Cd107a clone h4a3

Following BFA treatment for the final 4 h, cells were resuspended in 1:1000 dilution of Fixable Viability Stain (Life Technologies, Gloucester, UK) for LIVE/DEAD cell determination. Cells were subsequently resuspended in PBS with 1% BSA (Sigma-Aldrich) and treated with Fcγ block (Life Technologies). For extracellular cell staining, cells were labelled with fluorochrome-conjugated Abs against CD3 (clone OKT3; Life Technologies), CD69 (clone FN50; BD Biosciences, Wokingham, UK), CD86 (clone IT2.2; Biolegend), DC-SIGN (clone eB-h209; Life Technologies) and CD107a (clone H4A3; Biolegend). Cells were then fixed and permeabilized using the FIX and PERM Kit (Life Technologies) before intracellular staining with fluorochrome-conjugated Abs against IFNγ (clone 4S.B3; Biolegend), IL-17A (clone eBIO64 DEC17; Life Technologies), Granzyme B (clone GB11; Biolegend), perforin (clone B-D48; Biolegend), granulysin (clone DH2; Biolegend), and granzyme A (clone CB9; Biolegend). For apoptosis analysis, cells were stained with Abs against Annexin V (Biolegend) and with propidium iodide (PI) (Biolegend) before immediate acquisition. Flow cytometric data were acquired with a BD LSRFortessa (BD Biosciences) and analysed using FlowJo v10.6.2 (Treestar, Ashland, OR, USA). Fluorescence minus one controls were used for the setting of gates.

APC were seeded in 96-wells plates at a concentration of 2x10⁵/ml and pulsed with (glyco)-dendrimers or DMSO vehicle control in complete medium for 3 hours at 37^oC. MoDC were pulsed in presence or absence of 10µg/ml MPLA (Invivogen) and primary LCs of 20µg/ml Poly I:C (Invivogen). Pulsed APC were washed and co-cultured with the gp100_280-288 specific T cell clone. For T cell degranulation, moDC were washed two times at 900rpm to remove free products and co-cultured with gp100 T cells in a 3:1 effector to target ratio for 45 minutes at 37^oC. To measure degranulation cells were stained with a FVD, anti-human CD8 BV421 (clone RPA-T8, BD), CD107a (clone H4A3, Biolegend) and CD107b Fitc (clone H4B4, Biolegend).
For IFNγ production by T cells, moDC were pulsed for 30 minutes and LC for 3 hours at 37^oC and co-cultured in a 1:5 effector to target ratio for 16-21 hours. IFNγ production was measured in supernatant using human cytokine ELISA (IFNγ Ready-Set-Go kit, eBioscience) according to manufacturer's protocol. Incubation with a short peptide containing the HLA-A2 minimal epitope was used to set maximum activation levels per experiment.

Flow cytometry assays were performed on an LSRII or FACSCantoII (BD Bioscience, USA) and analyzed by FlowJo 7.6.1 software. The percentage of CAR-T cells was determined by staining with goat anti-mouse IgG, F(ab')₂ fragment specific antibody labeled with biotin (Jackson Immunoresearch, USA) for the detection of both GFP- and F(ab’)- positive cells. The following antibodies used in this study were purchased from Biolegend, USA, including CD22 (clone HI22), CD19 (clone HI19), CD3 (clone HIT3a), CD33 (clone WM53), CD34 (clone 561) and CD107a (clone H4A3).