Fig 1: Calnexin (CANX) interacts with T6BP through its cytosolic tail and stabilizes CD74 thus favoring MHC-II-restricted antigen presentation to CD4+ T cells A, BCANX co-immunoprecipitates with T6BP in model and professional APCs. Endogenous T6BP was immunoprecipitated from HeLa-CIITA, B (DG-75), and dendritic-like (KG-1, DC) cells. The input, the flow-through (FT), and the immunoprecipitation (IP) fractions were analyzed by Western blot using the indicated antibodies. Bottom panels, control IPs using the beads without antibody (IP CTRL) are presented for each IP. (B) As in (A) but using anti-CANX Ab for the IP. For A and B, one representative experiment out of three biological replicates is shown.CCANX interacts with T6BP using proximity ligation assay (PLA). 48 h post-transfection with the indicated siRNAs, HeLa-CIITA cells were fixed, stained with anti-T6BP and anti-CANX antibodies and proximity revealed using PLA (Duolink). Nuclei were stained using DAPI. Top panel, two representative fields are shown. Bottom panel, quantitative analysis using ImageJ displaying the number of PLA per cell. The continuous line indicates the mean (± SEM). PLA was quantified in at least 130 cells corresponding to three biological replicates. Scale bars: 10 µm. CTRL: control; Mann–Whitney's test; ***P < 0.0003.DCalnexin interacts with T6BP through its cytosolic tail. HeLa-CIITA cells were transfected with a plasmid encoding the GST-tagged transmembrane (TM) and cytosolic (CytoTail) domains of CANX (GST-TM-CytoTail-CANX) or the vector not encoding TM-CytoTail-CANX as control and immunoprecipitated with anti-GST antibodies. The input, FT, the wash, and the IP fractions were analyzed by Western blot and revealed using the indicated antibodies. One representative experiment out of three biological replicates is shown.ESilencing of T6BP does not influence CANX expression levels while silencing of CANX reduces the level of CD74 expression. HeLa-CIITA cells transfected with the indicated siRNAs and samples analyzed, 48 h post-transfection, by Western blot with the indicated antibodies. These western blot results are representative of at least three biological replicates.FCANX-silencing dampens MHC-II-restricted antigen presentation to CD4+ T cells. HeLa-CIITA cells were treated with the indicated siRNAs and transfected with a plasmid encoding pp65 HCMV antigen fused to GFP. HeLa-CIITA cells were then co-cultured with pp65-specific T cells. Left panel, a representative experiment is shown. The mean of three technical replicates (± SD) is shown. Middle, three biological replicates are combined and presented as the mean percentage (± SD) of activated cells producing IFN? normalized to CTRL conditions. Right panel, influence of T6BP silencing on peptide presentation by Hela-CIITA cells. The cognate peptide was added exogenously (pp65, 0,5 µg/mL) on siRNA-treated cells (2 h, 37°C), washed and T cell activation monitored using IFN?-ELISPOT. Three biological replicates are combined and presented as the mean percentage (± SD) of activated cells producing IFN? normalized to CTRL conditions. Data information: The background secretions of IFN? by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL—control. Wilcoxon's test; **P < 0.01; # P > 0.05. Source data are available online for this figure.