Hla dr bv650

Flow cytometry was performed using fluorescence-conjugates or specific mAb and their controls followed by species-specific conjugate (Supplementary Table 2) using a FACS CantoII flow cytometer (Beckman Coulter) or a LSRFortessa (Becton Dickinson) from the flow cytometer facility Tuebingen. Positive cells in percentage (%) were calculated as follows: Surface expression in percent obtained with the specific antibody—surface expression in percent obtained with isotype control. Platelets were preselected by CD41a⁺ and CD62P⁻ (resting) or CD62P⁺ (activated). Data analysis was performed using FlowJo software (v.10). In order to verify the reproducibility of our flow cytometry system, we performed a Bland–Altman analysis (Supplementary Fig. 8f). For immunophenotyping of PBMC subsets of lung cancer patients and healthy control donors were identified by counterstaining with CD3-PECy5 (BD biosciences, San Diego, CA), CD19-APC/Fire750, CD4-Pacific Blue, CD8a-BV605, CD56-PECy7, CD14-BV785, HLA-DR-BV650 (Biolegend, San Diego, CA) and CD16-FITC (invitrogen). PD-1 and PDL-1 expression as well as activation levels were analyzed using a PD-1-APC or PDL-1-APC and a CD69-PE antibody (BD biosciences), respectively. Isotype controls were obtained from BD biosciences. Dead cells were excluded from analysis with LIVE/DEAD™ Fixable Aqua (Thermo Fisher Scientific, Waltham, MA).

On days 0, 1, and 14 after the first immunization, the cellular composition of the PBMCs was analyzed using flow cytometry. Freshly isolated PBMCs were stained with Live/Dead Fixable Blue Dye (Life Technologies, cat# L-23105, 1:40 dilution) and FcR blocking reagent (Miltenyi Biotec, cat# 130-059-901, 1:20 dilution) followed by a panel of antibodies: CD40 FITC (5C3, Biolegend, cat# 334306, 1:20 dilution), NKG2A PE (Z199, Beckman Coulter, cat# IM3291U, 1:40 dilution), CD80 BV421 (L307.4, BD, cat# 564160, 1:40 dilution), CCR7 PE-Dazzle594 (G043H7, Biolegend, cat# 353236, 1:50 dilution), CD123 Per-CP-Cy5.5 (7G3, BD, cat# 558714, 1:80 dilution), CD3 APC-Cy7 (SP34-2, BD, cat# 557757, 1:80 dilution), CD66 APC (TET2, Miltenyi Biotec, cat# 130-118-539, 1:80 dilution), CD70 BV786 (Ki-24, BD, cat# 565338, 1:80 dilution), HLA-DR BV650 (L243, Biolegend, cat# 307650, 1:80 dilution), CD11c PE-Cy7 (3.9, Biolegend, cat# 301608, 1:160 dilution), CD16 AF700 (38 G, BD, cat# 560713, 1:160 dilution), CD20 BV605 (2H7, Biolegend, cat# 302334, 1:160 dilution) and CD14 BV510 (M5E2, Biolegend, cat# 301842, 1:160 dilution). After washing with PBS, samples were fixed using 1% paraformaldehyde (PFA) and acquired on a BD LSRFortessa cell analyzer. The data were analyzed using FlowJo software v.10.7.1 (FlowJo).

Out of the 104 mBC patients included in the mass cytometry analysis, tumor biopsies were obtained from 63 patients (HR⁺ BC, n = 42; TNBC, n = 21) and were subjected to flow cytometry. Briefly, freshly obtained tumor biopsies were mechanically chopped and then dissociated in DMEM F12 medium containing 10% FBS, collagenase-IV (1 mg/mL), hyaluronidase (1 mg/mL), & DNase-I (50 µg/mL) in a 37 °C water bath for 45 min. Single cell suspensions were then filtered, washed with PBS, and stained with antibodies CD45-A488, CD3-Pacific blue, CD4-PE, CD25-BV605, CD14-PE-Cy7, CD33-BV711, HLA-DR-BV650 (Biolegend), CD127-BV786 (BD Biosciences), and Fixable Viability Dye (eFluor780, ThermoFisher). Samples were acquired using LSR-II/FACSymphony A5 flow cytometer (BD Biosciences), and the data analyzed with FlowJo.

To generate the protein probes, the recombinant WA-1 S-2P and RBD proteins were biotinylated using the EZ-Link Micro Sulfo-NHS-LC Biotinylation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions [20 (link)]. Streptavidin-conjugated fluorophores (SA-PE, SA-APC or SA-BV421) and biotinylated proteins were coupled at a 4:1 molar ratio. The cryopreserved PBMCs were thawed and stained with 100 ng of the fluorescent protein probes for 20 min at 4 °C, followed by staining with 7-aminoactinomycin D (7-AAD, Thermo Fisher, Waltham, MA, USA) and a panel of antibodies, IgM PerCP-Cy5.5 (G20-127, BD, Franklin Lakes, NJ, USA), IgD FITC (polyclonal, Southern Biotech, Birmingham, AL, USA), CD3 BV510 (SP34-2, BD), CD14 BV510 (M5E2, BioLegend, San Diego, CA, USA), CD16 BV510 (3G8, BD), CD20 BV605 (2H7, BioLegend, San Diego, CA, USA), HLA-DR BV650 (L243, BioLegend, San Diego, CA, USA) and IgG BV786 (G18-145, BD, Franklin Lakes, NJ, USA), for another 20 min at 4 °C. After staining, the cells were washed with FACS buffer (PBS supplemented with 2% heat-inactivated fetal calf serum) and fixed with 1% formaldehyde solution. The samples were acquired using a BD LSRFortessa cell analyzer (BD, Franklin Lakes, NJ, USA). The data were analyzed using FlowJo software v.10.7.1 (FlowJo, Ashland, OR, USA).