat cell lysate (Fig. 3A) or culture supernatant (Fig. 3B). This suggested that Jurkat celldependent down-regulation of MHC class I on DCs was due to direct cell–cell interaction. To confirm this hypothesis, we fixed Jurkat cells with 2% formaldehyde and then co-cultured them with DCs. MHC class I molecules were indeed down-regulated in these DCs after co-culture with fixed Jurkat cells (Fig. 3C and D), suggesting that down-regulation of MHC class I was caused mainly by direct cell–cell interactions. Additionally, the expression of MHC class I on DCs co-cultured with fixed MCF-7 was barely modulated (data not shown). 3.4. mRNA and protein expression of APM components in DCs co-cultured with MCF-7 or Jurkat cells Changes in MHC class I expression may reflect alterations in transcriptional regulation of APM components, which play a critical role in the assembly and presentation of functional MHC class I molecules (Chang et al., 2005; Setiadi et al., 2007; Tomasi et al., 2006). Loss or dysfunction of molecules involved in antigen processing and presentation such as TAP1, TAP2, LMP2, LMP7, and tapasin, is known to decrease MHC class I expression (Ogino et al., 2006; Seliger et al., 2000). We therefore evaluated changes in mRNA expression of MHC class I molecules (HLA-A, HLA-B, and HLA-C), (2M, TAP1, TAP2, LMP2, LMP7, and tapasin. We found that mRNA expression levels of TAP1, TAP2, and LMP7 were decreased in DCs co-cultured with Jurkat cells, but not in those co-cultured with MCF-7 cells (Fig. 4A). We also investigated protein expression levels of TAP2, LMP7, and 2M after co-culturing DCs with cancer cells at a ratio of 1:4. DCs were isolated after 6, 12, 18, and 24 h of co-culture with cancer cells, and protein expression levels of TAP2, LMP7, and 2M were analyzed. We found down-regulation of LMP7, TAP2,
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Fig. 3. Surface expression of MHC class I on DCs. Dendritic cells were co-cultured with MCF-7 or Jurkat cells at a 1:4 ratio. (A) Expression levels of MHC class I on DCs treated for 24 h with lysate from MCF-7 and Jurkat cells were measured by flow cytometry. (B) Expression levels of MHC class I on DCs treated for 24 h with culture supernatant from MCF-7 cells, Jurkat cells, DCs + MCF-7 cells or DCs + Jurkat cells were measured by flow cytometry. (C) Expression of MHC class I on DCs co-cultured with 2% formaldehydefixed Jurkat cells and supplemented with lysate or culture supernatant from Jurkat cells was measured by flow cytometry. Data were assessed by FITC-conjugated anti-MHC class I antibodies (A–C), and APC-conjugated anti-CD11c antibody (C) for excluding potential contamination by cancer cells. (D) Data represent the M.F.I. from experiment (C).
and 2M in DCs that were co-cultured with Jurkat, but not with MCF-7 cells (Fig. 4B). We also investigated changes in TAP2, LMP7, and 2M expression in DCs after co-culture with Jurkat cells at a ratio of 1:1, 1:2, 1:4, and 1:8, for 24 h. Protein expression was found to be down-regulated in a cell number-dependent manner. Collectively, these results suggested that the down-regulation of LMP7 (proteasomes function), TAP2 (antigen transporter), and 2M contributed to the down-regulation of MHC class I expression in DCs co-cultured with Jurkat cells. 3.5. CD25 and CD69 expression, and cytokine production in CD8+ T cells co-cultured with DCs that had been previously exposed to HSV and cancer cells The down-regulation of surface MHC class I expression on DCs co-cultured with cancer cells may decrease the likelihood of active interactions between T cells and DCs at the first phase of immunological synapse formation, hence leading to a decrease in DC-dependent T cell activation and proliferation (Gao et al., 2008). We used HSV as a model antigen to examine functional and phenotypic changes of CD8+ T cells in response to the modulation of surface expression on DCs co-cultured with cancer cells. DCs were co-cultured with cancer cells for 24 h, isolated and then treated
with HSV at a MOI of 10 for 2 h. DCs were then co-incubated with autologous CD8+ T cells for an additional 48 h, followed by staining with either CD8-FITC and CD25-APC antibodies, or CD8-FITC and CD69-APC antibodies to assess CD8+ T cell activation. CD25 and CD69 were found to be highly expressed in CD8+ T cells upon co-cultured with HSV-treated DCs that had been previously cocultured with MCF-7 cells. However, the expression of CD25 and CD69 was down-regulated in HSV-treated DCs that had been previously co-cultured with Jurkat cells when compared to HSV-treated DCs without previous co-culture (Fig. 5). This might be due to the decreased expression of MHC I on DCs co-cultured with Jurkat cells being hard to transfer HSV antigen to CD8+ T cells. Upregulation of CD25 and CD69 could be primarily due to the up-regulation of CD80, CD83, CD86, and IL-12p40 in DCs co-cultured with MCF-7 cells. As expected, expression of CD25 and CD69 was hardly changed in CD8+ T cells co-incubated with DCs in the absence of HSV treatment. Next we carried out intracellular staining of IFN-␥ and granzyme B on the second day of co-incubation of CD8+ T cells and HSVtreated DCs that had been previously co-cultured with cancer cells. IFN-␥ and TNF-␣ production was also examined by ELISA after three days of co-incubation. IFN-␥ expression was the highest when CD8+ T cells were co-incubated with HSV-treated DCs, by both intracellular staining and ELISA (Fig. 6A and D). Expression
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Fig. 4. mRNA and protein expression of antigen processing components in DCs co-cultured with MCF-7 or Jurkat cells. (A) Dendritic cells were co-cultured with MCF-7 or Jurkat cells at a 1:4 ratio. After co-culture with cancer cells for 9 h, DCs were isolated and mRNA levels of MHC class I molecules (HLA-A, HLA-B and HLA-C), 2-microglobulin (2M), TAP1, TAP2, LMP2, LMP7, and Tapasin were analyzed by RT-PCR. (B) DCs were co-cultured with cancer cells (MCF-7 or Jurkat cells) at a 1:4 ratio for 6, 12, 18 and 24 h or at different ratios for 24 h. DCs were isolated and DC lysates were prepared using RIPA buffer for Western blotting. Specific antibodies to TAP2, LMP7, 2M, and -actin were used for the assay.
