A transplantable tumor model allowing investigation of NY-BR-1-specific T cell responses in HLA-DRB1*0401 transgenic mice
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NY-BR-1 has been described as a breast cancer associated differentiation antigen with intrinsic immunogenicity giving rise to endogenous T and B cell responses. The current study presents the first murine tumor model allowing functional investigation of NY-BR-1-specific immune responses in vivo.
A NY-BR-1 expressing tumor model was established in DR4tg mice based on heterotopic transplantation of stable transfectant clones derived from the murine H2 compatible breast cancer cell line EO771. Composition and phenotype of tumor infiltrating immune cells were analyzed by qPCR and FACS. MHC I binding affinity of candidate CTL epitopes predicted in silico was determined by FACS using the mutant cell line RMA-S. Frequencies of NY-BR-1 specific CTLs among splenocytes of immunized mice were quantified by FACS with an epitope loaded Db-dextramer. Functional CTL activity was determined by IFNγ catch or IFNγ ELISpot assays and statistical analysis was done applying the Mann Whitney test. Tumor protection experiments were performed by immunization of DR4tg mice with replication deficient recombinant adenovirus followed by s.c. challenge with NY-BR-1 expressing breast cancer cells.
Our results show spontaneous accumulation of CD8+ T cells and F4/80+ myeloid cells preferentially in NY-BR-1 expressing tumors. Upon NY-BR-1-specific immunization experiments combined with in silico prediction and in vitro binding assays, the first NY-BR-1-specific H2-Db-restricted T cell epitope could be identified. Consequently, flow cytometric analysis with fluorochrome conjugated multimers showed enhanced frequencies of CD8+ T cells specific for the newly identified epitope in spleens of immunized mice. Moreover, immunization with Ad.NY-BR-1 resulted in partial protection against outgrowth of NY-BR-1 expressing tumors and promoted intratumoral accumulation of macrophages.
This study introduces the first H2-Db-resctricted CD8+ T cell epitope-specific for the human breast cancer associated tumor antigen NY-BR-1. Our novel, partially humanized tumor model enables investigation of the interplay between HLA-DR4-restricted T cell responses and CTLs within their joint attack of NY-BR-1 expressing tumors.
KeywordsNY-BR-1 Differentiation antigen CTL epitope HLA transgenic mice
Adenoviral vector coding for NY-BR-1
Cluster of differentiation
Cytotoxic T lymphocyte
Murine breast cancer cell line
Clone #9 of NY-BR-1 stably transfected EO771 cells
Breast cancer antigen 1
NY-BR-1 epitope comprising amino acid 1242–1256
Although improved diagnostic tools and advanced therapy approaches have succeeded in reducing the standardized cancer mortality rate of breast cancer over the last decades, this tumor entity still remains the second most frequent cancer type among women, predicted to cause more than 93,000 deaths in the European Union in the year 2020 . New treatment strategies are thus needed in order to further improve the clinical outcome of breast cancer patients, particularly since standard therapy approaches are often associated with severe side effects  or can even induce therapeutic resistance . At this point, immunological treatment strategies exploiting the activity of autologous tumor-reactive T cells might offer an attractive complement or alternative to classical treatment regimens. In fact, adoptive T cell therapy, particularly in combination with checkpoint inhibitors, has yielded promising results in various clinical settings [4, 5]. The concept of this therapeutic approach relies on the notion that activated tumor antigen-specific T cells selectively attack tumor cells leaving healthy tissue unaffected. However, to fulfill this aim, target antigens need to be identified that are selectively expressed by the tumor, but absent or underrepresented in normal tissue. The breast cancer associated differentiation antigen NY-BR-1 meets this criterion as it was found to be strongly overexpressed in breast tumors originating from ductal epithelial cells .
