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k+ DCs grown from C3H/HeN, C3H/HeJ, and CBA/J (Jackson Labs, Bar Harbor, ME) marrow suspensions were pulsed with HEL (1 to 3 mg/ml) with or without LPS (1 to 10 ng/ml, E. coli 0111.84) for 1 to 3 hours, washed, and chased in HEL-and LPS-free media. Similar results were obtained with DCs from each strain. DC cultures contained supernatant from mGM-CSF-expressing J558L cells (from A. Lanzavecchia, Basel Institute, Basel, Switzerland). LPS was removed from HEL (Sigma) with Kuttsuclean adsorbent (Manuha Corporation, Ibaraki, Japan). Maturation stimuli included LPS, CD40L, tumor necrosis factor-α, or replating (22).
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k (10-2.16) and fluorescein isothiocyanate (FITC)-conjugated mAb to CD86 (CL1). Isotype controls were purchased from Pharmingen. Biotinylated mAbs were detected with streptavidin-phycoerythrin. Fluorescence analysis was done with a FACSCalibur and CellQuest software.
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For ICM, DCs were adhered to Alcian blue-coated glass coverslips for 20 min at 37°C in serum-free media. Cell were fixed in 4% (wt/vol) PFA and permeabilized (for intracellular staining) in 0.05% saponin, 10 mM Hepes (pH 7.4), 10 mM glycine (pH 8.0), and 10% goat serum in RPMI 1640. Antibody incubations were done as described (6). Purified C4H3 was used for microscopy. To detect HEL, we used the mouse mAb 1B12, provided by P. Allen (Washington University, St. Louis, MO). Rabbit anti-H-2M (ULM) and rabbit anti-mouse I-A (RIV) have been described elsewhere (6). Hybridomas TIB126 (anti-MHC I), TIB99 (anti-Thy1.2), and TIB93 (anti-I-Ak) were obtained from American Type Culture Collection. Rabbit anti-CD18 was a gift from P. Blier (Boehringer Ingelheim, CT). Several mouse anti-rat mAbs were the gift of J. P. Soullilou (University of Nantes, Nantes, France), including OX-18 (anti-MHC I), antiThy1.1, and OX-6 (anti-MHC II). Rat B7 was detected with a CTLA-4-human IgG fusion protein (a gift of P. Linsley, Bristol Meyers Squibb) in combination with a FITC-conjugated horse anti-human IgG. Mouse B7-2 was detected with CL1. Rat lysosomes were stained with rabbit Igp120 antiserum (24). Irrelevant isotype-matched mAbs, preimmune rabbit serum, or normal human serum were used as negative controls in all experiments. Coverslips were mounted in Mowiol with DABCO (Calbiochem, La Jolla, CA), and fluorescence was analyzed with conventional and confocal (0.5-to 1.0-μm optical sections) Zeiss microscopes.
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We thank members of the Mellman, Steinman, and Inaba laboratories for valuable advice. Supported by grants from NIH (AI-34098 to I.M.), the National Institute of Allergy and Infectious Diseases (AI-13013 and AI-39672 to R.M.S.), and the Ministry of Education, Science and Culture of Japan (grants 08282104, 10153226, and 10044268 to K.I.). I.M. is a member of the Ludwig Institute for Cancer Research.
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