of granzyme B (Fig. 6B) and TNF-␣ (Fig. 6C) was the highest for coincubation of CD8+ T cells and HSV-treated, MCF-7 co-cultured DCs. Because granzyme B is produced upon CD8+ T cell activation, we also examined mRNA expression in CD8+ T cells co-cultured with HSV-treated DCs, with or without previous co-culture of these DCs with cancer cells. We found that mRNA expression of granzyme B was clearly increased in CD8+ T cells co-cultured with either HSV-treated DCs, or with HSV-treated and MCF-7 co-cultured DCs. However, granzyme B mRNA levels did not increase considerably in CD8+ T cells upon co-culture with HSV-treated DCs that had been previously co-cultured with Jurkat cells (Fig. 6E). It was noting that the differences in the production of cytokines and granzyme B in CD8+ T cells between MCF-7 and Jurkat co-cultured DCs were obvious. The secretion of cytokines, IFN-␥ and TNF-␣, and generation of granzyme B were significantly (P < 0.05) decreased in CD8+ T cells cultured with HSV-treated DCs that had been co-cultured with Jurkat cells compared to those cultured with HSV-treated DCs without previous co-culture likely because of down-regulation of MHC class I. 4. Discussion Dendritic cells (DCs) are responsible for driving and orchestrating tumor antigen-specific immune responses, thus controlling both the quality and the magnitude of the response. Here we investigated the expression of surface markers and cytokine secretion by DCs co-cultured with different cancer cells in order to define the role of tumor-induced suppression on DCs. Potential factors that could mediate such suppression included: (1) the expression of MHC I-peptide complex; (2) the expression of co-stimulatory molecules; (3) cytokine secretion; and (4) cross-priming to CD8+ T cells. First, we showed that MHC class I molecules were downregulated in DCs co-cultured with A549, SW480, Raji and Jurkat cells, but not with HeLa or MCF-7 cells. This result indicated that
surface expression of MHC class I on DCs depended on the type of cancer cells that they had been co-cultured with. DCs co-cultured with MCF-7 cells showed increased expression of CD80, CD83, CD86, and IL-12p40 but not CD40 and MHC class II. Gao et al. (2008) reported a similar result where CD40 is down-regulated and CD86 is not altered in DCs co-cultured with TSA-lymphoma, but CD40 is upregulated when co-cultured with the Hepa 1–6 cell line, a derivative of the BW7756 mouse hepatoma. We therefore attributed the variation in MHC class I expression to the use of different types of cancer cells with varying antigenicity. Notably, our observation of the MCF-7 cell-dependent activation of DCs and subsequent high expression of costimulatory molecules as well as IL-12 secretion is a novel finding. We found that about a half of DCs showed the increased CD83 expression (Fig. 2A). It has been shown that CD83 exists intracellularly in monocytes, macrophages, and DCs at resting stage whereas it is expressed on the cell surface upon the activation (Cao et al., 2005). Although existence of heterogeneity in monocyte-derived DCs is possible, it is unlikely in this study since only CD11c+ cells were accounted and represented. It is probable that the expression of CD83 changes as cells divide and the status of the cells are not all homogenous. We showed that MHC class I molecules were down-regulated in DCs at different levels when A549, SW480, Jurkat and Raji cells were co-cultured. The surface expression of MHC class I is known to be organized and regulated by several APM components. Therefore, we expected that the expression of APM components could play a critical role in the antigen processing or presentation pathway in DCs. Each APM component is responsible for a distinct functional step and necessary for implementing an effective interaction with a targeted T cell (Campoli et al., 2002; Seliger et al., 2000). For example, alterations in TAP1 expression may lead to defective peptide loading on MHC class I molecules and interfere with further expression on the cell surface (Campoli and Ferrone, 2008). Thus, the down-regulation of APM components such as LMPs, TAP
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Fig. 5. Expression of activation markers in CD8+ T cells co-cultured with DCs treated under various conditions. DCs were co-cultured with MCF-7 or Jurkat cells at a ratio of 1:4. After 24 h, DCs were isolated using APC-conjugated anti-CD11c antibody and anti-APC microbeads, and treated with HSV at a MOI of 10 for 2 h, and then co-cultured with autologous CD8+ T cells. After 48 h, the expression of (A) CD25 and (B) CD69 was assessed by flow cytometry using APC-conjugated anti-CD25 or -CD69 antibody together with FITC-conjugated anti-CD8 antibody. Data are shown the mean positive percentage of (C) CD25 and (D) CD69 from three independent experiments ± SD (P < 0.05).
and tapasin could result in deficient MHC class I surface expression (Seliger, 2008). As shown Fig. 4A, mRNA levels of HLA-A, B, C were not changed whereas the expression of mRNA and protein levels in TAP1, TAP2, LMP7 and 2M were decreased in Jurkat cocultured DCs. It represents, indirectly, that the intracellular level of MHC class I was stable. But, the antigen processing and transporting system did not work properly, which made MHC class I molecules having hard time to exit from the ER and transit to cell surface. Obviously it is still possible that MHC I molecules are internalized and degraded for some reason instead of re-utilized and re-expressed onto the cell surface, which requires further investigation. Our results improve the understanding of the interaction between DCs and cancer cells, providing fundamental insights into the mechanisms of immune surveillance of tumors and their immune escape, and suggesting novel approaches to cancer therapy. Our data also provide important insights into the antigen-processing pathway for the generation and maintenance of tumor antigen-specific immune responses in human DCs. We found that tumors could potentially exert strong inhibitory effects on DCs. Patients with various malignancies, including breast cancer and multiple myeloma, show abnormalities in DC numbers and function (Fricke and Gabrilovich, 2006; Vuckovic et al., 2004). Reduced
DC counts in the peripheral blood of cancer patients have been associated with an accumulation of immunosuppressive immature myeloid cells (Almand et al., 2001; Serafini et al., 2004) or an alternative lineage negative, CD11c-negative “gap” population of DC-like cells, suggesting a defect in DC differentiation. Thus, DCs in cancer patients have both quantitative proportional and qualitative defects that are at least partially influenced by tumor-derived factors. These can be overcome by several strategies both in vitro and in vivo, making them very suitable to be used for therapeutic cancer vaccine. It is a critical, but underappreciated, the fact that this strategy requires that once activated appropriately in vitro, the injected DCs must retain their functional capacity in vivo. In conclusion, we showed that modulation of DC surface markers caused by co-culture with different types of cancer cells differently affects the expression levels of CD25 and CD69, and the production of cytokines and granzyme B in CD8+ T cells. For effective anti-cancer therapy, the proper recognition of cancer cells by DCs and the resulting induction of a cancer-specific T cell immune response should be considered and confirmed. Finally, the poor immune responses against cancer cells in cancer patients are due to weakened and/or deficient functional dendritic cells that could train CD8+ T cells. We provide the novel findings on how some cancer cells acquire immune evasion mechanism.
