In contrast, when injected with XCL1-OT-I plus poly(I:C), tumour growth was significantly inhibited at day 18 (Fig.?5b). fusion protein plus poly(I:C) showed suppressive effects on tumour growth in synergy with anti-PD-1 Ab. Conclusions Cancer Ag targeting to XCR1+ DCs should be a promising procedure as a combination anticancer?therapy with immune checkpoint blockade. mice were generated by knocking in the cDNA for a fluorescence protein, Venus, to the locus, and backcrossed with C57BL/6J mice more than 10 times.26,27 mice were generated by crossing the mice, and used as XCR1-deficient mice. C57BL/6J mice and 2-microgloblin (2m)-deficient mice were purchased from CLEA Japan and Jackson Laboratory, respectively. All mice used were healthy and 7C12-week old, and their body weight was 25??15?g. Under specific pathogen-free conditions, they were housed in plastic cages, with wood chips, which were changed every week, and each cage was kept to five or less heads without mixing gender. The dark/light cycle is 12/12?h, and room temperature is kept at 22??2?C. All mice were allowed free access to water and sterilised normal chow. The experimental protocols were made in accordance with The Regulations for Animal Experiments in Wakayama Medical University, which states replacement, refinement or reduction (the 3Rs), and approved by Wakayama Medical University Animal Care and Use Committee. Reagents Ovalbumin (OVA) and OVA-derived MHC class I-restricted 2-Keto Crizotinib peptide OVA253C264 peptide (SIINFEKL, OT-I peptide) were purchased from Worthington Biochemical and MBL, respectively. PolyinosinicCpolycytidylic acid (poly(I:C)) was purchased from InvivoGen. Anti-PD-1 (clone: G4) Ab was previously reported.28 Cell line and cell culture An OVA-expressing murine B16 melanoma cell line, B16-OVA (clone MO4), was kindly provided by Dr. Senju, Kumamoto University, Kumamoto, Japan.29 B16-OVA and human embryonic kidney (HEK) cell line, 293T, were maintained in Dulbeccos modified Eagle medium (DMEM) supplemented with 10% FBS. Generation of a fusion protein, XCL1-OT-I A fusion protein, XCL1-OT-I, was designed as follows (Fig.?1a). First, the cDNA for XCL1-OT-I was generated by ligating murine XCL1 cDNA with cDNA coding the OT-I Ag peptide (SIINFEKL, corresponding to 257th to 264th amino acids of OVA), which was flanked by 2-Keto Crizotinib a glycine-rich linker (GGGGS), and FLAG tag (DYKDDDDK). Then the XCL1-OT-I cDNA was cloned into the pHEK293 Ultra Expression Vector II (TaKaRa), and the expression plasmid for XCL1-OT-I was transfected into HEK293T cells together with the pHEK293 Enhancer Vector (TakaRa) using linear polyethyleneimine (PEI, Sigma-Aldrich). After 16C18?h, the culture medium was changed to serum-free DMEM medium supplemented with 1% Nutridoma (Roche) and 1% sodium pyruvate. XCL1-OT-I protein was purified with anti-FLAG Agarose Affinity Gel (Sigma-Aldrich) from culture supernatants. Purified XCL1-OT-I was subjected to SDS-PAGE electrophoresis with Coomassie Brilliant Blue staining and western blotting with anti-FLAG Ab (Sigma-Aldrich). The molecular weight of XCL1-OT-I protein was estimated as 12.7?kDa. Open in a separate window Fig. 1 Generation of a fusion protein, XCR1-OT-I.a Schematic representation of an amino acid structure of XCL1-OT-I. Murine XCL1 was fused with the OT-I 2-Keto Crizotinib peptide flanked by two glycine-rich linkers, GGGGS. FLAG tag, DYKDDDDK, was attached at the carboxy terminal. b, c Coomassie Brilliant Blue (CBB) staining (b) and western blotting (WB) with anti-FLAG Ab (c) of purified XCL1-OT-I. The band for XCL1-OT-I is indicated by arrows. Molecular weight of XCL1-OT-I is estimated as 12.7?kDa. d Chemotactic activity of XCL1-OT-I. Flt3L-induced BM DCs were added to the upper chambers of a Rabbit Polyclonal to GABRD 24-well Transwell plate, and XCL1 or XCL1-OT-I was added in the lower chambers at the indicated concentrations for 2?h. Cells that migrated into the lower chambers were collected and analysed by a flow cytometer. Numbers indicate percentages of XCR1+CD11c+ and XCR1?CD11c+ cells in migrated cells. Similar data were obtained from two independent experiments. Migration assay of XCR1+ DCs To obtain 2-Keto Crizotinib bone marrow (BM)-derived DCs (BM DCs), BM cells were cultured for 8 days in the presence of 100?ng/ml of recombinant human Flt3 ligand (Flt3L, 2-Keto Crizotinib PeptoTech). BM DCs generated by this culture include plasmacytoid DCs (pDCs) and cDCs.30,31 cDCs include CD11c+CD24highCD11bint and CD11c+CD24intCD11bhigh cells, which correspond.