Dendritic Cells in Granulomas Figure 9. Blockade with anti-PD-L1 and anti-PD-L2 mAbs, or PD1-Fc restores 10-week granuloma CD11c+ cells’ ability to restimulate allogenic CD4+ T cells to produce IFNc. DCs (26104) purified from 3- and 10-week granulomas were cocultured ex vivo with purified, primed Ag85B-specifc transgenic CD4+ T cells (16105). Intracellular IFNc production was measured four hours after DC pretreatment with isotype control or anti-PD-L1 and anti-PD-L2 pretreatment (A), and media or PD1-Fc pretreatment (B). FACS lots were derived from initial lymphocyte gate on SSC vs. FSC, followed by gating of CD4+Vb11+ population. Representative FACs plots are shown from groups of 3–7 mice per group. Data are represented as mean +/2 SEM. doi:10.1371/journal.pone.0011453.g009 OTII stimulation and P25 reactivation by DCs from acute and chronic granulomas DCs were isolated from 3- and 10-week granuloma preparations using a CD11c MicroBeads Cell Separation Kit from Miltenyi Biotec (Auburn, CA). Live transgenic OTII CD4+ T cells were sorted using a FACSVantageSE at the University of Wisconsin Comprehensive Cancer Center’s Flow Facility. Pooled lymph node CD4+ Ag85b-specific T cells from 3-week BCG infected were purified using a Dynal Mouse CD4 Negative Isolation Kit following manufactures protocol (Invitrogen, Carlsbad, CA). CD4+ T cells (16105) were co-cultured with sorted CD11c+ cells at a 5:1 ratio of T cells:DCs, respectively. For OTII activation, cells were cultured in a 96 well plate in complete RPMI in the presence of 5 mg/mL OVA for 2 days at 37uC with 5% CO2. Frozen supernatant from 2-day culture was used for IFNc ELISA, samples ran in quadruplicate. Ready-Set-Go! Mouse Interferon gamma ELISA kit was used following manufacture’s protocol (eBioscience, San Diego, CA). For P25 reactivation, sorted DCs were pre-cultured in a 96 well plate (26104) in complete RPMI with either media, both isotype controls (Rat IgG2a k and IgG2b k - eBioscience, San Diego, CA), both anti-PD-L1 (10F.9G2- BioLegend, San Diego, CA) and anti-PD-L2 (TY25- eBioscience, PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11453