Anti cd4 efluor 450 clone rm4 5

Mediastinal lymph nodes were removed and processed similarly to lung as described in Section “Cytokine Levels in Lung Tissues.” The cells were collected, and the remaining erythrocytes of the left lung were lysed (lysis buffer, eBioscience, San Diego, CA, USA) according to the manufacturer’s instructions. Single-cell suspensions from MLN and lung tissues were resuspended at 1 × 10⁶ cells/mL. The cells were labeled with the following monoclonal fluorochrome-conjugated antibodies: anti-CD3-Brilliant Violet 510 (clone 17 A2, BioLegend, San Diego, CA, USA), anti-CD4-eFluor 450 (clone RM4-5, eBioscience), anti-CD25-APC (clone PC61, BD Pharmigen), anti-OX-40-PE (clone OX-86, eBioscience), anti-PD-1-APC-eFluor 780 (clone J43, eBioscience), and anti-GITR-PE-Cyanine 7 (clone DTLA-1, eBioscience) for 30 min at 4°C in the dark. The cells were then washed in PBS and incubated with a fixation buffer (eBioscience) for 30 min at 4°C in the dark. After washing, the cells were resuspended in permeabilization solution 1× (eBioscience) and stained with anti-FOXP3-Alexa Fluor 488 (clone FJK-16s, eBioscience) for 30 min at 4°C in the dark. Flow cytometry was performed on a FacsCANTO II TM (BD, San Diego, CA, USA). At least 100,000 events were analyzed with FlowJo (Tree Star) or FACSDiva (BD Biosciences) software. Gating strategy is indicated in Figure S2 in Supplementary Material.

The intracellular levels of cytokines and transcription factors were assessed using anti-CD4-eFluor450 (clone RM4-5), anti-C-X-C chemokine receptor type 5 (CXCR5)–peridinin chlorophyll protein complex (PerCP)–eFluor710 (clone SPRCL5), anti-B cell lymphoma 6 (Bcl-6)–APC (clone BCL-DWN), anti-IL-17–PE (clone eBio17B7), anti-forkhead box P3 (Foxp3)–PE (clone FJK-16 s), anti-B220–APC (clone RA3-6B2), anti-CD19–PerCP–Cy5.5 (clone eBio1D3), anti-IL-10–APC (clone JES5-16E3), anti-IL-17–fluorescein isothiocyanate (FITC) (clone eBio17B7; eBioscience), anti-T and B cell activation marker (GL-7)–FITC (clone GL7; BD Pharmingen), anti-CD1d–PE (clone 1B1), and anti-CD5–FITC (clone 53-7.3; eBioscience) antibodies. In brief, the cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of GolgiStop. Next, the cells were incubated with fixable dye (eBioscience) for 30 min and permeabilized using Cytofix/Cytoperm solution (BD Pharmingen). Thereafter, the cells were reacted with the above-listed antibodies and analyzed using a CytoFLEX flow cytometer. Events were collected and analyzed with FlowJo software (Tree Star, Ashland, CA, USA).

Isolated CD4⁺ T cells were labeled with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE; Sigma) at 37°C for 20 minutes. Cells were washed in RPMI 1640 supplemented with 10% FCS and cultured as stated above. At the indicated time-points, cells were harvested and stained with anti-CD4-eFluor450 (clone RM4-5; eBioscience). Flow cytometric data were acquired using a BD LSRFortessa cell analyzer (Becton Dickinson, Oxford, Oxfordshire, UK and data analyzed using FlowJo software (Treestar version 3.2.1, Ashland, Oregon, USA).