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.
Fig 2: (Related to Fig 3): T6BP silencing affects the repertoire of peptides presented by MHC-II molecules and has a modest influence on the source of MHC-II ligands For each sample the % of MHC-II peptide ligand shared with the 3 other experimental conditions was determined and the mean % of shared MHC-II peptide ligand calculated and plotted (± SD). Comparing the mean % of shared peptides between mock treat cells (Mock1 or Mock2) and the cells transfected with the control (siCTRL) siRNA, no significant differences were observed. By contrast, the mean % of shared MHC-II peptide ligand was significantly different between siT6BP-treated cells and Mock1-, Mock2-, and siCTRL-treated cells. The statistical significance was calculated using a Kruskal–Wallis test followed by a Dunn's test (*P < 0.05).Cell component enrichment analysis of peptide sources. As in Fig 3, HeLa-CIITA cells were transfected with siCTRL and siT6BP siRNA, lysed, submitted to MHC-II immunoprecipitation using TÜ39 antibody, and the peptide ligands sequenced using mass spectrometry (LC–MS/MS). The diversity of protein sources was analyzed according to cell component enrichment using Funrich software. Only canonical pathways statistically enriched (P < 0.05) for each condition (siCTRL and siT6BP) are shown. The P-value for pathway enrichment was obtained using the right-tailed Fisher's exact test.
Fig 3: T6BP silencing leads to exacerbated CD74 (Ii) degradation ACD74 expression was assessed using Western Blot. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48 h post-treatment, the Iip33 CD74 isoform, a degradation product (Iip16), T6BP, and actin were detected using indicated antibodies. Left panel, a representative Western Blot experiment is shown. Right panel, expression levels of five biological replicates were quantified using ImageJ and are presented as mean ratios (± SD) of CD74 to actin used as control housekeeping gene expression.BAs in (A) assessing HLA-DR and actin expression levels. Top panel, a representative Western Blot experiment is shown. Bottom panel, in three biological replicates, HLA-DR expression was quantified using ImageJ and is presented as mean ratios to actin (± SD).CAs in (B) assessing HLA-DM and actin expression levels. Bottom panel, HLA-DM expression was quantified in three biological replicates and presented as mean ratios to actin (± SD).DCD74 expression is partially recovered by blocking lysosomal acidification. As in (A), following siRNA transfection of Hela-CIITA cells, CD74 expression was assessed. The last 16 h prior to harvesting, cells were treated with chloroquine (CQ) or epoxomicin (Epo). Left panel a representative experiment is shown, membranes were blotted using anti-T6BP, -CD74, and -actin antibodies. The different degradation fragments of CD74 are indicated (Iip33, Iip22, and Iip16). Right panel, expression levels of Iip33 (top) and Iip16 (bottom) were quantified in three biological replicates using ImageJ and are presented as mean ratios of each fragment to actin (± SD).E, FAnalysis of CD74 proteolysis. HeLa-CIITA cells were transfected with siCTRL and siT6BP. 48 h post-treatment, cells were pulsed for 30 min with 35S-Met/Cys, washed, and chased for 1, 2, and 4 h. HLA-DR/CD74 complexes were first immunoprecipitated using Tü36 antibody (E) and then reimmunoprecipitated with VICY1 antibody (F). Samples were boiled and analyzed using SDS–PAGE. The bands corresponding to CD74 isoforms (Iip41 and Iip33) and the cleavage products (Iip16) are indicated. (E right panel) Iip16 expression was quantified using ImageJ and presented as a percentage of Iip16 normalized to the highest quantity of Iip16 detected after 1 h of chase in the control condition. Results are representative of two biological replicates. Ii: invariant chain (CD74); CTRL: control. Data information: (A-C) Wilcoxon's test; *P < 0.05; # P > 0.05. (D) One-way ANOVA statistical test combined with the Bonferroni's multiple comparison test was applied. Dotted lines indicate statistically significant differences between conditions. Source data are available online for this figure.
