Anti cd27 bv421

Phenotypic analysis of human B cell subsets was performed with the following antibodies: anti-CD19-PE-Cy7 (Beckman Coulter, Marseille, France), anti-CD27-BV421 (BD Biosciences, Heidelberg, Germany), anti-IgM-PerCP/Cy5.5 (BioLegend, CA, USA), anti-IgM-BV605 (BioLegend), anti-CD38-PE (BD Biosciences), anti-TNFR1(CD120a)-FITC (Miltenyi Biotech), anti-TNFR2(CD120b)-APC (R&D Systems, Inc., Minneapolis, MN, USA), and murine IgG_2A-APC (R&D Systems, Inc.) as isotype control where indicated. Cells were incubated in the dark for 30 min at 4°C in PBS with 0.5% FCS. Samples were acquired using a FACS LSRII SORP (BD Biosciences, Heidelberg, Germany), and cytometry data (LMD files) were analyzed with Kaluza software (Beckman Coulter). The aqua fluorescent reactive dye (LIVE/DEAD Fixable dead Cell stain Kit, Invitrogen, CA, USA) was used for definition of live and dead cells.
For staining of IL-10-producing B cells we used the IL-10 Secretion Assay (Miltenyi Biotech, Bergisch-Gladbach, Germany). B cells were stimulated for 40 h in culture medium with 1 µM CpG ODN 2006. Staining with anti-IL-10 was performed according to the protocol provided by the manufacturer with a prolonged incubation of cells labeled with IL-10 catch reagent (6 h) in presence of 0.25 µM CpG for restimulation. Cells were subsequently stained for expression of other surface markers before measurement on a flow cytometer.

Mitochondrial function was assessed using MitoTracker probes (MitoTracker Green and MitoTracker Deep Red from Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Briefly, 2 × 10⁵ harvested cells were stained with Live/Dead Aqua Dead Cell Stain (Invitrogen Molecular Probes, Eugene, OR, USA) for 20 min at room temperature (RT) followed by anti-CD19-PE-Cy7 and anti-CD27-BV421 (both from BD Biosciences, San Jose, CA, USA) staining for 15 min at RT. Then, cells were washed with PBS and stained with MitoTracker Green and MitoTracker Deep Red for the evaluation of mitochondrial mass and mitochondrial membrane potential, respectively, for 30 min at 37°C.
Reduction of mitochondrial membrane potential is a hallmark of mitochondrial dysfunction. For this reason, viable (Live/Dead⁻) naïve (CD19⁺CD27⁻) and memory (CD19⁺CD27⁺) B cells (Figure 1Ai), containing mitochondria with low membrane potential, identified as MitoTracker Deep Red^low and MitoTracker Green⁺ (Figure 1Aii), were considered B cells with dysfunctional mitochondria (CDM). Fold increase in the percentage of CDM induced by each single stimulus related to the unstimulated sample was expressed as a ratio: (single stimulus % CDM)/(unstimulated % CDM).

PBMC and tumor single cell suspensions were stained with LIVE/DEAD Fixable Aqua (Invitrogen) according to the manufacturer’s instructions and then incubated with Fc Blocking Reagent (Miltenyi Biotec) for 10 min at room temperature followed by staining with anti-CD45 PerCP (BioLegend), anti-CD19 FITC (BD), anti-CD27 BV421 (BD) and anti IgD APC H7 (BD) (BioLegend) for 30 min at 4 °C. The samples were then washed in Phosphate-Buffered Saline (PBS) 2% FBS (FACS Buffer), resuspended in 200 µl of FACS Buffer and analyzed by multicolor flow cytometry using BD FACSCanto II (BD Biosciences). Tumors without detectable B cells (less than 10 per sample) were excluded from analyses. For Ig sequence analysis, CD27+IgD- B cells (CD45 + CD19+) were sorted on 96-well plates at 1, 5, and 10 cells/well directly into 10 µl of lysis buffer (Single cell lysis kit, Ambion-Thermo Fisher Scientific), using BD FACSAria II Cell Sorter (BD Biosciences) with a 70 µm nozzle.