Interestingly, NY-BR-1 is characterized by marked intrinsic immunogenicity as both antibody  and cytotoxic T cell (CTL) responses [8, 9] have been detected in breast cancer patients. CTLs recognize short peptides that are presented by MHC I molecules on the surface of the target cell, resulting in apoptosis induction within the recognized cell [10, 11]. Thus, most T cell based immunotherapy approaches have aimed at the induction of tumor-reactive CD8+ T cells, considered as ultimate effector cells due to their capacity of direct tumor cell killing. On the other hand, tumor antigen-specific CD4+ T cells have been demonstrated to represent essential contributors of CTL-mediated tumor attack , implying that epitopes presented by MHC II molecules to CD4+ T cells are of central importance in tumor immune surveillance . In a previous study we thus used HLA-DR4 transgenic (HLA-DR4tg) mice to identify the first NY-BR-1-specific CD4+ T cell epitopes that might help to induce and monitor tumor antigen-specific T cell responses in breast cancer patients . However, a NY-BR-1 expressing tumor model allowing investigation of NY-BR-1-specific T cell responses in vivo has been lacking so far. We thus set out to establish such a tumor model based on ectopic transplantation of a NY-BR-1 expressing mouse mammary carcinoma cell line onto HLA-DR4tg mice. Moreover, we introduce the first NY-BR-1-specific H2b-restricted CTL epitope and describe the composition of tumor infiltrating immune cell populations with particular focus on NY-BR-1-specific CD4+ T cells and HLA-DR4 positive tumor-associated macrophages. The NY-BR-1 expressing tumor model presented here, offers a valuable tool to investigate immune responses against NY-BR-1 expressing tumor cells in vivo.
Both murine tumor cell lines used were of C57BL/6 origin (H2b). The mammary adenocarcinoma cell line EO771 was purchased from TEBU-Bio (Offenbach, Germany) and RMA-S cells (a Rauscher virus induced T lymphoma) were kindly provided by Günter Hämmerling, DKFZ Heidelberg, Germany. RMA-S cells were propagated in complete RPMI medium containing RPMI 1640 supplemented with Glutamax (Life technologies / Thermo Fisher), 10% FCS (Biochrom, Berlin, Germany), and 1% penicillin-streptomycin (Life technologies / Thermo Fisher). EO771 cells were cultured in complete RPMI medium containing 1 mM HEPES buffer (Sigma-Aldrich, Saint Louis, MO).
B6.129S2-H2-Ab1tm1Gru Tg (HLA-DRA/H2-Ea,HLA-DRB1*0401/H2-Eb)1Kito mice expressing a chimeric HLA-DRA-IEdα/HLA-DRB1*0401-IEdβ molecule on a H2-IA0/0 background  (designated as HLA-DR4tg mice throughout this paper) were obtained from Taconic (Cologne, Germany) and further bred in the Centralized Laboratory Animal Facilities of the German Cancer Research Center Heidelberg. Animals were group housed in standard individually ventilated cages with wood chip embedding (LTE E-001, ABEDD, Vienna, Austria), nesting material, ad libitum diet (autoclaved mouse/rat housing diet 3437, PROVIMI KLIBA AG, Kaiseraugst, Switzerland) and autoclaved tap water.
In accordance with the Appendix A of des European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes from 18th March 1986 room temperature and relative humidity were adjusted to 22.0 ± 2.0 °C and 55.0 ± 10.0%, respectively. All animals were housed under strict specified pathogen-free (SPF) conditions according to the recommendations of the FELASA. The light/dark (L/D) cycle was adjusted to 14 h lights on and 10 h lights off with the beginning of the light and dark period set at 6.00 am and 8.00 pm, respectively.
All animal experimentation performed in this study was conducted according to the national guidelines and was reviewed and confirmed by the institutional review board/ethics committee of the German Cancer Research Center, Heidelberg). The animal experiments were finally approved by the responsible national authority, which is the Regional Authority of Karlsruhe (Germany; official approval ID 35–9158.81/G172–12).
Sample size calculation was performed by the Biostatistics Department of the DKFZ following standard procedures. Mice were randomized to the different treatment groups. Treatment was performed in random order. Health status of mice has regularly been tested by the Animal Core Facility. Only animals with approved health status were included in the experiments.
Generation of stable NY-BR-1 expressing transfectant clones
EO771 cells were transfected with 1.2 μg linearized pcDNA3.1(−)zeo-NY-BR-1 expression vector generated upon cloning of the NY-BR-1 encoding cDNA fragment from pcDNA3.1-NY-BR-1 (kindly provided by I. Zörnig) into pcDNA3.1(−)zeo (Invitrogen / ThermoFisher, Waltham, MA) via Kpn1/Not1 digestion. After selection with Zeocin (400 μg/mL), individual clones were raised by limiting dilution.
