Anti hladr l243

Functional analyses of neoantigens-specific CD4+ T-cell clones were performed by using intracytoplasmic IL-2 and IFN-γ staining (ICS). Briefly, after a 14 h stimulation period with or without 2 μM, T cells were labeled with fixable viability dye (FVD) (eBioscience, 65-0865-14), anti-CD3 (BD Biosciences, 558117), anti-CD4 (Diaclone, 954.031.010), anti-IFN-γ (BD Biosciences, 554702) using Cytofix/CytoPerm KIT (BD Biosciences, 554714). Stained cells were acquired on a BD FACS Canto II (BD Biosciences) and analyzed with the BD FACS DIVA software. The HLA restriction of the specific TCR was determined with CD4+ T cell clones treated with 10μg/mL anti-HLA-DP (B7/21) (Leinco, H260) or anti-HLA-DQ (Bio-rad, MCA3796) or anti-HLA-DR (L243) (BD Biosciences, 555809) antibodies for 30 min before addition of 2 μM of neopeptides for 14 h before IFN-γ ICS. To assess the avidity of T cell clones, 1.105 T cell were culture with 1.105 peptide loaded B-EBV cells (HLA-DRB1^*1501 and HLA-DPB1^*0401) for 14 h before IFN-γ ICS.

Bound 24F4A was detected using PE-labeled anti-human IgG1 mAb (indirect method). To assess maximal 24F4A binding to cynomolgus pDCs in pre-dose samples, blood was “spiked” with 10 μg/ml of 24F4A at 4°C. The blood was then lysed (Easy Lyse, Leinco Technologies) and stained with anti-CD123 (7G3), anti-CD20 (2H7), anti-CD14 (M5E2), and anti-HLADR (L243) (BD Biosciences) to detect pDCs, and anti-human IgG1-PE mAb (Life Technologies) to identify bound 24F4A. The same indirect staining method was used to detect 24F4A on pDCs in post-dose cynomolgus whole-blood samples. Cells were acquired on a LSRII and analyzed in FlowJo.

A phenotypic analysis of human moDCs was performed for each donor after the differentiation of monocytes. Cells were stained using Zombie NIR viability dye, anti-CD14 (M5E2, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_2687593), anti-CD209 (DCN46, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_394123), and anti-HLA-DR (L243, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_2738559). Cells were acquired using LSR II cytometers (BD Biosciences, Franklin Lakes, NJ, USA), and results were analyzed using FlowJo softwares (v10, Treestar, Woodburn, OR, USA). The activation state of moDC after their infection with rAAV was analyzed by flow cytometry using the following antibodies: anti-CD80 (L307.4, BD Biosciences, RRID:AB_10562564), anti CD86 (2331, BD Biosciences, Franklin Lakes, NJ, USA, RRID:AB_11153866).