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Fig. 6. Expression of cytokines, IFN-␥ and TNF-␣, and cytotoxic-associated molecule, granzyme B, in CD8+ T cells co-cultured with DCs that had previously been treated with cancer cells and HSV. DCs were co-cultured with MCF-7 or Jurkat cells at a ratio of 1:4. After 24 h, DCs were isolated and treated with HSV at a MOI of 10 for 2 h, and then co-cultured with autologous CD8+ T cells. After 48 h, intracellular expression of (A) IFN-␥ and (B) granzyme B was assessed by flow cytometry using APC-conjugated anti-IFN-␥ or granzyme B-Alexa 647 antibody together with FITC-conjugated anti-CD8 antibody. After 72 h, the supernatant from each treatment was harvested and used to test the expression of (C) TNF-␣ and (D) IFN-␥ by ELISA. The data in (C) and (D) are average values from three independent experiments + SD (P < 0.05). (E) mRNA expression of granzyme B was assessed in CD8+ T cells co-cultured for 12 h with DCs that had been treated with HSV and/or previously co-cultured with cancer cells.
Funding
Appendix A. Supplementary data
This research was supported by Basic Science Research Program through the National Research Foundation (NRF) (2010-0027222 and 2010-0003291), and the Agriculture Research Center program and GRCMVP (607002-05-2-HD360) for Technology Development Program of the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molimm.2011.04.018.
Acknowledgements We thank members of the Protein Engineering and Comparative Immunology lab., Seoul National University for excellent technical assistance and helpful discussion.
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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm
Interactions of dendritic cells with cancer cells and modulation of surface molecules affect functional properties of CD8+ T cells Min Ji Seo a , Gi Rak Kim a , Young Min Son a , Deok-Chun Yang b , Hyuk Chu c , Tae Sun Min d , In Duk Jung e , Yeong-Min Park e , Seung Hyun Han f , Cheol-Heui Yun a,g,∗ a
Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea Korean Ginseng Center and Ginseng Genetic Resource Bank, Kyung Hee University, Seocheon-dong, Kiheun-gu, Yongin, Kyunggi-do 449-701, Republic of Korea c Division of Zoonoses, Center for Immunology & Pathology, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul, Republic of Korea d National Research Foundation of Korea, Daejeon 305-350, Republic of Korea e Department of Microbiology and Immunology and National Research Laboratory of Dendritic Cell Differentiation and Regulation, Medical Research Institute, Pusan National University, College of Medicine, Busan, Republic of Korea f Department of Oral Microbiology and Immunology, BK21 Program, and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea g Center for Agricultural Biomaterials, Seoul National University, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 9 February 2011 Received in revised form 29 March 2011 Accepted 15 April 2011 Available online 1 June 2011 Keywords: Dendritic cells Cancer cells Major histocompatibility complex CD8+ T cells Antigen processing component
a b s t r a c t To understand the interaction of dendritic cells (DCs) with cancer cells, we investigated molecular changes in DCs following co-culture with cancer cells. DCs co-cultured with Jurkat cancer cells showed remarkable down-regulation of MHC class I molecules, while DCs co-cultured with MCF-7 cancer cells showed minimal changes. Interestingly, down-regulation of MHC class I on DCs was not observed upon treatment with Jurkat cell lysate or culture supernatant, suggesting the importance of direct cell–cell interactions. The expressions of CD40, CD80, CD83, MHC class II, and IL-12p40 on DCs co-cultured with Jurkat cells were only slightly affected. In contrast, DCs co-cultured with MCF-7 cells showed increased expressions of CD80, CD83, CD86, and IL-12p40. Furthermore, DCs co-cultured with Jurkat cells showed a downregulation of low molecular weight polypeptides (LMP) 7, and of transporter associated with antigen processing (TAP) 1 and 2 at the mRNA expression level. LMP7, TAP2 and 2-microglobulin (2M) were also down-regulated at the protein level. We further demonstrated how altered expression of MHC class I on DCs caused by co-culture with cancer cells affected autologous CD8+ T cells, using the model MHC class I-presented HSV antigen. We found that DCs that had been HSV-treated and co-cultured with Jurkat cells showed a reduced potency to activate CD8+ T cells. In contrast, HSV-treated DCs that had been cocultured with MCF-7 cells induced activation of CD8+ T cells, including high expression of CD25, CD69, granzyme B and cytokines, TNF-␣ and IFN-␥. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Dendritic cells (DCs) are heterogeneous professional antigenpresenting cells and are particularly important for the activation
Abbreviations: APM, antigen processing machinery; 2M, 2-microglobulin; CTL, cytotoxic T lymphocyte; DCs, dendritic cells; ERAAP, endoplasmic reticulum aminopeptidase associated with antigen processing; GM-CSF, granulocyte macrophage colony stimulating factor; HSV, herpes simplex virus; LMP, low molecular weight polypeptides; LPS, lipopolysaccharide; M.F.I., mean fluorescence intensity; PBMC, peripheral blood mononuclear cells; TAP, transporter associated with antigen processing. ∗ Corresponding author at: Protein Engineering and Comparative Immunology, Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea. Tel.: +82 2 880 4802; fax: +82 2 886 4805. E-mail address: [email protected] (C.-H. Yun). 0161-5890/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2011.04.018
