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cei12005-sup-0001-si.tif810K

Figure S1. Cytotoxic T lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) binds to dendritic cells (DCs) and down-regulates CD86 on both DCs and B cells in the draining lymph node after sensitization with dinitrofluorobenzene (DNFB). Groups of mice were treated with either CTLA-4-Ig or isotype control and sensitized with 0·5% DNFB the following day. Lymph node cells from the draining lymph node were stained with anti-human IgG1 and analysed by flow cytometry at days 3, 4 and 5 after sensitization for detection of binding of CTLA-4-Ig on lymph node cells. (A) %hIgG1+ cells of DCs gated as CD19T cell receptor (TCR)-βmajor histocompatibility complex II (MHC)II+CD11c+ cells 3, 4 and 5 days after sensitization. (B) %CD86+ cells of DCs. (C) Median fluorescence intensity (MFI) of CD86 phycoerythrin (PE) on CD19MHCII+CD11C+ cells. (D) %hIgG1+ cells of B cells gated as CD19+ cells. (E) %CD86+ cells of B cells gated as CD19+ cells. (F) MFI of CD86 PE on CD19+ cells. Data are depicted as mean ± standard error of the mean, *P < 0·05, **P < 0·01 and ***P < 0·001. ‘Grey box' : Isotype control-treated mice (hIgG1) (25 mg/kg); ‘black box' : CTLA-4-Ig-treated mice (25 mg/kg).

cei12005-sup-0002-si.tif541K

Figure S2. Cytotoxic T lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) treatment during challenge phase mediates a reduced release of interleukin (IL)-4 and macrophage inflammatory protein-2 (MIP-2). Donor mice were sensitized to dinitrofluorobenzene (DNFB) in the presence or absence of CTLA-4-Ig. After 5 days, cells from the draining lymph node were transferred to recipient mice which were treated with CTLA-4-Ig 24 h earlier where indicated. Mice were challenged 5 h later with DNFB and ear swelling measured 24 and 48 h later; 48 h after challenge homogenates of inflamed ear tissue were analysed for their content of IL-1β, IL-4, interferon gamma-induced protein (IP)-10 and MIP-2 (a). Ear swelling in the groups is shown in (b) after 24 h (upper) and as area under the curve (lower). +/−: CTLA-4-Ig treatment during sensitization phase alone; −/+: CTLA-4-Ig treatment during challenge phase alone; −/−: no treatment with CTLA-4-Ig. Data are depicted as mean ± standard error of the mean, *P < 0·05, **P < 0·01 and ***P < 0·001.

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