(B) Light microscopy image of CD4? MutuDC2s. that express the simian virus 40 Large T-oncogene in the DCs. The comparison of these DC lines with the vast variety of DC subsets described has shown that all the MutuDC lines that we have generated so far have phenotypic and functional features of type 1 conventional DCs (cDC1s). Parbendazole With the purpose of deriving DC lines with characteristics of type 2 conventional DCs (cDC2s), we bred a new Batf3?/? Mushi1 murine line in which the development of the cDC1 subset is severely defective. The new MutuDC line that we generated from Batf3?/? Mushi1 mice was phenotypically and functionally characterized in this work. Our results demonstrated that all the tested characteristics of this new cell line, including the expression of subset-determining transcription factors, the profile of cytokine production and the ability to present antigens, are comparable with the features of splenic CD4? cDC2s. Therefore, we concluded that our new cell line, that we named CD4? MutuDC2 line, represents a valuable model for the CD4? cDC2 subset. (100 ng/mL, tlrl-peklps, InvivoGen), ultrapure flagellin from (100 ng/mL, tlrl-pbsfla, InvivoGen), FSL-1 (100 ng/mL, tlrl-fsl, InvivoGen), Gardiquimod? (1 g/mL, tlrl-gdqs, InvivoGen), CpG ODN 1826 (1 M, TriLink BIOTECHNOLOGIES). In all the experiments each condition was plated in technical triplicate. The supernatants were analyzed by ELISA for the presence of IL-6, IL-10, IL-12/IL-23 p40, IL-12p70, and MCP-1(CCL2) using the following kits according to manufacturer’s instructions: Mouse IL-6 ELISA Set (555240, BD Biosciences) or Mouse IL-6 ELISA Ready-SET-Go! (88-7064, eBioscience), Mouse IL-10 ELISA Set (555252, BD Biosciences) or Mouse IL-10 (Interleukin-10) ELISA Ready-SET-Go! (88-7104, eBioscience), Mouse IL-12 (p40) ELISA Set (555165, BD Biosciences), Mouse IL-12 (p70) ELISA Set (555256, BD Biosciences), Parbendazole Mouse CCL2 (MCP-1) ELISA Ready-SET-Go! (88-7391, eBioscience). RNA extraction, cDNA synthesis, and Parbendazole RT-qPCR Total RNA from CD4? MutuDC2s and MutuDC1s was extracted with the RNeasy Plus Mini Kit (74134, QIAGEN) according to manufacturer’s instructions and stored in RNA secure (AM7005, Thermo Fisher SCIENTIFIC). The synthesis of cDNA was carried out using random nonamers and the M-MLV reverse transcriptase kit (M1701, Promega) or the SuperScript? Reverse Transcriptase kit (18064014, Thermo Fisher SCIENTIFIC) according to manufacturer’s instructions, with the addition of RiboLock RNase Inhibitor (EO0381, Thermo Fisher SCIENTIFIC). DNA/RNA hybrids were removed with RNase H (70054Y, Thermo Fisher SCIENTIFIC). cDNAs were purified using the QIAquick PCR Purification Kit (28104, QIAGEN). RNA and cDNA yields were quantified by Nanodrop spectrophotometry (Thermo Fisher SCIENTIFIC). RT-qPCR was carried out using KAPA SYBR? FAST qPCR kit for LightCycler?480 (KK4611, SIGMA-ALDRICH) on a LightCycler?480 (384-well Parbendazole plate, 5 L reaction) from Roche Diagnostics. The following primers were used at the final concentration of 500 nM: TLR3 FW (5-GCGTTGCGAAGTGAAGAA-3), TLR3 REV (5-TCGAGCTGGGTGAGATTT-3), TLR5 FW (5-CCTCATCTCACTGCATACC-3), TLR5 REV (5-TATTACCAACACGGGGCT-3), ACTB FW (5-CTGAACCCTAAGGCCAACCGTG-3), ACTB REV (5-GGCATACAGGGACAGCACAGCC-3). Every sample was analyzed in technical triplicates. T cell activation assays Ovalbumin-specific CD8+ and CD4+ T cells were isolated from spleens and lymph nodes (brachial, inguinal and mesenteric) of OT-I and OT-II mice, respectively, and purified using the following MACS or EasySep? kits: CD4+ T Cell Isolation Kit, mouse (130-104-454, Miltenyi Biotec), CD8a+ T Cell Isolation Kit, mouse (130-104-07, Miltenyi Biotec), EasySep? Mouse CD4+ T Cell Isolation Kit (19852, STEMCELL? TECHNOLOGIES), EasySep? Mouse CD8+ T Cell Isolation Kit (19853, STEMCELL? TECHNOLOGIES). The T cell isolation kits were used following manufacturer’s protocols except for the buffers that were prepared as follows: MACS buffer (0.5% FCS, 2 MCM2 mM EDTA in PBS), EasySep buffer (2% FCS, 1 mM EDTA in PBS). A fraction of the purified T cells was stained with fluorochrome-conjugated monoclonal antibodies specific for TCR chain (clone H57-597, Brilliant Violet 510, BioLegend) and for either CD4 (clone RM4-5, APC, BioLegend or eBioscience) or CD8 (clone 53-6.7, APC, eBioscience) and analyzed by flow cytometry to assess T cell purity. The purified T cells were stained with the cell proliferation dye eFluor? 670 (65-0840, eBioscience) or eFluor? 450 (65-0842, eBioscience). CD4? MutuDC2s and MutuDC1s were plated in 96-well round bottom plates at a Parbendazole density of 104 cells/well and incubated for 6C8 h with the ovalbumin-derived peptides OVA332?339 (Protein and Peptide Chemistry Facility, UNIL) or OVA257?264 (Protein and Peptide Chemistry Facility, UNIL) or with the full-length ovalbumin (vac-pova, InvivoGen) in the presence of CpG ODN 1826 (1 M, TriLink BIOTECHNOLOGIES). At the.