1. Volume 42, Issue 5, Pages 850-863 (May 2015)
The Translocon Protein Sec61 Mediates Antigen Transport from Endosomes in the Cytosol for Cross-Presentation to CD8+ T Cells 
Matthias Zehner, Andrea L. Marschall, Erik Bos, Jan-Gero Schloetel, Christoph Kreer, Dagmar Fehrenschild, Andreas Limmer, Ferry Ossendorp, Thorsten Lang, Abraham J. Koster, Stefan Dübel, Sven Burgdorf 
Immunity 
Volume 42, Issue 5, Pages (May 2015)
DOI: /j.immuni
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Immunity 2015 42, 850-863DOI: (10.1016/j.immuni.2015.04.008)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
Inhibition of the ERAD Machinery Prevents Antigen Translocation and Cross-Presentation
(A) BM-DCs were incubated with OVA in the presence or absence of 10 μg/ml ExoA for 2 hr. Then, BM-DCs were fixed and co-cultured with OT I T cells. T cell activation was determined by IL-2 ELISA.
(B) As in (A) using OT II T cells.
(C) BM-DCs were electroporated with OVA- or GFP-encoding mRNA and incubated in the presence or absence of 10 μg/ml ExoA for 3 hr, fixed, and co-cultured with OT I T cells.
(D) as in (A) using 10 nM SIINFEKL.
(E) BM-DCs were incubated with 5 mg/ml OVA or 10 nM SIINFEKL in the presence or absence of 10 μg/ml ExoA and stained with the 25.D1-16 antibody.
(F) BM-DCs were electroporated with OVA- or GFP-encoding mRNA, incubated in the presence or absence of ExoA and stained with the 25.D1-16 antibody.
(G) BM-DCs were treated with OVA in the presence of the proteasome inhibitor MG132 and in the presence or absence of 10 μg/ml ExoA. Cytosolic OVA was visualized after isolation of the cytosolic fraction by Western blot. All graphs depict representative results of at least three independent experiments. Data are represented as mean ± SEM. ExoA, ExotoxinA; MFI, mean fluorescence intensity. See also Figure S1.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2
Downregulation of Derlin-1 Does Not Influence Cross-Presentation, MHC II-Restricted Presentation, and T Cell Activation after Peptide Loading
(A) Downregulation of Derlin-1.
(B) BM-DCs were electroporated with siRNA. After 3 days, cells were incubated with OVA for 3 hr and co-cultured with OT I T cells. T cell activation was determined after 18 hr by ELISA.
(C) As in (B), using OT II T cells.
(D) As in (B) using 10 nM of SIINFEKL peptide. Data are represented as mean ± SEM. All graphs depict representative results of at least three independent experiments. See also Figure S2.
Immunity  , DOI: ( /j.immuni )
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5. Figure 3
Downregulation of Sec61α Prevents Antigen Export into the Cytoplasm and Cross-Presentation
(A) Expression of Sec61α1 and Sec61α2 in BM-DCs monitored by RT-PCR.
(B) Downregulation of Sec61α 24 hr after electroporation.
(C) BM-DCs were electroporated with siRNA. After 1 day, cells were incubated with OVA for 2 hr, fixed, and co-cultured with OT I T cells. T cell activation was determined after 18 hr by ELISA.
(D) As in (C) using OT II cells.
(E) siRNA treated DCs were electroporated with OVA- or GFP-encoding mRNA. After another 3 hr, cells were fixed and co-cultured with OT I T cells.
(F) As in (C) using 10 nM SIINFEKL peptide.
(G) 18 hr after electroporation, siRNA-treated DCs were incubated with 5 mg/ml OVA. After another 6 hr, cells were stained with the 25.D1-16 antibody and analyzed by flow cytometry.
(H) BM-DCs were electroporated with siRNA. After 24 hr, cells were incubated with 250 ng/ml fluorochrome-labeled OVA and analyzed by flow cytometry.
(I) siRNA-treated BM-DCs were incubated with OVA in the presence of the proteasome inhibitor MG132. After 45 min, cytosolic fractions were isolated and the amount of OVA determined by Western blot.