Western blot analysis
Cellular proteins (15–50 μg) of heat denatured cell lysates were separated by SDS PAGE using a 10% polyacrylamide gel, followed by electro-transfer onto nitrocellulose membranes. Membranes were incubated overnight at 4 °C with a murine monoclonal antibody (clone#2, diluted 1:1000) specific for NY-BR-1 in 0.5% non-fat milk in Tris buffered saline containing 0.1% Tween 20 (TBS-T buffer) on a shaking platform. Beta actin was detected using a monoclonal antibody (MP Biomedical, Solon, OH) diluted 1:10,000 in 0.5% non-fat milk in TBS-T buffer. Next, membranes were washed and incubated with horseradish peroxidase- conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, TX) diluted 1:10,000 in 0.5% non-fat milk in TBS-T buffer for 1 h at room temperature. Protein signals were detected using the enhanced chemiluminescence system (GE Healthcare, Munich, Germany) either by exposing blots to an X-ray film or by a CCD camera.
Peptide binding assay
Peptide binding assays were performed as described . Briefly, 2 × 105 RMA-S cells were incubated overnight with graded peptide concentrations in round bottom microtiter plates followed by indirect immunoflourescence staining using supernatants of hybridoma E3–25 or B22.249 specific for H2-Kb or H2-Db molecules, respectively. All hybridomas were kindly provided by G. Hämmerling.
IFNγ ELISPOT assay
IFNγ secretion of splenocytes from immunized mice or of established T cell lines was analyzed by IFNγ ELSIPOT assay as described previously , except that 5 μg/mL anti-mouse IFNγ capture antibody were used for membrane coating. ELISPOT results were analyzed using two ELISPOT reader devices from AID (Strassberg, Germany) or C.T.L. (Cleveland, OH), respectively. Statistical analysis was performed using Mann Whitney test.
IFNγ catch assay
Flow cytometric detection of IFNγ secreting T cell subpopulations was performed using the Mouse IFN-γ Secretion Assay – Detection Kit (Miltenyi Biotec GmbH, Gladbach, Germany) according to the optimized manufacturer’s protocol. Briefly, 2 × 106 - 2.5 × 106 spleen cells were stimulated overnight with 5 μg/mL peptide. Next day, cells were washed twice followed by incubation with IFNγ catch reagent for 2–3 h at 37 °C cells. Cells were then washed and stained with LIVE/DEAD® Fixable Yellow Dead Cell Stain or LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Invitrogen / Thermo Fisher) diluted 1:1000 in PBS for 30 min at 4 °C. Finally, cells were stained with fluorochrome-labelled anti-mouse CD4 antibody, anti-mouse CD8 antibody and anti-IFN-γ PE (Miltenyi Biotec GmbH, Gladbach, Germany). Data was acquired on a FACS Calibur1, FACS Canto II or LSR II and analyzed with FlowJo software. Statistical analysis was done using Mann Whitney test. Finally, cells were stained with fluorochrome-labelled anti-mouse CD4 antibody, anti-mouse CD8 antibody and anti-IFN-γ PE (Miltenyi Biotec GmbH, Gladbach, Germany). Data was acquired on a FACS Calibur1, FACS Canto II or LSR II and analyzed with FlowJo software. Statistical analysis was done using Mann Whitney test.
Tumor growth experiments
Harvested tumor cell lines were washed three times in PBS and adjusted to titers mentioned in the Results part. Tumor cells were suspended in PBS (100 μL) and injected subcutaneously into the right hind flank of 6–10 week-old female HLA-DR4tg mice without anaesthesia. Tumor growth was monitored by caliper measurements twice per week. Mice were killed by CO2 intoxication 30 days after tumor cell injection or when tumors reached a size of 15 mm in diameter, respectively.
Immunization with recombinant adenovirus
Recombinant, replication-deficient adenovirus encoding NY-BR-1 (Ad.NY-BR-1) and the empty control virus (Ad.Control) were purchased from GeneCust (Dudelange, Luxembourg). Mice were injected (i.p.) with 5 × 108 plaque-forming units (pfu) Ad.NY-BR-1 or Ad. Control, respectively, and killed 14 days later by CO2 intoxication for splenectomy, unless otherwise indicated.
