Icos pe

Spleen mononuclear cells were prepared as previously described [69 (link)]. For studies of cDCs, spleens were treated with deoxyribonuclease I (0.5mg/ml; Worthington Biochemical) and collagenase type 4 (1mg/ml; Worthington Biochemical) for 25 minutes at room temperature, in order to ensure maximal recovery of splenic cDCs. Fluorescently conjugated monoclonal antibodies, anti-mouse B220-Alexa Fluor 700 (RA3-6B2), B220-Pacific blue (RA3-6B2), CD19-FiTC (6D5), CD138-BV605 (281–2), IgD-APCCy7 (11-26c.2a), IgM-PECy7 (RMM-1), TCRβ-Alexa Fluor 700 (H57-597), TCRβ-APC/Cy7 (H57-597), CD4-BV605 (RM4-5), IFNγ-BV421 (XMG1.2), ICOS-PE (7E.17G9), Streptavidin-PE/Cy7, CD45.1-FiTC (A20), CD45.2-Alexa Fluor 700 (104), CD11c-APC (N418), MHCII-Pacific blue (M5/114.15.2), CD8α-PE/Cy7 (53–6.7), CD40-PE (1C10), CD80-PE (16-10A1), CD86-PE (GL1), ICOSL-PE (B7-RP1), PDL1-PE (MIH5), PDL2-PE (TY25), CD49d-Biotin (R1-2), CD11a-FiTC (M17/4), Ki-67-PE (16A8) and Zombie Aqua fixable viability dye were purchased from Biolegend (San Diego, CA). Anti-mouse CD95/Fas-BV421 (Jo2), CXCR5-biotin (2G8), and Bcl6-PerCP/Cy5.5 (K112-91) were purchased from BD Biosciences (Franklin Lakes, NJ). Anti-mouse T-bet-APC (eBio4B10), GL-7-APC (GL-7) and PD1-APC/Cy7 (J43) were purchased from eBioscience. Cell surface and intracellular IFNγ, T-bet and Bcl6 staining was performed as previously described [69 (link),70 (link)].

PBMCs were separated by a gradient centrifugation procedure on a lymphocyte separation medium (Secoll Separation Media, Mannheim, Germany). After separation, freshly isolated cells were stained with the following fluorophore-conjugated antibodies: CD4 APC-Cy7 (clone: RFT-4g), ICOS PE-Cy7 (clone: ISA-3, eBioscience, ThermoFisher), CXCR5 FITC (CD185, clone: REA303), CXCR3 APC (CD183, clone: REA232), PD-1 PE-Vio 770 (clone: PD-1.3.1.3, Miltenyi Biotec), CD45RA PE-Cy5 (clone: HI100, BioLegend), and CCR6 PE (CD196, clone: 11A9, BD Bioscience).
The freshly isolated thymocytes and the PBMCs obtained at the time of thymectomy were stained with the following fluorochrome-conjugated antibodies: CD4 FITC (clone: RPA-T4, Beckman Coulter), CD8 APC (clone: RPA-T8), CXCR3 eFluor660 (CD183, clone: CEW33D, eBioscience), CCR6 PE (CD196, clone: 11A9), PD-1 PE (CD279, clone: MIH4, BD Bioscience), ICOS PE (clone: C398.4A, BioLegend), and CXCR5 PE (CD185, clone: 51505, R&D Systems). The samples were analyzed on an Attune Flow Cytometry (Thermofisher, USA). Fluorescence minus one control, which contains all flurochromes in a panel except for the target markers measured, was used to identify and to gate the cells.