Anti-CD40-induced inflammatory E-cadherin+ dendritic cells enhance T cell responses and antitumour immunity in murine Lewis lung carcinoma
© Zhang et al.; licensee BioMed Central. 2015
Received: 6 November 2014
Accepted: 19 January 2015
Published: 5 February 2015
Agonistic CD40 antibodies have been demonstrated to activate antigen-presenting cells (APCs) and enhance antitumour T cell responses, thereby providing a new therapeutic option in cancer immunotherapy. In agonistic CD40 antibody-mediated inflammatory responses, a novel subset of E-cadherin + dendritic cells (DCs) has been identified, and little is known about the role of these DCs in tumour immunity. This study investigated the effect of anti-CD40-mediated inflammatory E-cadherin + DCs in murine Lewis lung carcinoma (LLC).
The phenotype and characteristics of anti-CD40-mediated inflammatory E-cadherin + DCs isolated from the anti-CD40 model were assessed in vitro. The antitumour activity of E-cadherin + DCs were evaluated in vivo by promoting the differentiation of effector CD4+ T cells, CEA-specific CD8+ T cells and CD103+ CD8+ T cells and assessing their resistance to tumour challenge, including variations in tumour volume and survival curves.
Here, we demonstrated that anti-CD40-mediated E-cadherin + inflammatory DCs accumulate in the lungs of Rag1 KO mice and were able to stimulate naïve CD4+ T cells to induce Th1 and Th17 cell differentiation and polarisation and to inhibit regulatory T cell and Th2 responses. Importantly, with the adoptive transfer of E-cadherin + DCs into the Lewis lung cancer model, the inflammatory DCs increased the Th1 and Th17 cell responses and reduced the Treg cell and Th2 responses. Interestingly, following the injection of inflammatory E-cadherin + DCs, the CD103+ CD8+ T cell and CEA-specific CD8+ T cell responses increased and exhibited potent antitumour immunity.
These findings indicate that anti-CD40-induced E-cadherin + DCs enhance T cell responses and antitumour activity in non-small cell lung cancer (NSCLC)-bearing mice and may be used to enhance the efficacy of DC-based peptide vaccines against NSCLC.
CD40 is a tumour necrosis factor receptor superfamily member that is expressed on antigen-presenting cells (APCs) such as dendritic cells (DC), B cells, monocytes and some tumour cells. Recently, agonistic CD40 antibodies were applied in clinical trials targeting advanced pancreatic ductal adenocarcinoma (CP-870,893) and diffuse large B cell lymphoma (dacetuzumab and Chi Lob 7/4). The CD40 agonistic antibody has displayed excellent antitumour activity in the patients in these trials [1,2]. Many subsets of DCs exist in the agonistic CD40 antibody-mediated tumour microenvironment or under sterile inflammatory response conditions. However, the mechanism and function of CD40-mediated inflammatory DCs in cancer immunity are unknown.
In CD40 agonistic antibody-mediated inflammatory responses, a novel subset of E-cadherin + DCs has been identified. Although CD40 signalling is critical for the differentiation of inflammatory monocytes into E-cadherin + inflammatory DCs and the promotion of anti-CD40-mediated colitis has been confirmed in Rag1 KO mice , little is known regarding the role of E-cadherin + inflammatory DCs in tumour immunity.
Precisely how inflammatory DCs with tumour antigen peptides can induce a T cell response in tumour immunity is poorly understood. Here, we identified the inflammatory E-cadherin + DCs that accumulate in the lung during the anti-CD40 antibody-mediated inflammatory response. The phenotypes of these DCs are the same as those of spleen-derived inflammatory E-cadherin + DCs that are present during anti-CD40-mediated colitis. The agonistic CD40 mAb has not been universally accepted as a novel cancer therapy. Concerns include cytokine release syndromes, autoimmune reactions , thromboembolic syndromes, hyperimmune stimulation leading to activation-induced cell apoptosis or tolerance [5,6] and tumour angiogenesis, possibly as a result of the CD40-dependent activation of tumour endothelial cells . These effects may cause unacceptable toxicity or promote tumour growth . This study aimed to investigate the effects of anti-CD40-induced E-cadherin + DCs on the T cell response and antitumour activity in the tumour microenvironment. We found that inflammatory E-cadherin + DCs were present only in anti-CD40-mediated innate immunity, not innate, adoptive and tumour immunity. Our study will address the disadvantages of agonistic CD40 mAb in tumour therapy and may provide novel therapeutic strategies, as well as explain the pathogenesis of non-small cell lung cancer (NSCLC).
Materials and methods
Additional materials and methods can be found in the Additional file 1.
