Anti tcrγδ

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by standard Ficoll-Hypaque (GE Healthcare) density gradient centrifugation. Dead cells were excluded using fixable viability dye Fixable Near-IR Dead Cell Stain Kit (Invitrogen; dilution 1:10). For surface marker staining, cells were incubated with antibodies at 4°C for 30 min and then washed and fixed. The following fluorescence antibodies were applied: anti-TCR Vγ9 (BioLegend; clone B3), anti-CR Vδ2 (BioLegend; clone B6), anti-TCRγδ (BD; clone B1), anti-TCR Vδ1, human (Miltenyi; clone REA 173), and anti-CD3 (Biolegend, clone UCHT1). All samples were acquired with a BD FACS CANTO A (BD Biosciences) and analyzed with FlowJo software.

For intracellular staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA; final concentration 25 ng/mL; Merck, Darmstadt, Germany) and ionomycin (final concentration 1 µg/mL; Merck) for 18 hours. After 2 hours, Brefeldin A (5µg/mL; Merck) and Monensin (1µg/mL; Merck) were added. First, surface antigens were stained as described above. Cells were then permeabilized with fixation/permeabilization solution (Cytofix/Cytoperm; BD Biosciences) and stained with fluorochrome-conjugated antibodies for 30 minutes at 4°C. The following anti-human monoclonal antibodies were used: anti-IL-2, anti-CD4, anti-IL-10, anti-TCR-γ/δ (BD Biosciences), anti-TCR-Vδ2 (Beckman Coulter Life Sciences), anti–IFN-γ, anti-TNF-α, anti-CD8, anti-TGF-β, anti-CD39, anti-Granzyme-B, anti-CD19, anti-CD3, anti-CD73, and anti-CD14 (all BioLegend), see also Supplementary Table 3.
For kinetic studies of CD39 surface expression, cells were stimulated as previously described with small adaptations (109 (link)). Briefly, cryopreserved PBMC were plated into 48-well plates and stimulated with rhIL-2 (20 U/mL; Miltenyi Biotec, Bergisch Gladbach, Germany), PMA (5 ng/mL), ionomycin (0,5 µg/mL), anti-CD3/CD28-Dynabeads (ratio 1:1; ThermoFisher Scientific, Waltham, USA) or combinations thereof. Cells were cultured for up to 6 days before FACS analysis.

Cells were washed with PBS containing 0.5% BSA and incubated for 30 minutes on ice with the following antibodies: anti-CD4 (RM-4.5; eBioscience), anti-CD8 (53.6.7; BD), anti-CD44 (IM.7; BD), anti-CD25 (PC61; BD), anti-CD62L (MEL14; Biolegend), anti-TCRβ (H57-597; eBioscience), anti-TCRγδ (BD), anti-CD69 (H1.2F3; eBioscience), anti-CD11b (M1/70; BD), anti-Gr1 (RB6-8C5; eBioscience) and anti-HSA (M1/69, eBioscience). Cells were then washed in PBS with 0.5% BSA and data was collected using a Fortessa cytometer (BD Bioscience) and analyzed using FlowJo software (Treestar, Ashland, OR).
In the case of intracellular staining, cells were fixed and permeabilized using the Foxp3 buffer staining kit (eBioscience) according to the manufacturer’s instructions prior to staining for intracellular Foxp3 expression using an anti-Foxp3 antibody (FJK-16s, BD), for 30 minutes.
For Annexin V staining, cells were washed with PBS and stained with BD Pharmingen Annexin V Apoptosis Detection Kit I according to the manufacturer’s instructions.
The detection of BrdU was performed using BD Pharmingen BrdU Flow Kit according to the manufacturer’s instructions.

Bone marrow (BM) was collected from WT or Brwd1^-/- mice, and cells were resuspended in the staining buffer (3% (vol/vol) FBS in PBS). Erythrocytes were lysed, and cells were stained with anti-CD11c (HL3), anti-NK1.1 (PK136), anti-TCRβ (H57-597), anti-CD71 (C2), anti-Ter119 (TER-119), anti-Mac-1 (M1/70), anti-Gr-1 (RB6-8C5), anti-CD34 (RAM34) (1:200), anti-Sca1 (Ly-6A/E, D7) (1:200), anti-cKit (CD117, 2B8) (1:200), anti-Flt3 (CD135, A2F10.1) (1:200), anti-IL7Rα (CD127, SB/199) (1:100), anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-CD25 (IL2Rα, 7D4), anti-CD44 (IM7), anti-TCRγδ (GL3), anti-CCR9 (CW1.2), anti-CXCR4 (2B11) (1:100), anti-CD43 (S7), IgM (R6-60.2), IgD (11-36), anti-CD19 (1D3), anti-B220 (RA3-6B2l), and anti-CD93 (AA4.1), CD21 (7G6), CD23 (B3B4) (all from BD Biosciences or Biolegend). Antibodies were directly coupled to fluorescein isothiocyanate, phycoerythrin, phycoerythrin–indotricarbocyamine, allophycocyanin, eFluor 450, or biotin and were used at 1:400 dilution, except otherwise mentioned. Pre-pro B cells (Lin^negCD19^negB220⁺IgM^neg), pro-B cells (Lin^negCD19⁺B220⁺CD43⁺IgM^neg), large pre-B cells ((Lin^negB220⁺CD43^negIgM^negFSC^hi), small pre-B cells (Lin^negB220⁺CD43^negIgM^negFSC^low), and immature B cells (Lin^negB220⁺CD43^negIgM⁺) were isolated by cell sorting with a FACSAriaII (BD).

For flow cytometric analysis, splenocytes (2 × 10⁵) were labelled with mouse anti-rat monoclonal antibodies (mAb) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophylla protein (PercP), allophycocyanin (APC) or APC-Cyanine (Cy)7, as in previous studies^{60 (link)}. In this case, the antibodies used were anti-CD4, anti-CD8α, anti-CD8β, anti-TCRαβ, anti-TCRγδ, anti-NKR-P1A, anti-CD25, anti-CD45RA (BD Biosciences, San Diego, USA), anti-CD62L, anti-CD103 (Biolegend, San Diego, CA, USA), anti-TLR-4 (Novus Biologicals, Littlon, CO, USA) and anti-Foxp3 (eBioscience). After staining with standard procedures^{20 (link)}, analyses were performed using a Gallios^TM Cytometer (Beckman Coulter Inc., Miami, FL, USA) at the CCiT-UB. All results were assessed by the Flowjo v.10 software (TreeStar, Inc., Ashland, OR, USA).