BMDCs were plated in 96-well plate (5×104 cells/well) for at least 2 h in DC media, then cultured in the presence of TLR agonists at doses indicated for 16 h, after which culture supernatants were collected. Cytokine concentrations in the culture supernatants were determined using mouse IL-12 p70, TNF, IL-6 and IL-10 ELISA kits (eBioscience) and VeriKine Mouse IFN-β ELISA kit (PBL interferon source) according to the manufacturer’s protocol. The OD450/570 was measured using a VERSAmax microplate reader and Softmax Pro software (Molecular Devices). Total RNA prepared by using RNeasy plus mini kit (QIAGEN) was reverse-transcribed with Superscript III Reverse Transcriptase (Invitrogen) using oligo

dT primer according to the manufacturer’s protocol. Quantitative PCR was performed using the Power SYBR Green PCR Master Mix (Applied mTOR inhibitor Biosystems) and 7900HT (Applied Biosystems) according to the manufacturer’s protocol. The sequences of IFN-α4, IFN-β, IL-12 p40 and IRF7 primers were as described previously 23, 47–49. HPRT was used as an internal control (Hprt-F: 5′-TGA AGA GCT ACT GTA ATG ATC AGT CAA C-3′; Hprt-AS: 5′-AGC AAG CTT GCA ACC TTA ACC A-3′). OVA-specific T-cell response induced by BMDCs was determined by CFSE dilution. Briefly, WT and TREM-2-deficient BMDCs were isolated by MACS after 6 days of culture and plated at 1×104 cells per well of a round bottom 96

well plate with OVA323–339 (2 or 0.5 μg/mL) and CpG DNA (100 or 25 nM) in the presence of GM-CSF (10 ng/mL) for 4 h. CD4+ T cells from spleen and

lymph node of OT-II NVP-BEZ235 order transgenic mice were isolated by using Dynal Mouse CD4 Negative Isolation Kit (Invitrogen) Ribose-5-phosphate isomerase and stained with CFSE (final 0.8 μM). After 4 h of DC culture, 1×105 CFSE-labeled CD4+ OT-II T cells were added into each well and incubated for 72 h. After culture, cells were stained with anti-CD4 mAb and we performed flow cytometry to detect CFSE dilution of gated CD4+ OT-II T cells. Data analysis to calculate the percentage of divided and division index was performed by Flowjo software (Treestar). Significant differences of each genotype of DCs in comparison with WT DCs were determined by using Prism 5 software (Graphpad). Specific statistical tests for each figure are indicated in the figure legends. The authors thank Dr. Marco Colonna for providing TREM-2-deficient mice, Dr. Takashi Saito for providing the FcRγ-deficient mice, J. P. Houchins for providing TREM-1-Fc and TREM-2-Fc reagents, Dr. Dan Campbell for providing OVA peptide and Dr. Estelle Bettelli for providing OT-II mice. They also thank Dr. Dan Campbell and members of our laboratory for helpful discussions and review of the manuscript. H. Ito is supported by an Irvington Institute Fellowship Program of the Cancer Research Institute. J. A. Hamerman is supported by a Cancer Research Institute Investigator Award and NIH AI073441 and AI081948.