We obtained 6- to 8-week-old C57BL/6 mice from the Wuhan University Centre for Animal Experiments. B6.129S7-Rag1tmiMom/JNju (Rag1−/−) mice (background: C57BL/6) were provided by the Mode Animal Research Centre of Nanjing University. These Rag1−/− mice were housed and maintained in individual ventilated cages (IVC) under specific pathogen-free conditions; C57BL/6 mice were housed in specific pathogen-free conditions but not under IVC conditions. All breeding was conducted in the Huazhong University of Science and Technology Centre for Animal Experiments according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Spleens were digested with collagenase VIII (Sigma) as previously described  and filtered using a 70-μm cell strainer (BD Biosciences) to obtain mononuclear spleen cells. Lung or lung tumour tissues were cut into pieces, and mononuclear cells were digested with collagenase V (Sigma) for 2 h at 37°C as described previously . The resulting spleen or lung cell suspensions from anti-CD40 model mice were stained using E-cadherin, CD11c, CD4, CD103 and 7-AAD. The cells were first sorted for 7-AAD−CD11c+ cells using a FACS AriaIII sorter (BD Biosciences); 7-AAD + CD11c- cells were discarded. The CD11c + cells were then sorted into E-cadherin + and E-cadherin- DCs (E-cadherin+/− CD11chigh CD4-CD103-7-AAD-, purity >98%), as described (Additional file 1: Figure S1). The DC subsets were cultured in DMEM (GIBCO, Invitrogen) supplemented with 10% foetal bovine serum (FBS, GIBCO, Invitrogen), LPS (1 μg/ml), streptomycin (100 μg/ml) and penicillin (100 U/ml) .
Naive CD4+ T cells (CD4 + CD62L + CD44low) and naive CD8+ T cells (CD8 + CD62L + CD44low) were prepared from cell suspensions isolated from the spleens of 6-8-week-old C57BL/6 mice. The cells were isolated using a FACSAriaIII sorter. The cells were first sorted for CD4+ T cells, which were then sorted for CD62L + CD44low cells. Similarly, CD8 + CD62L + CD44low cells were sorted from the CD8+ T cells, and naive CD8+ T cells were obtained. The naive T cell (CD4+/CD8+ T) purities exceeded 99% (Additional file 1: Figure S2).
T cell differentiation and polarisation assay
Cell suspensions were prepared from the spleens of the anti-CD40 model of Rag1−/− or C57BL/6 mice. Briefly, 3.2 × 104 E-cadherin + or E-cadherin- CD11chighCD4-CD103-7-AAD-cells (E-cadherin + or E-cadherin- DCs, respectively) were cultured with or without 5 μg/ml CEA421-435 peptide (Invitrogen) for 24 h; 3.5 × 105 sorted naive CD4+ T cells were then added to the E-cadherin + or E-cadherin- DCs in complete RPMI 1640 medium supplemented with 10% FBS, IL-2 (100 IU/ml, R&D Systems), plate-bound anti-CD3 and soluble anti-CD28 (5 μg/ml each, eBioscience) , either under Th1, Th2 and Th17 conditions for 96 h or Treg conditions for 5 days. Then, the Th1, Th2 and Th17 cells and the relevant cytokines were analysed at 72 h and 96 h. Treg cells and the relevant cytokines were analysed at 72 h and on day 5. Cell supernatants were collected and stored at −80°C for subsequent analysis of CD4+ T cell differentiation using the mouse Th1/Th2/Th17/Th22 13-plex kit (eBioscience) and the mouse TGF-β1 simplex kit (eBioscience). For Th1, Th2 and Th17 effector T cell polarisation analysis, multiplying system cells were collected on day 3 and analysed with the mouse Th1/Th2/Th17 phenotyping kit (BD Biosciences); for the Treg cell analysis, the mouse Th17/Treg phenotyping kit (BD Biosciences) was used after multiplying system cells collected at day 5.
For CD8+ T cell differentiation analysis, 2.3 × 104 E-cadherin + or E-cadherin- DCs that were cultured with 5 μg/ml CEA526-533 peptide (Invitrogen) for 24 h were cultured with 3 × 105 sorted naive CD8+ T cells, anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml). The naive CD8 + Tcells cultured with anti-CD3 (1 μg/ml) and anti-CD28 (5 μg/ml) (no DCs) were considered as a control. At 48 h, the supernatant was collected to detect IFN-γ secretion using the mouse IFN-γ ELISA Kit (R&D systems).