Isolation of tumor infiltrating leukocytes
Excised tumors were cut into small pieces followed by digestion with a mixture of Collagenase D (0.5 mg/mL) (Roche Diagnostics, Mannheim, Germany), DNAse I (10 μg/mL) (Sigma-Aldrich), TLCK inhibitor (0.1 μg/mL) (Sigma-Aldrich, Saint Louis, U.S) and HEPES buffer (10 mM) (Sigma-Aldrich) in HBSS (Sigma-Aldrich) for 1 h at 37 °C. The tumor pieces were then passed through a 70 μm cell strainer and the resulting suspension was centrifuged at 1400 rpm for 10 min. Cells were resuspended in 5 mL RPMI medium and separated by density gradient centrifugation (Lympholyte M, Cedarlane Labs, Burlington, Canada). Leukocytes were harvested from the interphase and used for subsequent experiments. The pellet consisting of tumor cells was washed with PBS and used for RNA and protein isolation as required.
Magnetic activated cell sorting (MACS) for positive selection
Cytotoxic T cells and tumor-infiltrating myeloid cells were enriched by magnetic cell sorting using magnetic antibody-coated (CD8a- or CD11b-specific) microbeads and MS or LS columns, respectively, according to the manufacturer’s instructions (Miltenyi Biotec).
Spleen cell suspensions prepared from immunized mice were adjusted to 1 × 106 cells/mL and incubated with LIVE/DEAD® Fixable Yellow Dead Cell Stain (Life Technologies / Thermo Fisher) diluted 1:1000 in PBS for 30 min at 4 °C. After washing with PBS, cells were incubated with APC-labeled H2-Db dextramers (Immudex, Copenhagen, Denmark) loaded with NY-BR-11241-1249 or with Human Papilloma Virus (HPV) 16 E749–57, diluted as indicated in 60 μL FACS buffer for 30 min at room temperature in the dark. Fluorochrome-labeled antibodies against CD3 (clone 17A2), CD8 (clone 53–6.7), CD4 (clone GK1.5) CD14 (clone Sa14–2) (all purchased from Biolegend, San Diego, CA), and the respective isotype controls were diluted 1:50 in 60 μL FACS buffer and added to the cells without washing off the dextramers so that the final dilution of the antibodies was 100-fold. Cells were incubated with the antibody mix at 4 °C for 30 min. The cells were then washed and analyzed by flow cytometry for viable CD3+CD14−CD8+ dextramer+ cells.
Flow cytometry and cell sorting
For flow cytometry, 1 × 106 cells were used per sample unless otherwise indicated. A mix of 0.05 mg/mL purified rat anti-mouse CD16/CD32 (BD Pharmingen), rat serum (GeneTex, Irvine, CA) and hamster serum (Jackson Immunoresearch, West Grove, PA) was used to block Fc receptors on tumor infiltrating leukocytes for 20 min at 4 °C. This step was omitted for tumor cell lines and splenocytes. The cells were then washed twice and 100 μL of LIVE/DEAD® Fixable Yellow Dead Cell Stain or LIVE/DEAD® Fixable Blue Dead Cell Stain Kit, for UV excitation (Life technologies / Thermo Fisher) diluted 1:1000 in PBS was added. After incubation for 30 min at 4 °C, washed cells were stained with antibodies (or isotype controls) listed in Additional file 1: Table S1. After incubation for 1 h at 4 °C, cells were washed and resuspended in 200–400 μL FACS buffer for flow cytometric sorting or analysis, respectively. For intracellular cytokine staining, cells were incubated in 100 μL fixation permeabilization solution (BD Biosciences, San Jose, CA) for 20 min at 4 °C, followed by two washing steps using BD Perm/Wash buffer. Cells were then incubated for 1 h at 4 °C with 100 μL BD Perm/Wash buffer containing antibodies or isotype controls, respectively, diluted 1:100. Finally, cells were resuspended in 200–400 μL FACS buffer for analysis.
Gene expression analysis by standard qRT-PCR using SYBR green was performed for the analysis of the polarization status of tumor-associated macrophages. Primer sequences are listed in Additional file 1: Table S2.
Generation of a NY-BR-1 expressing breast cancer cell line
Thus, s.c. transplantation of 2 × 105 NY-BR-1 expressing EONY#17 cells was sufficient to give rise to subcutaneous tumors containing elevated frequencies of CD8+ T cells and of TAMs.
