Lsr11

Flow-cytometric analysis was performed to characterize myeloid dendritic cell (mDC) and monocyte (Mo) frequencies in PBMCs. All antibodies were purchased from BD Biosciences (San Jose, CA). Cells were stained according to BD protocols using the following mouse anti-human antibodies: CD3 (clone SP34-2), CD14 (clone M5E2), CD16 (clone 3G8), CD20 (clone 2H7), CD33 (clone P67.6), HLA-DR (clone G46.6), and CD11c (clone S-HCL-3). MDC frequencies were reported as percentage of mononuclear cells (MNC). Monocytes were further defined by gating as traditional monocytes (CD14⁺⁺CD16⁻), inflammatory monocytes (CD14⁺⁺CD16⁺) and patrolling monocytes (CD14^dim CD16⁺⁺) (see Additional file 1: Figure S1). Samples were acquired on the LSR11 (BD; San Jose, CA) using FACS DIVA software and analysed with FlowJo (TreeStar, Inc., Ashland, OR).

For tetramer staining, cells from secondary lymphoid tissue were pooled and stained for 1 hour at room temperature with 10 nm PE-conjugated 2W1S:I-A^b. For in vivo peptide experiments, 10 μg/mL brefeldin A was added at this stage. All cell surface staining was done at 4°C for 30 min, with the exception of CXCR5 that was stained for 1 hour at room temperature. Enrichment for 2W1S:I-A^b-specific T cells was performed at d2, d3, d4 and d28, as described previously, using anti-PE MicroBeads (Miltenyi Biotech) and MACS enrichment 29 (link). Enriched and run-through fractions were stained with the same cocktail of surface Abs to enable calculation of cell frequencies. Samples were run using an LSR11 (BD Biosciences) and analysed using FlowJo software (Tree Star). For intracellular cytoplasmic staining, cells were fixed and permeabilised with Cytofix/Cytoperm Plus (BD Biosciences) according to manufacturer's instructions. Intracellular cytokines were stained with IL-2 488 and IFN- γ PECy7 (BD Bioscience).