Anti cd19 apc h7

Fresh PBMCs were isolated from whole blood by density-gradient centrifugation using the BD Vacutainer CPT Mononuclear Cell Preparation Tubes with Sodium Heparin (BD Biosciences, Franklin Lakes, NJ, USA). The cells were first stained using the Zombie Yellow™ Fixable Viability Kit (BioLegend, San Diego, CA, USA) and then with combinations of the following monoclonal antibodies against human cell-surface antigens for 30 min on ice: anti-CD11c-Alexa700, anti-HLADR-V500, anti-CD19-APC-H7 (all from BD Biosciences), anti-CD14-ECD, anti-CD56-APC, (both from Beckman Coulter, Brea, CA, USA), anti-CD123-FITC, anti-CD3- PerCPCy5.5, anti-CD56-BV421 (all from BioLegend), and anti-CD19-PE (TONBO Biosciences, San Diego, CA, USA). pDCs were identified as CD3^-CD19^-CD14^-CD56^-HLADR⁺CD11c^-CD123⁺(Additional file 2: Figure S1). Data were acquired on a FACS LSR Fortessa (BD Biosciences) and the percentages of each cell population and mean fluorescence intensity were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA).

Stromal cells were analyzed using anti-CD45-PE-Cy7, anti-CD21/35-APC (BD Biosciences, Heidelberg, Germany), anti-Podoplanin-FITC (gp38; Biozol, Eching, Germany), anti-CD31-APC (Biolegend, San Diego, USA), and anti-Lyve-1 (kindly provided by R. Förster, Institute of Immunology, Hannover Medical School, Germany) detected by a Cy3-coupled goat anti-rabbit antibody (Dianova, Hamburg, Germany). Activation of stromal cells was determined using anti-MHCII-PE, anti-CD54-PE (BD Biosciences), and anti-CD106-PE (Serotec).
Cell suspensions from mLN and the small intestine were prepared as described above. In addition, 1x10⁶ cells from mLN and the small intestine were incubated with anti-CD3-FITC, anti-F4/80-APC (both from Biolegend), anti-CD8-PE-Cy7, anti-CD4-APC, anti-IgA-PE (all from Serotec, Oxford, UK), anti-CD19-APC-H7, anti-LPAM1-PE, anti-CD11c-FITC (all acquired from BD Biosciences), and anti-CCR9-PE (eBiosciences). All FACS analyses were performed on a FACSCanto (BD Biosciences) and analyzed using Diva software (BD Biosciences) or Kaluza software (Beckmann Coulter).

CD8⁺ T cell lines were generated as previously described^{25 (link),58 (link)}. In brief, PBMCs were incubated with 1 μM of individual peptide (NQK-Q8 or NQK-A8) and cultured for 10–14 days in RPMI-1640 supplemented with 2 mM MEM non-essential amino acid solution (Sigma-Aldrich), 100 mM HEPES (Sigma-Aldrich), 2 mM l-glutamine (Sigma-Aldrich), penicillin–streptomycin (Life Technologies), 50 mM 2-ME (Sigma-Aldrich) and 10% heat-inactivated fetal bovine serum (Bovogen). The cultures were supplemented with 10 IU IL-2 2–3 times weekly. CD8⁺ T cell lines were used fresh for subsequent analysis. For the double tetramer staining experiments 0.5 × 10⁶ cells from the CD8⁺ T cell lines were stained with a single PE-conjugated tetramer (HLA-B*15:01-NQK-Q8 or HLA-B*15:01-NQK-A8) or double stained with both tetramers (PE-conjugated NQK-A8 and APC-conjugated NQK-Q8 tetramer) for 1 h at room temperature. Cells were washed and surface-stained with anti-CD3-BV480 (dilution 1:100), anti-CD8-PerCP-Cy5.5 (1:50), anti-CD4-BV650 (1:100), anti-CD14-APCH7 (1:200) and anti-CD19-APCH7 (1:100) antibodies (all BD Biosciences) and Live/Dead Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were single-cell sorted into PCR plates (Eppendorf) using the BD Aria Fusion system.

Expanded A68/NP₁₄₅⁺ CD8⁺ T cells were stimulated with 1 µM peptide (DATYQRTRALVR, DTTYQRTRALVR, DVTYQRTRALVR or pool of DATYQRTRALVR, DTTYQRTRALVR, and DVTYQRTRALVR) and cultured for 5 h in the presence of 10 U/ml rIL2 and Golgi Stop (BD Biosciences). Following activation, cells were surface stained for 30 min with human anti-CD3-BV510 (1:200, Biolegend #317332), anti-CD4-BV650 (1:200, BD Horizon #563875), anti-CD14-APC-H7 (1:100, BD Pharmingen #560180), anti-CD19-APC-H7 (1:100, BD Pharmingen #560177), anti-CD8-PerCPCy5.5 (1:100, BD Pharmingen #565310), Live/Dead near-infrared (1:800, Invitrogen), and PE-streptavidin-conjugated A68/NP₁₄₅ (DATYQRTRALVR) tetramer. Cells were fixed with BD Fix-Perm buffer (BD Biosciences) for 20 min, before intracellular staining for 30 min with human anti-IFN-γ-V450 (1:100, BD Horizon #560371) in perm wash buffer (BD Biosciences). Cells were washed, acquired on the BD Fortessa (BD Biosciences) and analyzed using the Flowjo software (Treestar, OR, USA).

Characterization of immune cell subsets was performed essentially as described previously (20 (link)). Samples were acquired with a LSRII/Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software Vx (Treestar). All antibodies and secondary reagents were titrated to determine optimal concentrations. CompBeads (BD) were used for single-color compensation to create multi-color compensation matrices. For gating, fluorescence minus one controls were used. The instrument calibration was controlled daily using Cytometer Setup and Tracking beads (BD). For characterization of immune cell subsets, the following antibodies were used: anti-CD3-PE-CF594, anti-CD4-BV711, anti-CD11c-AlexaFluor700, anti-CD19-APC-H7, anti-CD326-BV711, anti-Ly-6C-PerCP-Cy5.5, anti-NK1.1-BV510 (all from BD Biosciences), anti-CD8-BV650, anti-CD11b-BV605, anti-F4/80-PE-Cy7, anti-GITR-FITC, anti-Ly-6G-APC-Cy7 (from BioLegend), anti-CD31-PE-Cy7, anti-CD117-APC-eFluor780 (from eBioscience), anti-CD45-VioBlue, and anti-HLA-DR-APC (from Miltenyi).