Identification of a NY-BR-1-specific H2-Db-restricted CD8+ T cell epitope in HLA-DR4tg mice
NY-BR-1-specific H2-Db-restricetd CTL epitopes predicted by the SYFPEITHI database (www.syfpeithi.de)
Prediction score (Db)
Next, we made use of a fluorochrome-conjugated H2-Db dextramer displaying the newly identified CTL epitope enabling us to detect NY-BR-11241-1249-specific CD8+ T cells directly ex vivo. Using this multimer, we found 0.3% ± 0.1% CD8+ dextramer+ spleen cells in HLA-DR4tg mice immunized with Ad.NY-BR1, as compared to 0.036% ± 0.005% in spleens of control mice. In contrast, incubation with a control dextramer displaying an irrelevant epitope did not stain spleen cells at all, thus proving specificity of our dextramer (not shown). When gating on CD3+ CD14− splenocytes, we detected 3.0 to 3.8% CD8+ dextramer+ T cells among spleen cells of mice immunized with Ad.NY-BR-1, whereas among spleen cells from control mice or from immunized mice treated with control dextramer only 0.18 and 0.29%, of the cells appeared double positive, respectively (Fig. 5c). A representative set of dot plots showing NY-BR-1-specific CTLs among spleen cells of a control mouse and an immunized mouse is depicted in Fig. 5b.
Immunization with Ad.NY-BR-1 mediates partial tumor protection in HLA-DR4tg mice and induces accumulation of TAMs with reduced expression of Cxcl10
Thus, immunization with replication-deficient, NY-BR-1-encoding adenovirus had a suppressive, though not fully inhibitory effect on outgrowth of EONY#17-derived tumors and promoted accumulation of TAMs with enhanced HLA-DR4 surface expression, accompanied by decreased expression of the M1-associated marker Cxcl10.
The current paper presents the first NY-BR1 expressing tumor model based on ectopic transplantation of the C57BL/6-derived mammary adenocarcinoma cell line EO771, stably transfected with a NY-BR-1-encoding expression vector, onto H2-compatible HLA-DR4tg mice. As NY-BR-1 represents a xeno-antigen in mice, it was not unexpected that the transplanted NY-BR-1 expressing tumor cells showed immunogenic potential promoting intra-tumoral CD8+ T cell infiltration and delayed tumor outgrowth. In fact, various tumor models based on xeno-antigens have been described. The most common ones use chicken ovalbumin (OVA) as target antigen, for example, in tumor models of melanoma , lymphoma  or breast cancer . In some of these examples, OVA expressing transfectants showed delayed tumor growth compared to the parental tumor cell line in vivo [23, 24, 25] as observed in our NY-BR-1 expressing model, although OVA expressing tumor models without effects on tumor outgrowth have been described as well . In our system, clone EONY#17 showing intermediate NY-BR-1 expression gave rise to tumors in all mice transplanted, providing the basis for the performance of tumor protection experiments. Immunization with recombinant adenovirus induced NY-BR-1-specific CTL as well as CD4+ T cell responses and reduced outgrowth of the NY-BR-1 expressing tumor cell line EONY#17 in DR4tg mice. Optimization of the immunization protocol, for example by inclusion of adjuvants such as CpGs or antibodies against checkpoint inhibitors in combination with adoptive T cell transfer [27, 28, 29], might further improve this protective effect, providing the basis for preclinical immunotherapy studies in this NY-BR-1 expressing tumor model.
In our study we identified the first H2-Db-restricted, NY-BR-1-specifc CTL epitope NY-BR-11241-1249, almost entirely embedded within the HLA-DR4-restricted epitope NY-BR-11242-1256, recently identified by us . Thus, immunization experiments using constructs or synthetic peptides encompassing a minimal stretch of 16 amino acids should enable simultaneous analysis of NY-BR-1-specifc CD8+ CTL and HLA-DR4-restricted CD4+ T cell responses in this mouse strain. Current HLAtg mouse strains are often engineered to lack expression of endogenous MHC molecules to prevent interference between T cell responses that are restricted by the transgenic HLA-molecule and endogenous H2 molecules respectively [30, 31]. In fact, EO-NY-derived β2m knock-out variants have been established  that could be used as parental cell lines to generate stable cell transfectant clones co-expressing NY-BR-1 in combination with the transgenic HLA molecule, resulting in NY-BR-1 expressing breast cancer cell lines that might allow performance of tumor protection and regression experiments in HLA-double transgenic mice co-expressing HLA-DR4 and HLA-A2 .
