Cd40 5c3

In some coculture experiments, blocking antibodies (final concentration: 10 µg/mL) against CD54 (HCD54) (BioLegend, San Diego, CA), CD58 (TS2/9) (BioLegend, San Diego, CA), CD40 (5C3) (BioLegend, San Diego, CA), and CD40L (24–31) (BioLegend, San Diego, CA) and an isotype control (T8E5) (InVivoGen, San Diego, CA) were added to KCs cocultured with naive T cells in serum-free medium (XVIVO-15, Lonza, Basel). T cell activation was assessed after 24 h by analyzing the expression of the activation markers CD25 and CD69.

DCs were harvested 20 h poststimulation by pipetting the cells up and down. Cell death was assessed using the LIVE/DEAD fixable aqua dead cell stain kit (Life Technologies, Inc., Carlsbad, CA) as well as annexin V (BD Biosciences, San Jose, CA). Fc receptor blocking was performed with human TruStain FcX (BioLegend, San Diego, CA). DCs were stained with fluorochrome-conjugated antibodies against surface markers for 20 min on ice. The antibodies used were directed against CD3 (catalog number HIT3α), CD11c (3.9), HLA-DR (L243), CD86 (IT2.2), CD83 (HB15e), CD80 (2D10), and CD40 (5C3) and were all purchased from BioLegend (San Diego, CA). Anti-CD14 (M5E2) was obtained from eBioscience (San Diego, CA), and anti-CD19 (HIB19) was from BD Biosciences (San Jose, CA). DCs were then washed, samples were acquired on a BD LSR II apparatus, and data were analyzed using FlowJo software (version 9.9.3). DCs analyzed for costimulatory molecule expression were gated on the live, CD3/CD19-negative, CD11c-positive population.