Anti cd86 clone it2

CD8 T cells were harvested and incubated with HLA-multimers for 15 min at 4 °C and washed three times with cold PBS/BSA prior to staining with antibodies. Cells were incubated with anti-CD3 (clone SK-7, BD), anti-CD4 (clone SK-3, BD), anti-CD8 (clone SK-1, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. T cell activation was measured by intracellular IFNγ staining (XMF1.2, BioLegend) using an intracellular cytokine staining kit (BioLegend) according to manufactures protocol. moDC’s were stained with anti-CD1a (clone HI149, BD), anti-CD14 (clone M5E2, BD), anti-CD80 (clone L307.4, BD), anti-CD83 (clone HB15e, BD), anti-CD86 (clone IT2.2, BioLegend), and anti-HLA-DR (clone G46-6, BD) antibodies for 30 min at 4 °C and washed three times with cold PBS/BSA. Samples were acquired using a BD LSRFortessa™ flow cytometry machine and analyzed using FlowJo software (Tree Star).

Cells were washed 2 times with cold PBS, scraped and collected by centrifugation and resuspended in FACS buffer (PBS supplemented with 0,5% bovine serum albumin and 5mM of EDTA). Cells were stained with anti-CD11b (clone ICRF44, FITC conjugated; Biolegend) or isotype control (mouse IgG1 FITC conjugated biolegend);, anti-CD14 (clone M5e2, APC conjugated; Biolegend) or isotype control (mouse, IgG2a, κ, APC conjugated, Biolegend), anti-CD206 (clone 15-2, APC/cy7 conjugated; Biolegend) or isotype control (mouse IgG1, APC/cy7 conjugated, Biolegend), anti-CD86 (clone IT2.2, Alexa Fluor 488 conjugated; Biolegend) or isotype control (mouse IgG2b, κ, Alexa Fluor 488 conjugated, Biolegend) and anti-CD163 (clone RM3/1, Alexa Fluor 647 conjugated; Biolegend) or isotype control (mouse IgG1, κ, Alexa Fluor 647 conjugated, Biolegend) Cells were analyzed on a BD LSRFortessa X-20 Cell Analyzer (BD Bioscience) with post-processing in FlowJo software (Tree star Inc). Cell populations were gated on forward and side scatter to select intact single cells. The gating strategy and a representative flow diagram is shown in S2 Fig.