Breast cancer is known to be highly infiltrated by macrophages generally correlating with poor prognosis [33, 34, 35, 36, 37]. In our model we saw similar results with CD11b+F4/80+ macrophages representing up to 70% of CD45+ immune cells within the tumor. This is reminiscent to TAM infiltration rates reported in the BALB/c-derived 4 T1 breast tumor model where CD11b+ cells constituted up to 86% of tumor infiltrating leukocytes . Performing detailed gene and protein expression analyses we found that the CD11b+F4/80+ immune cell population in NY-BR-1 expressing tumors showed enhanced expression of both, certain M1 markers genes as well as M2-associated genes. In fact, macrophages showing overlapping phenotypes thus representing neither M1 nor M2, have been described in a murine breast cancer model , possibly explaining the heterogenic gene expression pattern observed among TAMs in our tumor model. Alternatively, M1- and M2-like TAMs might be distributed differentially within the tumor tissue, depending on local oxygen supply with M1 accumulating preferentially in normoxic tumor areas, whereas M2-like TAMs could occur mainly at hypoxic sites . We cannot judge which of these explanations might actually apply to our tumor model since our analysis was performed on the entire CD11+F4/80+ myeloid population and not on isolated M1/M2 subpopulations. Tumor antigen-specific immunization with Ad.NYBR1 induced TAMs showing upregulated HLA-DR4 surface expression levels pointing towards a possible M1-like phenotype, likely due to the induction of an IFNγ-producing Th1 response. As immunization with Ad.NY-BR-1 induced NY-BR-1-specific CD4+ T cells in EONY#17 tumor- bearing mice and since tumor antigen-specific CD4+ T cells have been reported to stimulate upregulation of MHC II expression on TAMs in the tumor microenvironment [41, 42], this might explain why the TAMs in the EONY tumors of immunized mice showed preferential expression of M1-associated markers as compared to TAMs from parental tumors.
In summary, we established a NY-BR-1 expressing tumor model in HLA-DR4tg mice and introduce the first NY-BR-1-specific, H2-Db-resctricted CD8+ T cell epitope, thus allowing investigation of NY-BR-1 as a target for therapeutic vaccination approaches against breast cancer in vivo.
We thank Elke Dickes for technical assistance, Dr. Noemi Bender (DKFZ) for helpful advice regarding PEC isolation, Ulrike Träger (DKFZ) for providing antibodies, and Prof. Günter Hämmerling (DKFZ) for RMA-S cells and hybridoma supernatants. We gratefully acknowledge the help of the FACS core facility of the DKFZ in FACS sorting and analysis as well as the support by the Center for Preclinical Research at the DKFZ for animal care taking.
WO and SBE designed the study; KD generated the NY-BR-1 expressing cell line; MV identified the CTL epitope; KD and DE performed the in vivo experiments and analyzed the data; KMD and AH supported the design of transplantation experiments; AKS performed and interpreted the statistical analysis; AH and MF supported multi-color FACS experiments; DJ and IZ provided expertise on protein analysis; WO, KD and SBE wrote the manuscript. All authors read and approved the final manuscript.
DE and KD were supported by a fellowship from the Helmholtz International Graduate School for Cancer Research. The funding source had no role in the study design, data collection or analyses, the decision to publish, or the preparation of this manuscript.
Ethics approval and consent to participate
The physiological interactions between T cells and tumor-associated macrophages can only be investigated within the mammalian animal model, as it provides a complete immune system including the entirety of its complex interactions. Still, performance of the animal experiments followed the 3R principle, in order to minimize the number of animals recruited for the experiments and to reduce their burden.
The mice were housed and treated at the DKFZ animal facility. Animal experiments were approved by the District Government (Regierungspräsidium Karlsruhe) in Karlsruhe, Germany (approval ID 35–9158.81/G172–12).
Consent for publication
The authors declare that they have no competing interests.
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