of naïve T cells (Jang et al., 2009; Son et al., 2008). DCs play a key role in the induction of tumor-specific immune responses by enabling cross-priming where tumor antigens are presented by MHC class I to CD8+ cytotoxic T lymphocytes (CTLs) (Armstrong et al., 1998; Huang et al., 1994). However, tumor cells utilize a variety of mechanisms to escape immune recognition and attack during development and progression (Marincola et al., 2000). For example, tumor cells are necessary and sufficient to convert DCs into tolerogenic TGF--secreting cells that induce CD4+ CD25+ regulatory T cells (Ghiringhelli et al., 2005; Ha, 2009). A previous study showed that functional changes, in particular alterations in the ability to process and present cancer-specific antigens to T cells, are associated with DC maturation (Banchereau and Steinman, 1998; Steinman, 1996) and with exposure of DCs to tumor cells (Esche et al., 1999; Katsenelson et al., 2001; Pirtskhalaishvili et al., 2000). However, the molecular mechanisms of the modulation of MHC class I expression and association with the antigen processing
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machinery (APM) have not been well defined in DCs that interact with cancer cells. The APM is composed of multiple molecular species organized to efficiently process antigens entering the pathway (Campoli et al., 2002; Seliger et al., 2000). MHC class I heavy chains are initially assembled with 2-microglobulin (2M), followed by recruitment into the peptide-loading complex in the endoplasmic reticulum. Endogenous peptides, generated in the cytoplasm through the action of proteasomes and other peptidases, are transported into the endoplasmic reticulum by transporter associated with antigen processing (TAP). Endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP) mediates the final amino-terminal trimming of peptides, either before or after their initial binding to MHC class I molecules. Components of the peptide loading complex promote peptide loading and exchange, providing a ‘quality-control’ mechanism for the preferential export of kinetically stable peptide-MHC class I complexes to the cell surface (Hammer and Shastri, 2007; Jensen, 2007; Paulsson, 2004; Peaper and Cresswell, 2008; Zhang and Williams, 2006). Previous studies on the involvement of APM in DCs demonstrated that tumor cells down-regulate the expression of several APM components in immature DCs (iDCs) in a transwell system, indicating the importance of cancer cell-derived secretory molecules (Whiteside et al., 2004). However, it is unclear whether a direct interaction between cancer cells and DCs is required to modulate the molecular changes of DCs and CD8+ T cells. Here we analyzed functional properties of CD8+ T cells in response to the regulation of surface markers and the antigen presenting capacity of DCs co-cultured with MCF-7 or Jurkat cells. 2. Materials and methods 2.1. Cell lines, reagents and antibodies A549 (lung cancer), SW480 (colorectal cancer), HeLa (cervical cancer), MCF-7 (breast cancer), Jurkat (T cell lymphoma), and Raji (B cell lymphoma) cell-lines were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS), and 1% antibiotics (all from Invitrogen, NY, USA). For flow cytometiric analysis, fluorescence-labeled monoclonal antibodies to cell surface and intracellular molecules of lymphocytes (CD8-FITC, CD25-APC, CD69-APC, IFN-␥-APC, and granzyme BAlexa 647) and DCs (CD40-FITC, CD80-PE, CD83-FITC, CD86-APC, MHC class I-FITC, MHC class II-APC, and CD11c-APC) were purchased from BD Biosciences (CA, USA). Western blot analysis was performed with primary antibodies specific for TAP2, 2M, actin (Santa Cruz Biotechnology, CA, USA) and LMP7 (R&D Systems, MN, USA), and secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) or HRP-conjugated mouse anti-rabbit IgG (Chemicon, IL, USA). 2.2. DC generation Peripheral blood mononuclear cells (PBMCs) were obtained from human blood by density gradient centrifugation using FicollPaque PlusTM (Amersham Healthcare, Aylesbury, UK). Monocytes were isolated from the PBMCs using a magnetic bead-based positive selection kit (IMagTM ) and anti-human CD14 antibody (BD Biosciences). This procedure routinely yielded over 90% pure CD14positive cells as verified by flow cytometry (data not shown). To generate immature DCs (iDCs), CD14-positive cells were treated with 500 U/ml human recombinant Interleukin-4 (hrIL-4; R&D Systems, Minneapolis, MN, USA) and 800 U/ml human recombinant granulocyte-macrophage colony stimulating factor (hrGM-CSF;
