Dendritic Cells in Granulomas Figure 8. Na¨ıve CD4+ OTII T cell activation by CD11c+ cells from acute and chronic granulomas. A, Experimental schema. B, CD11c+ cells from three- and ten-week BCG infected animals were purified and cultured with purified na¨ıve OTII CD4+ T cells at 5:1 ratio of T cell reponders:APCs, respectively. Cells were co-cultured ex vivo for 48 hours, followed by a five hour recall with anti-CD3. Cells were stained for intracellular cytokines IFNc (left FACS plots) and IL2 (right FACS plots). B right graph, IFNc ELISA of supernatant following 48 hours ex vivo co-culture. Cells shown were from CD4+Vb5+ lymphocyte gate. Granuloma cells from 5–8 mice were pooled for each time point. doi:10.1371/journal.pone.0011453.g008 Flow cytometry A total of 106 cells were incubated for 30 min on ice with saturating concentrations of labeled Abs with 40 mg/mL unlabeled 2.4G2 mAb to block binding to Fc receptors and washed 3 times with staining buffer (PBS plus 1% BSA). Fluorochrome-labeled Abs against CD11c (HL3), CD11b (Mac-1), CD4 (RM5-4), LFA-1 (2D7), B220 (RA3-6B2), MHCII I-Ab (AF6-120.1), CD40 (3/23), CD80 (16-10A1), CD86 (GL1), IFNc (XMG1.2), IL-2 (JES6-5H4), and Vb11 (RR3-15) were purchased from BD Biosciences (San Jose, CA). Fluorochrome-labeled Abs against CD8a (53-6.7), PD-L1 (MIH5), and PD-L2 (TY25) were purchased from eBioscience (San Diego, CA). Anti-CD16/CD32 (2.4G2), anti-CD3 (145– 2C11) and anti-GR-1 (RB6) Abs were produced from hybridomas. For intracellular cytokine staining, single-cell suspensions from various tissues were cultured at 37uC in complete RPMI 1640 supplemented with GolgiStop (BD Biosciences) in the presence of anti-CD3 Ab for 5 h. After surface staining with anti-LFA-1 and anti-CD4 Abs, cell suspensions were fixed and permeabilized by Cytofix/ Cytoperm solution (BD Biosciences), followed by staining with anti-IFNc and/or IL-2 Ab. Cell surface staining was acquired on a FACSCalibur or LSRII (BD Biosciences, San Jose, CA) and analyzed with FlowJo (Tree Star) software version 5.4.5. Generation of recombinant PD1-Fc construct PD1-Fc was generated by fusing the extracellular domain of PD1 to a human IgG1 Fc fragment. PD1 was amplified from spleen-derived cDNA using oligonucleotides specific for the signal sequence (EcoR1PD1, atc gaa ttc acc tga gat gtc ttc ttt tgc) and the membrane proximal region of the extracellular region (hingePD1, ggc atg tgt gag ttt tgt cac cag att tgg gct caa ctt tgc ctt gaa acc ggc ctt ctg g). The Fc region of human IgG1 was amplified from the vector pHC-huIgG1 [83]. PCR products of PD1 and of Fc were fused by overlap-extension PCR, where the hinge region served as the overlapping sequence. The fused product was digested by EcoRI and XhoI and ligated into the pHC-huIgG1 plasmid from which IgG1 was excised by EcoRI and XhoI. The fusion protein was purified from supernatant of transfected CHO-K cells using protein G affinity chromatography. PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11453