Anti mhc 1

Prior to fluorochrome staining, FcRIII/II blocking was performed using the TrueStain fcX™ antibody (Biolegend, London, UK). Cell surface staining was done with anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone 53–6.7), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-CD19 (clone 6D5), anti-CD26 (clone H194–112), anti-CD45 (clone 30-F11), anti-CD69 (clone H1.2F3), anti-CD172a (clone P84), anti-CD206 (clone C068C2), anti-EpCAM (clone G8.8), anti-F4/80 (clone BM8), anti-Ly6C (clone HK1.4), anti-Ly6G (clone 1A8), anti-MHC-I (clone AF6–88.5), anti-MHC-II (clone AF6–120.01), anti-NK1.1 (clone PK136), anti-PD-1 (clone 29F.1A12), anti-PD-L1 (clone 10F.9G2), anti-CD86 (clone GL-1), anti-CD40 (clone 3/23), anti-XCR1 (clone ZET; all BioLegend, London, UK) and anti-CD204 (clone 2F8, Biorad, Munich, Germany) antibodies, and Fixable Viability Dye (Thermo Fisher Scientific, Karlsruhe, Germany) was used to exclude dead cells. The gating strategy is depicted in Additional file 1: Figure S1. Intracellular staining was done for arginase-1 (Polyclonal Sheep IgG; R&D Systems, Minneapolis, USA) using the eBioscience™ FoxP3/Transcription Factor Staining Buffer Kit (Thermo Fisher Scientific, Karlsruhe, Germany). Data were acquired on a BD LSRFortessa system (BD Bioscience, Heidelberg, Germany) and analyzed with FlowJo X software (FLOWJO LLC, Ashland, OR, USA).

Anti-MHC-I (Biolegends); anti-EEA1 (Novus Biologicals); anti-ARF6 (Santa Cruz Biotechnology); anti-Rab5 (BD transduction Laboratories); anti clathrin (Sigma); anti-M6PR kindly provided by J. Gruenberg University of Geneva, Switzerland; anti-HSP90 (SPA-830, Stressgene); Alexa-488-conjugated Phalloidin, Cy3-conjugated transferrin, Alexa488- and 543-conjugated secondary antibodies (Invitrogen); Alexa488-conjugated Anti-MHC-I (Santa cruz Biotechnology). Antibodies against rNadA were previously described in [53] (link). Alexa-488-conjugated and Alexa-633 conjugated rNadA were obtained by labelling with Alexa Fluor Microscale Protein Labeling Kit (Molecular Probes-Invitrogen) following manufacturer instructions. cDNA expressing the wild type and ARF6-Q67L mutant were kindly provided by J. Donaldson (to M.S. and A.L.), National Institutes of Health, Bethesda, USA. 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and Wortmannin were from Sigma. FITC-GA was synthesized by labogen (Milan - Italy). The myc-AP180C plasmid containing the C-terminal portion of the AP180 gene fused in frame with N-terminal myc tag sequence was generated as described elsewhere [54] (link). Generation of plasmids coding for C-terminal EGFP fusion Rabs was performed as described previously [55] (link).

Cells were suspended in blocking buffer (0.5 % BSA/ 2 mM EDTA, in PBS) and then labeled with fluorochrome-conjugated antibodies: PE-conjugated anti-Fas (BD Biosciences, San Jose, CA, USA), PE-conjugated anti-FasL (Biolegend, San Diego, CA) (1 μg/ml), or anti-MHC-I (Biolegend, San Diego, CA), anti-RAE-1δ (Biolegend, San Diego, CA), anti-Rae-1αβγδε (scbt, CA, USA) (1:50), followed by APC-conjugated anti-mouse IgG (scbt, CA, USA) or APC-conjugated anti-rabbit IgG (scbt, CA, USA) (1:50) secondary antibodies, as appropriate. Cell membrane expression of these molecules was assessed by Flow Cytometry (FACS Aria III, BD Biosciences). Raw data was further analyzed by using Flow Jo software (Tree Star, Inc. Ashland OR).