Anti cd3 pb

ErcDCs and macrophages were sorted from ccRCC tissue-cell-suspensions stained with CD45-PeCy7, CD11c-APC, CD3-PB, CD209-PE (all BD Biosciences), CD14-PerCPCy5.5 (eBioscience, San Diego, CA, USA) and LIVE/DEAD^® Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific). Sorting gates were set on CD209⁺CD14⁺ cells (ercDCs) and CD209⁻CD14⁺ cells (macrophages), among pre-gated CD45⁺, live, single CD11c⁺ CD3⁻ cells. CD1c⁺ DC and slanDCs were sorted from B- and NK-depleted PBMCs of healthy donors (HD) using anti-CD11c-PE, anti-CD3-PB (all BD Biosciences), anti-CD56-APC (Beckman Coulter, Brea, CA, USA), anti-CD19-PB (Dako by Agilent, Santa Clara, CA, USA), anti-CD1c-PeCy7 (Biolegend, San Diego, CA, USA), anti-slan-FITC (Miltenyi Biotec) and LIVE/DEAD^® Fixable Near-IR Dead Cell Stain Kit. The gating strategy and instrument parameters are in Supplemental Figure S1. Gates were set very strictly, not covering the whole population, to avoid contamination with other cell populations. Cell population purity varied between 98–100%. Cells were directly sorted into 250 µL of RLT lysis buffer with ß-mercaptoethanol (RNeasy Micro Kit by Qiagen, Venlo, The Netherlands) using FACSAria IIIu (BD Biosciences), then homogenized (QIAshredder by Qiagen) and stored at −80 °C. Details about sorted cell types and numbers of biological replicates are listed in Table S2.

PBMCs from H7N9 patients were stained with A2-M1₅₈ tetramer conjugated to PE (1:200) in FACS buffer (PBS with 1% bovine serum albumin). Cells were stained with antibodies: anti-CD3-PB (BD Cat #558117, UCTH1, 1:200), anti-CD8-FITC (BD Cat #347313, SK1, 1:50), anti-CD27-PE-Cy7 (eBioscience Cat #25-0279-42, O323, 1:25), anti-CD45RA-APC-Cy7 (BD Cat #560674, HI100, 1:50), anti-HLA-DR-ECD (Beckman Coulter Cat#IM3636, Immu-357, 1:25), anti-CD38-PerCPCy5.5 (Biolegend, Cat#303522, HIT2; 1:50), anti-PD-1-APC (Biolegend #329908, EH12.2H7, 1:25), and Live/Dead-aqua 525 (Invitrogen, 1:800). Lymphocytes were washed, acquired on a FACSAria II sorter with FACS Diva software (Becton Dickinson) and analyzed with FlowJo software (Treestar).

Splenic CD4⁺ T cells were enriched from naïve mice using magnetic cell sorting (MACS) following manufacturer’s instructions (Miltenyi Biotec). The purity comprised between 90–95%. CD11b⁺ cells were purified using MACS from spleens of infected and naïve mice previously digested with collagenase D; the purity of the samples was 80–90%. Microtest 96 well plates (Sarstedt) were coated with/without 200μl of PBS containing 1 μg/ml anti-mouse CD3 (eBiosiences) and incubated for 90 min at 37°C. Plates were then washed twice with PBS and equilibrated for 15 min at 37°C with 100μl per well of RPMI-1640 medium (Life technologies), supplemented with 10% fetal bovine serum (FBS), pen/strep and L-glutamine. Th1 polarization was induced as follows: CD4⁺ T cells were seeded at 2 × 10⁵/well in anti-CD3-coated 96-well plates with anti-CD28 (2 μg/ml), rIL-12 (30 ng/ml), and rhIL2 (0.5 μg/ml) (eBiosciences). CD11b⁺ cells enriched as described above were added or not to the culture at a 1:1 ratio. Cells were then incubated at 37°C in a 5% CO₂ incubator and 5 days later stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin in the presence of Brefeldin A (BD Biosciences). Production of IFNγ was analyzed by FACS using anti-CD4-FITC, anti-CD3-PB, and anti-IFN_ϒ-APC (BD-Bioscience). 50,000 events were acquired on a BD LSRFortessa cell analyzer and analyzed using the FlowJo software.

2x10⁶ splenocytes from each sample were plated in a 96-well plate. After centrifugation, cells were resuspended and incubated in culture medium (R10) consisting of RPMI 1640 containing 10% FBS (Mediatech, Herndon, VA), 2mM L-glutamine, 0.01 M HEPES buffer, 100mg/ml gentamicin (Mediatech), and 5×10^-5M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO). To test intracellular cytokine, cells were stimulated with 30 mg/ml PMA and 400 ng/ml ionomycin in the presence of GolgiStop (BD Pharmingen) for 4 hours at 37°C.
After incubation and stimulation, cells were surface-stained with anti-CD3-PB (BD), anti-CD4-PerCP (BD), anti-CD8-PO (Biolegend). Then cells were permeabilized using fixation and permeabilization solution (BD). We used anti-IL-2-FITC (BD), anti-TNF-PE-Cy7 (Biolegend) and anti-IFN-γ-Alexa 700 (BD) for intracellular cytokine staining. Samples were analyzed on an LSRII flow cytometer (BD) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).