Pd1 pacific blue

Peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood of patients using Ficoll density gradients as described previously [16 (link)]. Isolated PBMCs were stained for surface markers, fixed, permeabilised with IntraPreReagent (Beckman Coulter, Fullerton, CA) and further stained with antibodies directed against intracellular markers. Leukocytes were stimulated with Leukocyte Activation Cocktail (BD Bioscience, USA) at 37 °C for 4 h prior to intracellular staining using the manufacturer’s staining protocol. Anti-human mAbs against CD3-PE-CF594, CD56-FITC, NKG2D-PE, NKp46-PE-CY7, NKp-30-APC, NKp44-PE, NKG2A-APC, CD69-PE-CY7, PD1-Pacific blue, Tim-3-APC, perforin-APC, Granzyme B-BV421, IFN-γ-PE, and TNF-α-PE with corresponding isotype-matched controls were purchased from BD Biosciences (San Jose, CA, USA). Data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analysed using FlowJo software (Flow jo, LCC, USA).

Lymphocytes from local lymph nodes were collected and stained with different fluorescence-labeled antibody cocktails as we previously described [27 (link)] to detect the percentages of CD4⁺ T cells (CD3-FITC, CD4-PE), CD8⁺ T cells (CD3-FITC, CD8a-APC), IFN-γ secreting cells (CD3-FITC, CD8-APC, IFN-γ-PE-Cy5-5), Tfh (T follicular helper) cells (CD45-APC-Cy7 (BD Pharmingen, San Diego, CA, USA), CD4-FITC (BD Pharmingen, USA), CD185-APC, PD-1-Pacific Blue (BD Pharmingen, USA)), germinal center (GC) B cells (B220-APC-Cy7, CD45-APC-Cy7 (BD Pharmingen, USA), CD95-PE, GL-7-APC), and plasma cells (B220-Pacific Blue, CD27-PE-Cy7, CD138-PE). Except where indicated, the remaining antibodies were purchased from BioLegend, San Diego, CA, USA. The stained cells were assessed by flow cytometry (BD LSRFortessa ^TM Cell Analyzer, San Jose, CA, USA), followed by FlowJo software analysis.