Cd69 fitc h1.2f3

For measurement of surface receptor expression, B cells were harvested after 24 hours of culture for flow cytometry. All antibodies used for flow cytometry were purchased from eBioscience (San Diego, CA) unless otherwise stated. After blocking with purified anti-CD16/CD32 (clone 93), cells were stained using cocktails of: CD19-APC (clone 1D3), CD80-FITC (clone 16-10A1), CD86-PE (clone GL1), CD25-APC (clone PC61), MHC class II-APC (clone M5/114.15.2), CD40-PE (clone 1C10), CD69-FITC (H1.2F3), CD138-APC (clone 281–2; BD Bioscience), CD267-PE (TACI; clone ebio8F10-3). Cells were acquired using a FACSCalibur^® (BD Bioscience) and data was analyzed using WinList 7.0 software (Verity Software, Topsham, ME).

Flow cytometry was performed with CantoII (BD) with monoclonal antibodies CD4-FITC (RM4-5, eBioscience), CD4-PerCP (RM4-5, BD Bioscience), CD25-APC (PC61, BD Bioscience), CD62L-FITC (MEL-14, BD Bioscience), CD69-FITC (H1.2F3, BD Bioscience) and FoxP3-PE (FJK-16, eBioscience). For Fig 1A, whole blood cells were obtained via the submandibular vein 7 days after administration of anti-CD25 antibody (which would be the time point for peptide immunization). Red blood cells were lysed with ACK (Ammonium-Chloride-Potassium) buffer. Remaining cells were stained for CD25 and the percentage of CD25⁺ cells was determined for the entire population of blood cells. For Fig 2, single cell suspensions of splenocytes were stained for CD4 in combination with CD25, FoxP3, CD69, or CD62L.

Murine GM-CSF was from Peprotech (Neuilly-sur-Seine, France). IL-6 and LPS were from Sigma-Aldrich (Saint-Quentin Fallavier, France). CFDA-SE (CFSE) was from Molecular Probes (Montluçon, France). OVA (SIINFEKL) and Smcy (KCSRNRQYL) peptides were from PolyPeptide (Strasbourg, France). Anti mouse CD11b biotin (M1/70) (used with streptavidin APC or streptavidin APC-Cy7), CD11b APC-Cy7 (M1/70), CD11c PE-Cy7 (HL3), I-A^b FITC (AF6-120.1), Gr1 PE (Ly6C/G, RB6-8C5), CD45.1 APC (A20), CD45.2 APC-Cy7 (104), CD45.2 PerCP-Cy5.5 (104), CD19 APC (1D3), NK1.1 PE (PK136), CD3ε PerCP-Cy5.5 (145-2C11), CD3ε Pacific Blue (500A2), CD3ε FITC (145-2C11), CD4 PE-Cy7 (RM4-5), CD8α Pacific blue (53-6.7), CD8α APC-Cy7 (53-6.7), CD8α PerCP-Cy5.5 (53-6.7), FoxP3 Alexa Fluor647 (MF23), CD25 PE (704), CD69 FITC (H1.2F3), and CD86 FITC (B7.2, GL1) were from BD PharMingen (Le Pont de Claix, France). Male antigen UTY-specific CD8⁺ T cells were detected using a PE labelled Pro5 MHC Pentamer (H-2D^b, WMHHNMDLI) (ProImmune Limited, Oxford, UK).