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R&D Systems) at 1 × 106 cells/ml in RPMI 1640 (Invitrogen), supplemented with heat-inactivated 10% FBS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 2 mM l-glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 g/ml streptomycin (all from Invitrogen) and 0.05 mM 2-mercaptoethanol (Sigma–Aldrich, MO, USA) in 100 mm tissue culture plates (Costar Corp., MA, USA) for 5 days at 37 ◦ C under 5% CO2 with a media change after 3 days. All experiments involving human blood were approved by the Institutional Review Board of Seoul National University (IRB no. 0705/001-002). 2.3. Co-culture of DCs with cancer cells To examine cancer cell number-dependent modulation of DCs, human DCs generated from PBMCs were co-cultured with each cancer cell-line for 24 h at 37 ◦ C, in a 12-well plate for a 1:1 (DCs:cancer cells) ratio, 6-well plate for 1:2 ratio and 60 mm culture dish for 1:4 ratio. Cancer cells were seeded and incubated in a 37 ◦ C CO2 incubator for 8 h to allow attachment before the addition of DCs. DCs cultured in the absence of cancer cells were used as a control. 2.4. Isolation of DCs after co-culture with cancer cells For the isolation of DCs, co-cultured cells were stained with antihuman CD11c-APC monoclonal antibody (mAb) for 10 min at 4 ◦ C. The cells were washed twice with phosphate buffered saline (PBS) and incubated with anti-APC microbeads for 15 min at 4 ◦ C. After washing with buffer (PBS pH 7.2, 0.5% bovine serum albumin (BSA), and 2 mM EDTA), the cell suspension was loaded onto a MACS column, and placed in the magnetic field of a MACS separator (Miltenyi Biotec, CA, USA). The magnetically labeled cells were eluted and confirmed as DCs (data not shown). 2.5. RT-PCR analysis DCs at 1 × 106 cells were disrupted in 1 ml TRI Reagent (Invitrogen) and RNA was extracted using chloroform and isopropyl alcohol. Samples were kept frozen at −80 ◦ C until use. Total RNA was quantitated by A260 measurements (NanoDrop® ND-1000 Spectrophotometer, Amersham Bioscience). RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase and oligo(dT) (Invitrogen), and 2 g of each reaction mixture was then used for PCR. Sense and antisense oligonucleotide primers were designed for RT-PCR using DNA sequence information obtained from the Genome Database (NCBI). Primers were synthesized by Bioneer (Korea), with the following sequences: 5 -gatcacactgacctggcagc-3 , 5 -ggtgcctttgcagaaacaaa-3 for HLA-A; 5 -accaccccatctctgaccat-3 , 5 -tctcggtccctcacaagaca-3 for HLA-B; 5 -ggaccgggagacacagaagt-3 , 5 -gtcagtgtgatctccgcagg-3 for HLA-C; 5 -ctcgcgctactctctctttctgg3 , 5 -gcttacatgtctcgatcccacttaa-3 for beta2-microglobulin; 5 cagaatctgtaccagccc-3 , 5 -ctggctgtttgcatccagg-3 for TAP1; 5 tacctgctcgtaaggagggtgc-3 , 5 -attgggatatgcaaaggagacg -3 for TAP2; 5 -ttgtgatgggttctgattcccg-3 , 5 -cagagcaatagcgtctgtgc-3 for LMP2; 5 -tcgccttcaagttccagcatgc-3 , 5 -ccaaccatcttccttcatgtgg-3 for LMP7; and 5 -caaagtgtccctgatgccagc-3 , 5 -ggtgaattcgacaggcatagcg-3 for tapasin. PCR was performed in a thermal cycler (Bio-Rad Laboratories, CA, USA) under the following conditions: pre-denaturation at 95 ◦ C for 4 min, followed by 30 cycles of denaturation at 95 ◦ C for 30 s, annealing at 55 ◦ C for 30 s and extension at 72 ◦ C for 1 min, and finally an additional elongation step for 5 min at 72 ◦ C. The PCR products were separated on a 1.5% agarose gel containing ethidium bromide. 2.6. Isolation of T cells CD8+ T cells were isolated from PBMCs using a magnetic bead-based positive selection kit (IMagTM , BD Biosciences) and
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Fig. 1. Expression of MHC class I on dendritic cells co-cultured with different cancer cell-lines. DCs were co-cultured with each cancer cell-line. (A) Expression levels of MHC class I on the surface of DCs alone (shaded area) and DCs with cancer cells (ratio of 1:1 for dashed line, 1:2 for dotted line, 1:4 for bold line) were assessed by flow cytometry using APC-conjugated anti-CD11c and FITC-conjugated anti-MHC class I monoclonal antibodies. (B) The data are shown as the mean of five independent experiments ± SD (P < 0.05).
anti-human CD8 antibodies. CD8+ T cells were cultured in RPMI 1640, supplemented with heat-inactivated 10% FBS and 1% antibiotics. It should be noted that the cells were treated with 2.5 ng/ml of hrIL-2 (R&D Systems), which is the minimal dose for maintaining the survival of T cells (data not shown).
2.7. Western blot analysis DCs treated with different stimuli were washed three times with cold PBS and lysed in cold RIPA lysis buffer containing 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris pH 7.4, a protease inhibitor cocktail (Roche, Mannheim, Germany), 2 mM NaF, 0.1 mM sodium orthovanadate and 2 mM glycerol phosphate. After centrifugation at 28,000 × g for 7 min at 4 ◦ C, the insoluble material was removed and the protein concentration was measured by Bradford Assay (Bio-Rad Laboratories) with BSA as a standard. Twenty micrograms of lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride microporous membrane (Amersham Bioscience). Membranes were blocked with 3% BSA in TBST (0.1 M Tris, 0.9% NaCl, and 0.1% Tween 20) for 60 min at room temperature before being probed with primary antibodies. Primary antibodies were detected using HRP-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) or HRP-conjugated mouse anti-rabbit IgG (Chemicon) and visualized by the enhanced chemiluminescence system (GE Healthcare, Chalfont, UK).
2.8. ELISA to detect IL-12p40, TNF-˛, and IFN- DCs were co-cultured with MCF-7 or Jurkat cells at a ratio of 1:4, or stimulated with lipopolysaccharide (LPS) as a positive control for 24 h. The culture supernatant was then harvested to detect the level of IL-12p40 secretion. After co-culture with cancer cells, DCs were isolated using APC-conjugated anti-CD11c mAb and anti-APC microbeads. DCs were then treated with herpes simplex virus (HSV) at a multiplicity of infection (MOI) of 10 for 2 h at 37 ◦ C, and subsequently co-cultured with autologus CD8+ T cells. After 3 days, the production of TNF-␣ and IFN-␥ was measured by enzyme-linked immunosorbent assay (ELISA) kits (R&D system) according to the manufacturer’s instructions.
