Clone rpa t8

In a subset of patients (n = 63), freshly isolated cells were lysed using the Immunoprep Reagent System (Beckman Coulter) and stained with CD45-FITC/CD56-PE/CD19-ECD/CD3-PC5 and CD45-FITC/CD4-PE/CD8-ECD/CD3-PC5 antibody mixes (all Tetrachrome, Beckman Coulter, Krefeld, Germany), according to manufacturer´s instructions. Flow-cytometry analysis was carried out and analyzed using NAVIOS cytometer and analysis software (Beckman Coulter).
In a small patient subpopulation, from which pretreatment PBMCs were available (n = 6, patient characteristics in Supplementary Table 1), expression of membrane bound LAG3 was quantified in T lymphocytes, key players of antitumoral response. For this instance, priorly isolated and cryopreserved PBMCs were thawed and stained for flow cytometry analysis using fluorescently labeled mAbs directed towards CD3, CD4, CD8 and LAG3, in order to quantify LAG3 positive Helper T lymphocytes (HTLs) (CD3 + CD4 + CD8-LAG3 + ) and cytotoxic T lymphocytes (CTLs) (CD3 + CD4-CD8 + LAG3 + ) (CD3: Clone UCHT1, Fluorophor APC-R700 ; CD4: Clone SK3, Fluorophor BUV737 ; CD8: Clone RPA-T8, Fluorophor BV786; LAG3: Clone T47-530, Fluorophor Alexa 647; all originating from BD Biosciences, San Jose, CA, USA). A LSR Fortessa (BD Biosciences, San Jose, CA, USA) was used for cytometry analysis and FlowJo 10.6 software (TreeStar, Ashland, OR, USA) for data analyses.

Cells harvested from overnight stimulation cultures were incubated with live/dead aqua V510 for 15 min on ice. The cells were then surface stained for 30 min on ice using anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4-APC-H7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 560158), and anti-CD8-PerCPCy5.5 (BD Biosciences, clone RPA-T8, 1:200, Cat# 560662) antibodies. After fixation and permeabilization with Cytofix and Perm (BD Biosciences, Cat# 554714) on ice for 15 min, intracellular staining was performed on ice for 30 min using anti-TFNα-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD Pharmingen™, clone B27, 1:200, Cat# 554702) antibodies. After the final wash step, the cells were resuspended in 200 µL of FACS buffer. Samples were acquired using an FACSAria III instrument (BD Biosciences, San Diego, CA, USA) and analysed using the FlowJo software (Treestar, San Carlos, CA, USA).

The cells were stained and erythrocytes were lysed by researchers who were blinded to the clinical data. According to the manufacturer’s recommendations, monoclonal antibodies and their isotype controls were used: BV421-labeled anti-PD-1 (5 μl, clone EH12.1; BD Bioscience, San Jose, CA, USA), APC-H7 labeled anti-CD3 (5 μl, clone SK7; BD Bioscience), FITC-labeled anti-CD4 (5 μl, clone OKT4; eBioscience, San Diego, CA, USA), FITC-labeled anti-CD8 (20μl, clone RPA-T8; BD Bioscience) per 100μl of whole blood. Samples were measured on the Gallios^™ Flow Cytometer (Beckman Coulter, Inc.) and analyzed by Gallios Software Version 1.0 (Beckman Coulter, Inc.). Lymphocytes were gated by forward scatter and side scatter, and T cells subsets were further distinguished by CD3⁺ and CD4⁺ staining, or CD3⁺ and CD8⁺ staining. At least 3,000 CD4⁺ T cells or CD8⁺ T cells were analyzed from each sample. Results are expressed as percentage of PD-1⁺CD4⁺ and PD-1⁺CD8⁺ T cells (Fig 1)