Cd4 percp cy5

To determine T cell subsets in the lung, freshly isolated cells were blocked for 2 min at 4˚C using anti-mouse CD16/CD32 antibodies (Mouse BD Fc Block; BD Pharmingen, San Jose, CA, USA) and resuspended in FACS buffer (1% bovine serum albumin (BSA) in PBS). Subsequently, cells were washed and stained using combination of different antibodies against CD4-PerCP Cy5 (cat no. 45-0042, clone RM4-5), CD69-FITC (cat no. 11-0691, clone H1.2F3), GATA3-PE (cat no. 12-9966, clone TWAJ), Tbet-eFLUOR660 (cat no. 50-5825, clone eBio4D10), RORγt-PE (cat no. 12-6988, clone AFKJs-9), Foxp3-APC cat no: 17-5773, clone FJK-16s (eBioscience, San Diego, USA), CD8a-APC Cy7 (cat no. 557654, clone 53-6.7) and CD25-FITC (cat no. 553071, clone 7D4CD25 FITC) (BD, Breda, The Netherlands) for 60 min at room temperature. Viable cells were determined by a fixable viability Dye-eFluor^® 780 (eBioscience). For intracellular staining, cells were fixed and permeabilized using a fixation/permeabilization buffer set, according to manufacturer’s protocol (eBioscience). Flow cytometric acquisition was conducted with FACS Canto II (BD Biosciences) and results were analyzed using Flowlogic Software (Inivai Technologies, Victoria, Australia).

Aspecific background was blocked using PBS blocking buffer containing 1% BSA and 5% FCS for 30 min. 5 × 10⁵ cells were plated per well and incubated at 4°C for 30 min with different antibodies against CD4-PerCP Cy5 (cat no. 45-0042, clone RM4-5), CD69-FITC (cat no. 11-0691, clone H1.2F3), GATA3-PE (cat no. 12-9966, clone TWAJ), Tbet-eFLUOR660 (cat no. 50-5825, clone eBio4D10), RORγt-PE (cat no. 12-6988, clone AFKJs-9), Foxp3-APC cat no: 17-5773, clone FJK-16s (eBioscience, San Diego, USA), CD8a-APC Cy7 (cat no. 557654, clone 53-6.7) and CD25-FITC (cat no. 553071, clone 7D4CD25 FITC) (BD, Breda, The Netherlands) and matching isotype controls were used. Cells were permeabilized for intracellular staining using fixation/ permeabilization buffer set, according to manufacturer's protocol (eBioscience). Flow cytometry was conducted using FACS Canto II (BD) and analyzed using Flowlogic Software (Inivai Technologies, Victoria, Australia) (32 (link)).

For analysis of PD-1, cells were washed in serum-free PBS and stained with a fixable viability dye, eFluor506, CD4-PerCPCy5.5, PD1-PECY7, CD8-APCeFluor780 (eBioscience), incubated for 15 min at room temperature in the dark, and then washed in PBS buffer containing 1% FBS and sodium azide. Cells were stained for Treg cell markers using a FoxP3 staining buffer set (eBioscience) and accompanying protocol. Treg cell markers included CD39-FITC, FoxP3-PE, CD73-PerCPeFluor710, CD25-PECY7, CTLA-4-APC, CD127-APCeFluor780, Ki67-eFluor450 (eBioscience), and CD4-V500 (BD Biosciences). An intracellular staining kit (Fix and Perm kit, Invitrogen) was used to analyze cytokine production after restimulation with PMA/ionomycin. Cells were stained with IL-17A-AlexaFluor488, IL-10-PE, TNF-α-PerCPCy5.5, CD45RA-PECY7, CD8-APCeFluor780, FoxP3-eFluor450 (all eBioSciences), CD45 AlexaFluor700 (BioLegend), IFN-γ-APC, CD3-V500, and IL-2-PE-CF594 (BD Biosciences). Due to PMA/ionomycin-mediated reduction in CD4 expression, CD4⁺ T cells were identified as CD3⁺CD8⁻ T cells for cytokine analysis. Cells were acquired on a BD LSRFortessa flow cytometer and analyzed using FlowJo software (Flowjo LLC).

The predicted peptides were synthesized (Genscript Corporation). PBMCs were cultured in complete RPMI (Core Media Preparation Facility MSKCC) with peptides at 1 μg/mL, peptide vehicle (DMSO, Sigma-Aldrich) and CEF peptide pool (2 μg/ml, C.T.L) for 21 days with peptide restimulation at day 7 and day 14. IL-2 (Proleukin, Chiron) and IL-15 (Peprotech, cat#200-15) were added every 3 days at 10 IU/mL and 10 ng/mL respectively. Intracellular Cell Staining (ICS) was performed at day 14, and day 21 after 6 hr re-stimulation in the presence of monensin for 5 hr (GolgiStop, BD). Cells were then stained for 15 min with viability dye (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, ThermoFisher) at 4°C followed by 30 min incubation with CD45-APC-H7 (BD PharMingen, clone 2D1), CD3-Pacific Blue (BD PharMingen, clone UCHT1), CD4-PerCP-Cy5.5 (eBioscience, clone OKT4), CD8-PE (BD Biosciences, clone SK1). Cells were then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C and washed with BD Perm/Wash (BD Biosciences). The ICS was performed in BD Perm/Wash with IFN-γ-FITC (eBioscience, clone GZ-4) and TNF-α-PE-Cy (eBioscience, clone MAb11) at 4°C for 30 min. Samples were acquired on a BD LSRII flow cytometer (BD Biosciences) and the analysis was performed on FlowJo software (FlowJo, LLC).