2.9. Statistical analysis The mean value with standard deviation was determined for each treatment group in a given experiment, and all experiments were performed at least three times. Treatment groups were compared with the appropriate control group, and statistical significance was measured using a two-tailed paired t-test. Differences were considered significant when P < 0.05. 3. Results 3.1. Expression of MHC class I on dendritic cells co-cultured with various cancer cell-lines We evaluated the expression of MHC class I on DCs co-cultured with A549, SW480, HeLa, MCF-7, Jurkat, or Raji cells at a ratio of 1:1, 1:2 or 1:4 for 24 h at 37 ◦ C. DCs co-cultured with cancer cells were stained with APC-conjugated CD11c and FITC-conjugated MHC I antibodies. Only CD11c-positive cells were then gated and assessed for the expression of MHC class I. The surface levels of MHC class I on DCs were down-regulated when co-cultured with A549, SW480, Jurkat, or Raji, but not with HeLa and MCF-7 cells (Fig. 1A). These results suggested that expression of MHC class I on DCs depended on the type of cancer cells that they were co-cultured with. The expression of MHC class I on DCs co-cultured with Jurkat cells was decreased the most, showing more than 50% down-regulation for a 1:4 ratio when compared to DCs without cancer cells. These results indicated that down-regulation of the expression level of MHC class I on DCs upon exposure to Jurkat cells was cell number-dependent. We therefore fixed the co-culture setting at a ratio of 1:4 in all further experiments. For these findings to be relevant, we presented the data on several donors and examined the variability from donor to donor. As shown in Fig. 1B, the expression of MHC class I on DCs was decreased in similar pattern as observed in Fig. 1A. 3.2. Expressions of CD80, CD83, CD86 and IL-12p40 are increased in DCs co-cultured with MCF-7 cells, but not with Jurkat cells We also investigated the expressions of other DC surface molecules including CD40, CD80, CD83, CD86, and MHC class II. For this, we selected two cancer cell lines: Jurkat cells, which downregulated the expression of MHC class I molecules on DCs the most; and MCF-7 cells, which hardly affected MHC class I expression. DCs
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Fig. 2. Expression of surface markers on dendritic cells co-cultured with MCF-7 or Jurkat cells. Dendritic cells were co-cultured with MCF-7 or Jurkat cells at a 1:4 ratio. (A) Expression of CD40, CD80, CD83, CD86, and MHC class II (shaded area, DCs alone; dashed line, DCs co-cultured with MCF-7 cells; dotted line, DCs co-cultured with Jurkat cells) was assessed by flow cytometry. Data represent one of the three independent experiments. (B) Culture supernatants were harvested at 72 h after incubation and used for the detection of IL-12p40 in DCs alone, DCs co-cultured with MCF-7 cells, DCs co-cultured with Jurkat cells, and DCs treated with 100 ng/ml LPS (as a positive control), by ELISA. The data are representative of three independent experiments (P < 0.05).
were co-cultured with either MCF-7 or Jurkat cells for 24 h at a ratio of 1:4, followed by assessment of the expression of CD40, CD80, CD83, CD86, and MHC class II in CD11c-positive cells. Expression levels of CD80, CD83 and CD86 changed minimally on DCs after coculture with Jurkat cells, but were increased on DCs after co-culture with MCF-7 cells (Fig. 2A). On the other hand, expression levels of CD40 and MHC class II on DCs were not affected by co-culture with either MCF-7 or Jurkat cells (Fig. 2A). To ensure this, we conducted three independent experiments for investigating the expression of CD40, CD80, CD83, CD86 and MHC class II (Supplementary Fig. 1). IL-12 is mainly produced by DCs and is one of the key mediators for inducing IFN-␥ and TNF-␣ production from T cells. IL-12 also mediates enhancement of the cytotoxic activity of CD8+ T lymphocytes (Shurin et al., 2009). IL-12 is heterodimer form of IL-12p40 and IL-12p35, which is IL-12p70 active form. It has been shown that IL-12p40 expression could be a criterion for the activation of dendritic cells (Jelinek et al., 2011; Maroof et al., 2009). The functional changes in DCs co-cultured with cancer cells were examined by testing IL-12p40 secretion. As expected, the highest level of IL12p40 secretion was detected in DCs treated with 100 ng/ml LPS, and used as a positive control (Fig. 2B). Untreated DCs, and DCs cocultured with Jurkat cells hardly secreted any IL-12p40. However, DCs co-cultured with MCF-7 cells showed a greater than 100-fold increase in IL-12p40, indicating that MCF-7 cells could strongly activate DCs (Fig. 2B). 3.3. Direct contacts between DCs and Jurkat cells mediate down-regulation of MHC class I molecules on DCs To elucidate the cause of down-regulation of MHC class I on DCs co-cultured with Jurkat cells, we treated DCs with either lysates or culture media from these cells. MHC class I expression on DCs
was not affected by treatment with Jurkat cell lysate (Fig. 3A) or culture supernatant (Fig. 3B). This suggested that Jurkat celldependent down-regulation of MHC class I on DCs was due to direct cell–cell interaction. To confirm this hypothesis, we fixed Jurkat cells with 2% formaldehyde and then co-cultured them with DCs. MHC class I molecules were indeed down-regulated in these DCs after co-culture with fixed Jurkat cells (Fig. 3C and D), suggesting that down-regulation of MHC class I was caused mainly by direct cell–cell interactions. Additionally, the expression of MHC class I on DCs co-cultured with fixed MCF-7 was barely modulated (data not shown). 3.4. mRNA and protein expression of APM components in DCs co-cultured with MCF-7 or Jurkat cells Changes in MHC class I expression may reflect alterations in transcriptional regulation of APM components, which play a critical role in the assembly and presentation of functional MHC class I molecules (Chang et al., 2005; Setiadi et al., 2007; Tomasi et al., 2006). Loss or dysfunction of molecules involved in antigen processing and presentation such as TAP1, TAP2, LMP2, LMP7, and tapasin, is known to decrease MHC class I expression (Ogino et al., 2006; Seliger et al., 2000). We therefore evaluated changes in mRNA expression of MHC class I molecules (HLA-A, HLA-B, and HLA-C), (2M, TAP1, TAP2, LMP2, LMP7, and tapasin. We found that mRNA expression levels of TAP1, TAP2, and LMP7 were decreased in DCs co-cultured with Jurkat cells, but not in those co-cultured with MCF-7 cells (Fig. 4A). We also investigated protein expression levels of TAP2, LMP7, and 2M after co-culturing DCs with cancer cells at a ratio of 1:4. DCs were isolated after 6, 12, 18, and 24 h of co-culture with cancer cells, and protein expression levels of TAP2, LMP7, and 2M were analyzed. We found down-regulation of LMP7, TAP2,
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Fig. 3. Surface expression of MHC class I on DCs. Dendritic cells were co-cultured with MCF-7 or Jurkat cells at a 1:4 ratio. (A) Expression levels of MHC class I on DCs treated for 24 h with lysate from MCF-7 and Jurkat cells were measured by flow cytometry. (B) Expression levels of MHC class I on DCs treated for 24 h with culture supernatant from MCF-7 cells, Jurkat cells, DCs + MCF-7 cells or DCs + Jurkat cells were measured by flow cytometry. (C) Expression of MHC class I on DCs co-cultured with 2% formaldehydefixed Jurkat cells and supplemented with lysate or culture supernatant from Jurkat cells was measured by flow cytometry. Data were assessed by FITC-conjugated anti-MHC class I antibodies (A–C), and APC-conjugated anti-CD11c antibody (C) for excluding potential contamination by cancer cells. (D) Data represent the M.F.I. from experiment (C).