(J) siRNA-treated BM-DCs were incubated with 200 ng/ml OVA and 9 mg/ml Cyt c. After 8 hr, cells were harvested, stained with fluorochrome-labeled AnnV, and analyzed by flow cytometry.
(K) siRNA-treated BM-DCs were incubated with CCF4 for 1 hr, treated with 1 mg/ml β-lactamase for another 2 hr, and analyzed by flow cytometry. β-lactamase activity was determined by flow cytometry.
(L and M) As in (C and D) using beads coated with different ratios of OVA and BSA.
(N–P) Identical to (I–K) using bead-bound OVA, bead-bound Cyt c, and bead-bound β-lactamase. All graphs depict representative results of at least three independent experiments. Data are represented as mean ± SEM. MFI, mean fluorescence intensity. See also Figure S3.
Immunity  , DOI: ( /j.immuni )
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6. Figure 4 Sec61 Expression in Antigen-Containing Endosomes
(A) Transmission electron microscopy image of BM-DCs transfected with GFP-Sec61β and incubated with OVA. Sections were immuno labeled with a GFP-specific antibody and protein A-coated 15 nm gold beads. Scale bar represents 150 nm. ER, endoplasmic reticulum; M, mitochondrium; N, nucleus; E, endosome, PM, plasma membrane.
(B and C) Correlative light and electron microscopy (CLEM) of BM-DCs transfected with GFP-Sec61β and incubated with fluorochrome-labeled OVA. Immuno-labeling of Sec61 was performed as in (A). OVA is depicted in red, GFP-Sec61β in green, and the nucleus stained with DAPI in blue. Arrows indicate gold label associated with endosomes. Scale bar represents 150 nm. CLSM, confocal laser scanning microscopy; TEM, transmission electron microscopy.
(D) Quantitative analysis of Sec61 in OVA-positive endosomes. Beads in all OVA+ endosomes from 14 GFP-Sec61-negative (78 endosomes) or 25 GFP-Sec61-positive (115 endosomes) cells from the same sample were counted.
(E) BM-DCs were transduced with lentivirus expressing GFP or GFP-Sec61β. After incubation with fluorochrome-labeled OVA, endosome fractions were isolated and analyzed by flow cytometry. Right histograms depict OVA+ endosomes after gating as indicated.
(F) DC2.4 cells were transduced with lentivirus expressing GFP or GFP-Sec61β. After incubation with fluorochrome-labeled OVA, endosome fractions were isolated and stained with a Rab5-specific antibody. All graphs depict representative results of at least three independent experiments. Data are represented as mean ± SEM. MFI, mean fluorescence intensity. See also Figure S4.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5 Isolation and Purification of a Sec61-Specific Intrabody
(A) Isolation of different antibody clones against Sec61α55–76 by phage display.
(B) Binding of purified IB1 to Sec61α55–76 or control peptides depicted by ELISA.
(C) Immune precipitation from cell lysates of BM-DCs using purified IB1. Samples were loaded on an SDS-page and stained with Coomassie blue.
(D) Immune precipitation from cell lysates of BM-DCs using purified IB1 or control IB.
(E) HEK293T cells were transfected with 6His-tagged IB1. Cell lysates were subjected to Ni-NTA-affinity chromatography and analyzed by Western blot.
(F) Immune fluorescence microscopy images of BM-DCs transfected with IB1 or control IB. Cells were stained using antibodies for Sec61β, Calnexin, and intrabodies. Nuclei (blue) were stained with DAPI. Co-localization was analyzed by the Pearson correlation coefficient (PCC) and the Mander’s overlap coefficient (MOC). Scale bars represent 10 μm.
(G) DC2.4 cells were transduced with lentivirus expressing IB1 or control intrabody and with GFP or with GFP-Sec61β. Cells were treated with fluorochrome-labeled OVA before the endosome fraction was isolated and analyzed by flow cytometry.
(H) DC2.4 cells were transduced with lentivirus expressing GFP or a GFP-Sec61β and treated with OVA. Endosomes were stained with antibodies against calnexin.