In vivo experiments
For the in vivo experiments, 5 × 105 E-cadherin + CD11chighCD103−CD4-7AAD- DCs from the spleens of the Rag1−/− anti-CD40 mouse model were transferred into the orthotopic or subcutaneous lung tumour model by tail vein injection at day 7, day 14 or day 21 after the tumour model was established (the day of tumour cell injection was day 0). The same number of E-cadherin-CD11chighCD103-CD4-7AAD- DCs and 200 μl of PBS were injected as controls. The E-cadherin + and E-cadherin- CD11chigh cell fractions were cultured with 5 μg/ml CEA526-533 peptide (Invitrogen) for 24 h prior to i.v. injection into the LLC tumour-bearing mice. At day 28, the orthotopic lung tumour-bearing mice were sacrificed, and the tumour tissues were harvested and prepared to investigate the effects of the E-cadherin + DCs on the helper T cell response using PCR and western blotting analyses. Lung tissue from orthotopic tumour-bearing mice was also prepared as a cell suspension to assay CD103 expression in CD8+ T cells. Spleen lymphocytes of orthotopic tumour-bearing mice were stained for tetramer staining.
All data are expressed as the mean ± SD. Significant results were assessed using analysis of variance. Statistical significance between alues was determined using Student’s t test, and a statistically significant difference between two test groups was defined as P < 0.05.
The phenotype of inflammatory E-cadherin + DCs in the lung
The effects of E-cadherin + DCs on effector T cells (Th1 and Th2)
E-cadherin + DCs promote the Th17 response and decrease the percentage of Treg cells
E-cadherin + DCs enhance the T cell response in orthotopic Lewis lung cancer model
To investigate the effect of CD40-mediated E-cadherin + DCs on CD4+ T lymphocytes of lung tumours, tumour tissue was harvested for qRT-PCR and western blotting. In this tumour immunity microenvironment model, with E-cadherin + DC treatment carrying the CEA526-533 antigen peptide, the transcription of T-bet and RORγt was enhanced compared with mice that received E-cadherin- DCs and the PBS control mice, whereas FOXP3 transcription was reduced (Figure 4D). The protein levels of IFN-γ and IL-17 were increased in the E-cadherin + DC group, whereas TGF-β was decreased. The transcription of GATA3 and the protein level of IL-4 were reduced in the tumour tissue exposed to E-cadherin + DCs relative to that exposed to E-cadherin- DCs (Figure 4D-E).
The effects of E-cadherin + DCs on antitumour activity in Lewis lung cancer model
A subset of DCs not found in the steady state occurs as a consequence of inflammation or antibody-mediated sterile inflammation. These DCs have been termed inflammatory DCs. Inflammatory monocytes are the main precursors of inflammatory DCs . These precursors, such as Gr1 + CD115+ inflammatory monocytes, can differentiate into E-cadherin + DCs through GM-CSF mediation . Although a few DCs express E-cadherin in normal mice, the inflammatory E-cadherin + DCs detected in the lungs of the lung tumour model are not detected. Here, we demonstrated that E-cadherin + DCs accumulated in the lung and spleen during CD40 mAb-mediated innate immunity, indicating that the provisions of the CD40 signalling pathway are sufficient to drive the accumulation of inflammatory DCs expressing E-cadherin.
Monocyte-derived inflammatory DCs play an early role in adaptive immunity . However, these cells have not been shown to prime naive T cells in vivo, and their functions in tumour immunity in vitro and in vivo are unknown. The differences in the activities of DC subsets on naive T cells depend on the phenotype of the DCs. CD8α + DCs could play a role in tolerance induction and restrict the immune response, whereas CD8α- DCs could be stimulatory to the T cell response . CD40-mediated inflammatory E-cadherin + DCs do not express CD8α and CD4. Surprisingly, Pulendran et al. and Maldonado-Lopez et al. demonstrated that CD8α + DCs lead to Th1 differentiation and that CD8α- DCs induce a Th2-type response [17,18]. Although E-cadherin + and E-cadherin- BM-DCs failed to promote IFN-γCD4+ T cell generation, due to increased overall cell numbers, E-cadherin+, but not E-cadherin-, BM-DCs enhanced the Th1 and Th17 responses. E-cadherin + BM-DCs derive from inflammatory Gr1+ monocytes and express Gr1 . Blood-derived Gr1+ inflammatory DCs can induce a Th1 response to infection in vivo . Here, CD40-mediated inflammatory E-cadherin + DCs promoted the Th1 response, whereas Th2 responses were decreased when these DCs were carrying the CEA antigen peptide. As IFNγ and IL-12 dominate, the enhanced Th1 response can inhibit Th2 cell development through a reduction in IL-4 expression . The Th1 response promotes host responses to tumours because IFN-γ and IL-2 can prime the CD8+ T cell response, thereby protecting the host by monitoring against tumour development [21-23]. Nevertheless, the effect of the Th2 response on the tumour is controversial. In the 1990s, IL-4 was identified as a potent anti-tumour factor . Recently, the Th2 response has been considered a factor promoting tumour growth that affects CD8+ T cells. IL-4 was found to stimulate CD8+ T cells to differentiate into non-lytic CD4−CD8− T cells and to reduce the susceptibility of human CD8+ T cells to activate induced cell death .
