Anti il 10 apc clone jes5 16e3

Leukocytes were extracted from murine spleen, salivary glands and lungs as described previously [13 (link),60 (link)]. For CD4⁺ functional responses, isolated leukocytes were stimulated for 2 hours with 3 μg/ml m09, (GYLYIYPSAGNSFDL), M25 (NHLYETPISATAMVI), m139 (TRPYRYPRVCDASLS), and m142 (RSRYLTAAAVTAVLQ) MCMV MHCII peptides (Genscript). Brefeldin A (Sigma) was then added and cells incubated for a further 4 hours. For CD8⁺ functional responses, leukocytes were stimulated with 2 μg/ml m139, IE3, M38, and M45 MCMV MHCI peptides in the presence of anti-mouse CD107a-FITC (Biolegend) with monensin (BD Biosciences) and Brefeldin A for 6 hours. Cells were subsequently stained with Zombie Aqua fixable viability dye (Biolegend) or LIVE/DEAD-Aqua (Life-Technologies), stained with anti-CD16/CD32 Fc-block (Biolegend) and then with either anti-CD4 Pacific-Blue or PercP, (clone RM4-5, Biolegend) or with anti-CD8 PercP (clone 53–6.7, Biolegend) and anti-CD262 (TRAILR DR5, clone MD5.1, eBioscience). Cells were fixed, saponin permeabilised and stained for anti-IFNγ FITC or Pacific-Blue (clone XMG1.2, Biolegend) and anti-IL-10 APC (clone JES5-16E3, eBioscience). Data was acquired using a BD FACSCantoII or a BD LSR II fortessa flow cytometer (BD Biosciences) and analysed with FlowJo software (TreeStar).

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).