and 2M in DCs that were co-cultured with Jurkat, but not with MCF-7 cells (Fig. 4B). We also investigated changes in TAP2, LMP7, and 2M expression in DCs after co-culture with Jurkat cells at a ratio of 1:1, 1:2, 1:4, and 1:8, for 24 h. Protein expression was found to be down-regulated in a cell number-dependent manner. Collectively, these results suggested that the down-regulation of LMP7 (proteasomes function), TAP2 (antigen transporter), and 2M contributed to the down-regulation of MHC class I expression in DCs co-cultured with Jurkat cells. 3.5. CD25 and CD69 expression, and cytokine production in CD8+ T cells co-cultured with DCs that had been previously exposed to HSV and cancer cells The down-regulation of surface MHC class I expression on DCs co-cultured with cancer cells may decrease the likelihood of active interactions between T cells and DCs at the first phase of immunological synapse formation, hence leading to a decrease in DC-dependent T cell activation and proliferation (Gao et al., 2008). We used HSV as a model antigen to examine functional and phenotypic changes of CD8+ T cells in response to the modulation of surface expression on DCs co-cultured with cancer cells. DCs were co-cultured with cancer cells for 24 h, isolated and then treated
with HSV at a MOI of 10 for 2 h. DCs were then co-incubated with autologous CD8+ T cells for an additional 48 h, followed by staining with either CD8-FITC and CD25-APC antibodies, or CD8-FITC and CD69-APC antibodies to assess CD8+ T cell activation. CD25 and CD69 were found to be highly expressed in CD8+ T cells upon co-cultured with HSV-treated DCs that had been previously cocultured with MCF-7 cells. However, the expression of CD25 and CD69 was down-regulated in HSV-treated DCs that had been previously co-cultured with Jurkat cells when compared to HSV-treated DCs without previous co-culture (Fig. 5). This might be due to the decreased expression of MHC I on DCs co-cultured with Jurkat cells being hard to transfer HSV antigen to CD8+ T cells. Upregulation of CD25 and CD69 could be primarily due to the up-regulation of CD80, CD83, CD86, and IL-12p40 in DCs co-cultured with MCF-7 cells. As expected, expression of CD25 and CD69 was hardly changed in CD8+ T cells co-incubated with DCs in the absence of HSV treatment. Next we carried out intracellular staining of IFN-␥ and granzyme B on the second day of co-incubation of CD8+ T cells and HSVtreated DCs that had been previously co-cultured with cancer cells. IFN-␥ and TNF-␣ production was also examined by ELISA after three days of co-incubation. IFN-␥ expression was the highest when CD8+ T cells were co-incubated with HSV-treated DCs, by both intracellular staining and ELISA (Fig. 6A and D). Expression
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Fig. 4. mRNA and protein expression of antigen processing components in DCs co-cultured with MCF-7 or Jurkat cells. (A) Dendritic cells were co-cultured with MCF-7 or Jurkat cells at a 1:4 ratio. After co-culture with cancer cells for 9 h, DCs were isolated and mRNA levels of MHC class I molecules (HLA-A, HLA-B and HLA-C), 2-microglobulin (2M), TAP1, TAP2, LMP2, LMP7, and Tapasin were analyzed by RT-PCR. (B) DCs were co-cultured with cancer cells (MCF-7 or Jurkat cells) at a 1:4 ratio for 6, 12, 18 and 24 h or at different ratios for 24 h. DCs were isolated and DC lysates were prepared using RIPA buffer for Western blotting. Specific antibodies to TAP2, LMP7, 2M, and -actin were used for the assay.
of granzyme B (Fig. 6B) and TNF-␣ (Fig. 6C) was the highest for coincubation of CD8+ T cells and HSV-treated, MCF-7 co-cultured DCs. Because granzyme B is produced upon CD8+ T cell activation, we also examined mRNA expression in CD8+ T cells co-cultured with HSV-treated DCs, with or without previous co-culture of these DCs with cancer cells. We found that mRNA expression of granzyme B was clearly increased in CD8+ T cells co-cultured with either HSV-treated DCs, or with HSV-treated and MCF-7 co-cultured DCs. However, granzyme B mRNA levels did not increase considerably in CD8+ T cells upon co-culture with HSV-treated DCs that had been previously co-cultured with Jurkat cells (Fig. 6E). It was noting that the differences in the production of cytokines and granzyme B in CD8+ T cells between MCF-7 and Jurkat co-cultured DCs were obvious. The secretion of cytokines, IFN-␥ and TNF-␣, and generation of granzyme B were significantly (P < 0.05) decreased in CD8+ T cells cultured with HSV-treated DCs that had been co-cultured with Jurkat cells compared to those cultured with HSV-treated DCs without previous co-culture likely because of down-regulation of MHC class I. 4. Discussion Dendritic cells (DCs) are responsible for driving and orchestrating tumor antigen-specific immune responses, thus controlling both the quality and the magnitude of the response. Here we investigated the expression of surface markers and cytokine secretion by DCs co-cultured with different cancer cells in order to define the role of tumor-induced suppression on DCs. Potential factors that could mediate such suppression included: (1) the expression of MHC I-peptide complex; (2) the expression of co-stimulatory molecules; (3) cytokine secretion; and (4) cross-priming to CD8+ T cells. First, we showed that MHC class I molecules were downregulated in DCs co-cultured with A549, SW480, Raji and Jurkat cells, but not with HeLa or MCF-7 cells. This result indicated that
surface expression of MHC class I on DCs depended on the type of cancer cells that they had been co-cultured with. DCs co-cultured with MCF-7 cells showed increased expression of CD80, CD83, CD86, and IL-12p40 but not CD40 and MHC class II. Gao et al. (2008) reported a similar result where CD40 is down-regulated and CD86 is not altered in DCs co-cultured with TSA-lymphoma, but CD40 is upregulated when co-cultured with the Hepa 1–6 cell line, a derivative of the BW7756 mouse hepatoma. We therefore attributed the variation in MHC class I expression to the use of different types of cancer cells with varying antigenicity. Notably, our observation of the MCF-7 cell-dependent activation of DCs and subsequent high expression of costimulatory molecules as well as IL-12 secretion is a novel finding. We found that about a half of DCs showed the increased CD83 expression (Fig. 2A). It has been shown that CD83 exists intracellularly in monocytes, macrophages, and DCs at resting stage whereas