The effect of inflammatory E-cadherin + DCs to the blance of Th17/Treg is unknown in tumour immune microenvironment. As is well-known that Treg cells contribute to the progression of cancer by suppressing antitumor immunity [26,27]. Even though it is protective factors in colitis, with suppressive functions through IL-10 and FOXP3 . Inflammatory E-cadherin + DCs from the CD4+ CD45RBhigh T cell model demonstrate a potent ability to induce T cell-mediated colitis with Th17 responses; the effect on Treg cells was not significant with E-cadherin- DCs . However, we found that CD40-mediated E-cadherin + DCs enriched the Th17 response and significantly inhibited the Treg response when the DCs were carrying the CEA peptide. Treg-mediated suppression is a crucial component of CD8+ T cell repopulation . Th17 cells are related to IL-17 and mediated by Th17-stimulated CD8+ T cells in the induction of preventive and therapeutic antitumour immunity . However, IL-17 and IL-23 have been found to drive tumour growth in colorectal cancer with Th17-mediated intestinal inflammation . With Treg cell development impaired by E-cadherin+ DCs and the increased number of Th17 cells, CEA-specific CD8+ T cell responses are enhanced in the in vivo lung tumour model. Tumour volume decreased, and the survival time of tumour-bearing mice was prolonged significantly following the injection of CD40-mediated inflammatory E-cadherin+ DCs compared with injection of E-cadherin− DCs. These results may be relevant to the comprehensive effects of Th1 and Th17 on the proliferation of tumour antigen-specific CD8+ T cell responses. The promotion of tumour growth by CD4+ helper T cells and CD8+ T cells is subdued with the intervention of CD40-mediated inflammatory E-cadherin + DCs. In particular, as the CD40 signalling pathway is activated, the expression of CD40 in the E-cadherin+ DCs is upregulated, and naive CD4+ and CD8+ T cells are primed because helper T cells and the generation of CTLs by cross-priming are mediated by signalling through CD40 on the antigen-presenting cell .
Interestingly, the results of transferring CD40-mediated E-cadherin + DCs into the Lewis lung tumour model suggest that the development of helper T cells in vivo is similar to that in the in vitro experiments. Furthermore, the tumour-specific CD8+ T cell responses were significantly enhanced following the addition of E-cadherin + DCs carrying CEA in vivo. Additionally, when E-cadherin + DCs were cultured with naïve CD8+ T cells, the production of IFN-γ was greater than when E -cadherin− DCs were used (Additional file 1: Figure S3). In the Th1 response, CD8 cytotoxic-T cell responses increased, Treg responses decreased, and the tumour volume was decreased. CD40-mediated inflammatory E-cadherin + DCs therefore possess an excellent antitumour ability via enhancement of the anti-tumour T cell response and suppression of Treg cell development. Recently, CD103 + CD8+ T cells have been shown to be effective in inhibiting breast cancer and glioma progression. These CD103+CD8+ T cells can also upregulate CD8+ T cell cytotoxic mediators when in contact with their specific antigen. CD103+CD8+ T cells also contribute to protecting the human lung against viral infection by producing IFN-γ and other Th1 cytokines, such as IL-2. In addition, these cells have an effector or memory phenotype . In our vivo experiments, we demonstrated that E-cadherin + DCs are able to enhance the development and accumulation of CD103+CD8+ T cells. The ligand for CD103 is E-cadherin; thus, E-cadherin-expressing DCs are more likely to be recognised and combined by CD103+CD8+ T cells during antigen peptide capture, thereby activating these CD8+ T cells.
Taken together, these results strongly suggest that Anti-CD40-induced inflammatory E-cadherin + DCs promote T cell responses and antitumour activity in murine Lewis lung carcinoma. It is unknown why inflammatory E-cadherin+ DCs accumulate in the presence of CD40 signalling pathway activation only in innate immunity. A further understanding of the mechanisms of CD40 signalling pathway-mediated inflammatory E-cadherin + DC differentiation in innate immunity and the tumour microenvironment may provide novel therapeutic strategies and insight into the pathogenesis of NSCLC.
We thank Pr. Antonius Rolink (Basel Institute for Immunology, Basel, Switzerland) for providing the FGK45 cells and the mouse anti-mouse CD40 antibody. This work was supported by 2 grants from the National Natural Science Foundations of China (81171979, 81372260) and 1 grant from Doctoral fund (20110142110005).
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