Fig 4: T6BP function in autophagosome maturation. (A) HeLa cells transfected with the indicated short interfering RNAs (siRNAs) for 48 h, were lysed, and the expression of relevant proteins was probed by Western blotting; (B) GFP-LC3 HeLa cells were transfected or co-transfected with the indicated siRNAs for 48 h and fixed for analysis by confocal microscopy. Representative profiles are shown along with a graph expressing the relative fold induction of the dot number compared with control cells; (C) mRFP-GFP-LC3 HeLa cells were transfected with the indicated siRNAs for 48 h and were treated or not treated during the last 2 h of culture with chloroquine. Representative profiles of autophagosomes (RFP+GFP+ dots) and autolysosomes (RFP+GFP- dots) per cell section assessed by confocal microscopy are shown and were quantified. Results are expressed as absolute numbers of individual vesicles (total autophagic vesicles = all RFP+ dots); (D) Results in (C) are shown as the percentage of total autophagic vesicles; (B,C) were each carried out three times in duplicates. GFP: green fluorescent protein; RFP: red fluorescent protein; WB: Western blot; Ctrl: control; CQ: chloroquine.
Fig 5: T6BP silencing decreases endogenous viral Ag presentation and CD4+ T cell activation Schematic representation of the experiment. Hela-CIITA cells were transfected with siRNAs targeting ARs and 24 h later with plasmids encoding the antigens: Gag, Gag-LC3, Gag-LC3 G120A, or pp65. 24 h post-DNA transfection, the HeLa-CIITA cells were co-cultured with antigen-specific CD4+ T cells and T cell activation was assessed using IFN?-ELISPOT. AR: Autophagy Receptor; D: day; TCR: T cell receptor.48 h post-transfection of HeLa-CIITA cells with siRNAs targeting NDP52, T6BP, OPTN, and p62, AR expression was analyzed using Western Blot. Tubulin was used as control. The results are representative of at least 3 independent experiments and correspond to AR expression levels of the experiment in (C), left panel.Monitoring of Gag-specific CD4+ T cell activation. HeLa-CIITA cells were treated as indicated above and transfected with a plasmid encoding Gag-LC3. Left panel, a representative experiment is shown (± SD of three technical replicates). Right panel, three biological replicates are combined and presented as mean percentage (± SD). The y-axis represents the relative percentage of IFN? spots reported to the secretion of IFN? by the CD4+ T cell clones incubated with the siCTRL-treated HeLa-CIITA and set to 100%. The mean T cell activation levels of the three experiments using siCTRL or siT6BP were (100, 195, 119) and (35, 53, 38) IFN?+ spots, respectively. 5,000 T cell clones were seeded per well in technical triplicates.Left panel, as in (C) right panel, but using DNA encoding Gag-LC3, Gag-LC3G120A, or Gag. With Gag-LC3, Gag-LC3 G120A or Gag antigens, the mean T cell activation levels using siCTRL or siT6BP were (199, 435, 384), (91, 141, 119) and (343, 415, 385) or (98, 273, 171), (27, 50, 19) and (97, 148, 131) IFN?+ spots, respectively. 5,000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (± SD). Right panel, influence of T6BP silencing on peptide presentation by Hela-CIITA cells. The cognate peptide was added exogenously (gag2, 0,1 µg/mL) on siRNA-treated cells (2 h, 37°C), washed and T cell activation monitored using IFN?-ELISPOT. The mean T cell activation levels using siCTRL or siT6BP were (100, 45, 42) and (71, 45, 33) IFN?+ spots, respectively. 1,000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (± SD).As in (D) but using cDNA encoding HCMV pp65 antigen (left panel) or pp65 peptide (0,5 µg/mL; right panel) and a pp65-specific CD4+ T cell line. Results of three independent experiments are represented. The mean T cell activation levels using siCTRL or siT6BP and pp65 DNA were (66, 106, 99) and (39, 50, 33) IFN?+ spots, respectively. For the peptide: siCTRL or siT6BP: (185, 317, 415) and (163, 292, 381) IFN?+ spots, respectively. 1,000 or 2000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, 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; the symbols correspond to **P < 0.01 and # P > 0.05 comparing each experimental conditions solely with its internal control (siCTRL). Source data are available online for this figure.
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