Anti cd62l pecy7 mel 14

4-amino-5-(4-chlorophenyl)-7-(dimethylethyl) pyrazolo [3,4-d] pyrimidine (PP2) was purchased from Calbiochem. The anti-CD3ε mAb (clone145-2C11) and the CTLA4–Fc fusion protein were supplied by Becton Dickinson. The antibodies used for FACS analysis were as follows: anti-CD28-PE (37.51) and anti-CD80-PE (16-10A1) from eBioscience, anti-CD80-APC (16-10A1), anti-CD54-APC (YN1/1.7.4), anti-CD4-Pacific Blue (GK1.5), anti-CD25-APC (PC61) and anti-CD62L-Pecy7 (MEL-14) from BioLegend, anti-CD44-PE (IM7), anti-I-A^k-PE (11-5.2) and anti-CD69-FITC (H1 2F3), anti-CD11a-PE (Clone 2D7) from BD Biosciences.

Single‐cell suspensions from spleens and lymph nodes of OVA‐immunized mice were incubated for 15 min at 4°C with anti‐CD16/CD32 Fc‐receptor block (2.4G2, eBiosciences), followed by incubation for 45 min at 4°C with anti‐CXCR5‐PE (L138D7, Biolegend), anti‐CD62L‐PE‐Cy7 (MEL‐14, Biolegend), anti‐PD‐1‐BV605 (J43, BD Biosciences), anti‐CD4‐FITC (GK1.5, Biolegend), anti‐CD44‐BV785 (IM7, Biolegend), and anti‐ICOS‐BV421 (7E.17G9, BD Biosciences) antibody, all diluted in PBS containing 2% FCS. Dead cells were excluded using 7‐Aminoactinomycin D (7AAD, life technologies). Single T cells from defined subpopulations were isolated into 384‐well plates containing 2 µL/well of ice‐cold Sort/RHP mix (4 mM DTT, 0.69% NP‐40, 20 ng/µL random‐hexamer‐primers (RHP, Roche), 1.87 U/µL RNAsin (Promega) in 0.25× PBS) with an AriaIII cell sorter and index‐sort option (BD Biosciences).