The Autophagy Receptor TAX1BP1 (T6BP) improves antigen presentation by MHC-II molecules. (2022)
Figure 7

Figure 7. Calnexin (CANX) interacts with T6BP through its cytosolic tail and stabilizes CD74 thus favouring MHC-II-restricted presentation to CD4+ T cells.

(A-B) CANX 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.

(C) CANX interacts with T6BP using proximity ligation assay (PLA). 48h 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 were quantified in at least 130 cells corresponding to three biological replicates. Scale bars: 10μm. CTRL: control; Mann-Whitney's test; ***: p<0.0003.

(D) Calnexin 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.

(E) Silencing 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, 48h post transfection, by Western blot with the indicated antibodies. These western blot results are representative of at least three biological replicates.

(F) CANX 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 IFNγ normalised to CTRL conditions. Right panel, influence of T6BP silencing on peptide presentation by Hela-CIITA cells. The cognate peptide was added exogenously (pp65, 0,5µg/mL) on siRNA-treated cells (2h, 37°C), washed and T cell activation monitored using IFNγ-ELISPOT. Three biological replicates are combined and presented as mean percentage (+/- SD) of activated cells producing IFNγ normalised to CTRL conditions. Data information: The background secretions of IFNγ 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.

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