it is expressed on the cell surface upon the activation (Cao et al., 2005). Although existence of heterogeneity in monocyte-derived DCs is possible, it is unlikely in this study since only CD11c+ cells were accounted and represented. It is probable that the expression of CD83 changes as cells divide and the status of the cells are not all homogenous. We showed that MHC class I molecules were down-regulated in DCs at different levels when A549, SW480, Jurkat and Raji cells were co-cultured. The surface expression of MHC class I is known to be organized and regulated by several APM components. Therefore, we expected that the expression of APM components could play a critical role in the antigen processing or presentation pathway in DCs. Each APM component is responsible for a distinct functional step and necessary for implementing an effective interaction with a targeted T cell (Campoli et al., 2002; Seliger et al., 2000). For example, alterations in TAP1 expression may lead to defective peptide loading on MHC class I molecules and interfere with further expression on the cell surface (Campoli and Ferrone, 2008). Thus, the down-regulation of APM components such as LMPs, TAP
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Fig. 5. Expression of activation markers in CD8+ T cells co-cultured with DCs treated under various conditions. DCs were co-cultured with MCF-7 or Jurkat cells at a ratio of 1:4. After 24 h, DCs were isolated using APC-conjugated anti-CD11c antibody and anti-APC microbeads, and treated with HSV at a MOI of 10 for 2 h, and then co-cultured with autologous CD8+ T cells. After 48 h, the expression of (A) CD25 and (B) CD69 was assessed by flow cytometry using APC-conjugated anti-CD25 or -CD69 antibody together with FITC-conjugated anti-CD8 antibody. Data are shown the mean positive percentage of (C) CD25 and (D) CD69 from three independent experiments ± SD (P < 0.05).
and tapasin could result in deficient MHC class I surface expression (Seliger, 2008). As shown Fig. 4A, mRNA levels of HLA-A, B, C were not changed whereas the expression of mRNA and protein levels in TAP1, TAP2, LMP7 and 2M were decreased in Jurkat cocultured DCs. It represents, indirectly, that the intracellular level of MHC class I was stable. But, the antigen processing and transporting system did not work properly, which made MHC class I molecules having hard time to exit from the ER and transit to cell surface. Obviously it is still possible that MHC I molecules are internalized and degraded for some reason instead of re-utilized and re-expressed onto the cell surface, which requires further investigation. Our results improve the understanding of the interaction between DCs and cancer cells, providing fundamental insights into the mechanisms of immune surveillance of tumors and their immune escape, and suggesting novel approaches to cancer therapy. Our data also provide important insights into the antigen-processing pathway for the generation and maintenance of tumor antigen-specific immune responses in human DCs. We found that tumors could potentially exert strong inhibitory effects on DCs. Patients with various malignancies, including breast cancer and multiple myeloma, show abnormalities in DC numbers and function (Fricke and Gabrilovich, 2006; Vuckovic et al., 2004). Reduced
DC counts in the peripheral blood of cancer patients have been associated with an accumulation of immunosuppressive immature myeloid cells (Almand et al., 2001; Serafini et al., 2004) or an alternative lineage negative, CD11c-negative “gap” population of DC-like cells, suggesting a defect in DC differentiation. Thus, DCs in cancer patients have both quantitative proportional and qualitative defects that are at least partially influenced by tumor-derived factors. These can be overcome by several strategies both in vitro and in vivo, making them very suitable to be used for therapeutic cancer vaccine. It is a critical, but underappreciated, the fact that this strategy requires that once activated appropriately in vitro, the injected DCs must retain their functional capacity in vivo. In conclusion, we showed that modulation of DC surface markers caused by co-culture with different types of cancer cells differently affects the expression levels of CD25 and CD69, and the production of cytokines and granzyme B in CD8+ T cells. For effective anti-cancer therapy, the proper recognition of cancer cells by DCs and the resulting induction of a cancer-specific T cell immune response should be considered and confirmed. Finally, the poor immune responses against cancer cells in cancer patients are due to weakened and/or deficient functional dendritic cells that could train CD8+ T cells. We provide the novel findings on how some cancer cells acquire immune evasion mechanism.
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Fig. 6. Expression of cytokines, IFN-␥ and TNF-␣, and cytotoxic-associated molecule, granzyme B, in CD8+ T cells co-cultured with DCs that had previously been treated with cancer cells and HSV. DCs were co-cultured with MCF-7 or Jurkat cells at a ratio of 1:4. After 24 h, DCs were isolated and treated with HSV at a MOI of 10 for 2 h, and then co-cultured with autologous CD8+ T cells. After 48 h, intracellular expression of (A) IFN-␥ and (B) granzyme B was assessed by flow cytometry using APC-conjugated anti-IFN-␥ or granzyme B-Alexa 647 antibody together with FITC-conjugated anti-CD8 antibody. After 72 h, the supernatant from each treatment was harvested and used to test the expression of (C) TNF-␣ and (D) IFN-␥ by ELISA. The data in (C) and (D) are average values from three independent experiments + SD (P < 0.05). (E) mRNA expression of granzyme B was assessed in CD8+ T cells co-cultured for 12 h with DCs that had been treated with HSV and/or previously co-cultured with cancer cells.
Funding
Appendix A. Supplementary data
This research was supported by Basic Science Research Program through the National Research Foundation (NRF) (2010-0027222 and 2010-0003291), and the Agriculture Research Center program and GRCMVP (607002-05-2-HD360) for Technology Development Program of the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.molimm.2011.04.018.
Acknowledgements We thank members of the Protein Engineering and Comparative Immunology lab., Seoul National University for excellent technical assistance and helpful discussion.
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