Pd l1 clone 29e 2a3

For experiments with AMG 330, cells were incubated at 37 °C (in 5% CO₂ and air) in 96-well round bottom plates (BD Falcon) at 5–10 × 10³ cells/well in culture medium containing various concentrations of the BiTE antibody construct (kindly provided by Amgen, Amgen Research GmbH, Munich, Germany) as well as T-cells at different E/T cell ratios.^{8 (link)} In some experiments, blocking PD-L1 (clone 29E.2A3),^{14 (link)} PD-L2 (clone MIH18)^{15 (link)} or CD86 (clone IT2.2)^{16 (link)} antibodies, or an activating CD28 (clone CD28.2; all from BioLegend, San Diego, CA, USA) antibody were added at 1 μg/ml. After 48 h, cell numbers and drug-induced cytotoxicity, using 4′,6-diamidino-2-phenylindole (DAPI) to detect nonviable cells, were determined using a LSRII cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo (Tree Star, Ashland, OR, USA). Leukemic cells were identified by forward/side-scatter properties and negativity for CellVue Burgundy dye.^{8 (link)} For experiments with other cytotoxic agents, tumor cell lines were incubated in medium containing various concentrations of cytarabine or mitoxantrone (Sigma-Aldrich, St Louis, MO, USA) for 72 h, after which cell numbers and drug-induced cytotoxicity were quantified by flow cytometry.

Ex vivo and cultured PBMC were stained with APC-H7 (Tonbo Biosciences) or Brilliant Violet 405 (Molecular Probes) viability dye in the presence of different panels of monoclonal antibodies against lineage (Lin) (CD3 clone HIT3a, CD19 clone SJ25C1, CD20 clone 2H7, and CD56 clone 5.1H11), CD14 clone M5E2, CD16 clone 3G8, CD40 clone 5C3, CD86 clone IT2.2, ILT4 clone 42D1, HLA-DR clone L243, CD11c clone 3.9, CD1c clone L161, CD141 clone M80, CD64 clone 10.1, and PDL1 clone 29E.2A3 (BioLegend). Samples were analyzed on a Fortessa cytometer (BD Biosciences) at Centro Nacional de Investigaciones Cardiovasculares (CNIC, Madrid, Spain). Analysis of individual and multiparametric flow cytometry data was performed using FlowJo software (Tree Star Inc.).
For the transcriptional studies, viable human Lin^–CD14^–CD11c⁺HLADR⁺CD1c⁺ cDC, CD14^–CD11c⁺HLADR⁺CD141⁺ cDC, and total CD14⁺ Mo were sorted using a FACS Aria II sorter (BD Biosciences) from either PBMC from n = 4 untreated patients with RA and n = 4 HC, or SF from n = 3 individuals with RA and n = 3 suffering mechanic CPPD crystal–associated arthritis.

Cells were blocked with human Fc receptor (Miltenyi Biotech, DE), stained and fixed with 4% paraformaldehyde (PFA) before proceeding for acquisition. For each donor and each condition, three technical replicates were processed. Following antibodies were used: CD3 (clone UCHT1), CD8 (clone HIT8a), CD11b (clone ICRF44), CD33 (clone WM53), CD14 (clone M5E2), CD15 (clone H198), Lineage cocktail (CD3, CD19, CD20 and CD56; clones UCHT1, HIB19, 2H7 and 5.1H11, respectively), Ki-67 (clone Ki-67) and PD-L1 (clone 29E.2A3) from BioLegend, CA, USA; CD4 (clone RPA-T4), HLA-DR (clone G46-6) and AnnexinV from BD Biosciences, NJ, USA; live/dead staining kit from Thermo-Fisher Scientific, MA, USA. In selected studies, MDSCs, MDMs or T cells were labeled with 5 μM CFSE (CellTrace, Invitrogen, CA, USA) following manufacturer's instructions. All samples were acquired in the BSL3 facility on a CytoFLEX flow cytometer (Beckman Coulter, CA, USA).