(I) As in (H) using TAP-specific antibodies.
(J) Confocal laser scanning microscopy on OVA-treated JAWSII cells expressing GFP-Sec61β and control IB or IB1. In merged images, OVA is depicted in red, Sec61 in green, and intrabody in blue. Scale bars represent 10 μm in large panel and 1 μm in enlarged views.
(K) Quantification of co-localization between OVA and GFP-Sec61 in 15 random cells expressing control IB or IB1.
(L) HEK293T cells stably expressing a split Venus protein (293T.FluERAD) were transfected with IB1 or control intrabody. After 48 hr, cells were treated with 5 μM of the proteasome inhibitor MG132. Fluorescence of the Venus protein was determined by flow cytometry.
(M) BM-DCs were transduced with lentivirus expressing IB1 or control IB and transfected with mRNA encoding a fusion protein between the TCRα and the SIINFEKL epitope. MHC I-restricted presentation was determined after co-culture with OT I T cells by ELISA.
(N) Identical to (M) for a fusion protein between CD3δ and SIINFEKL. All graphs depict representative results of at least three independent experiments. Data are represented as mean ± SEM. SA, streptavidin, MFI, mean fluorescence intensity. See also Figure S5.
Immunity  , DOI: ( /j.immuni )
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8. Figure 6
Exclusion of Sec61 from Antigen-Containing Endosomes Prevents Antigen Translocation into the Cytosol and Cross-Presentation
(A) BM-DCs were transduced with lentivirus expressing IB1 or control IB, incubated with OVA for 2 hr, fixed, and co-cultured with OT I T cells. T cell activation was determined by ELISA.
(B) As in (A) using OT II cells.
(C) BM-DCs were transduced with lentivirus expressing IB1 or control IB and electroporated with GFP- or OVA-encoding mRNA. After 3 hr, cells were fixed and co-cultured with OT I T cells.
(D) As in (A) using 10 nM SIINFEKL peptide.
(E) BM-DCs were transduced with lentivirus expressing IB1 or control IB and incubated with OVA in the presence of the proteasome inhibitor MG132. After 45 min, cytosolic fractions were isolated and the amount of OVA determined by Western blot.
(F) BM-DCs transduced with lentivirus expressing IB1 or control IB were incubated with 200 ng/ml OVA and 9 mg/ml Cyt c. After 8 hr, cells were harvested, stained with fluorochrome-labeled AnnV, and analyzed by flow cytometry.
(G) BM-DCs transduced with lentivirus expressing IB1 or control IB were incubated with CCF4 for 1 hr and treated with 1 mg/ml β-lactamase for another 2 hr. β-lactamase activity was determined by flow cytometry.
(H) BM-DCs were transduced with lentivirus expressing IB1 or control IB. Cells were incubated with latex beads coated with different ratios of OVA and BSA for 3 hr, fixed and co-cultured with OT I T cells.
(I) As in (H) using OT II cells.
(J–L) As in (E–G) using bead-bound OVA, bead-bound Cyt c, or bead-bound β-lactamase.
(M–O) Immune fluorescence microscopy images of BM-DCs transfected with IB1ΔKDEL. Cells were stained using antibodies for intrabodies, Calnexin (M), Sec61 (N), and the Golgi marker GM130 (O). Nuclei (blue) were stained with DAPI. Scale bars represent 10 μm.
(P) DC2.4 cells were transduced with lentivirus expressing IB1ΔKDEL or control intrabody and with GFP or a GFP-Sec61β. Cells were treated with fluorochrome-labeled OVA before the endosome fraction was isolated and analyzed by flow cytometry.
(Q) BM-DCs were transduced with lentivirus expressing IB1ΔKDEL or control IB. Cells were incubated with OVA for 2 hr, fixed, and co-cultured with OT I T cells.
(R–T) As in (E–G) expressing IB1ΔKDEL instead of IB1. All graphs depict representative results of at least three independent experiments. Data are represented as mean ± SEM. MFI, mean fluorescence intensity. See also Figure S6.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
Impaired Recruitment of Sec61, Antigen Translocation and Cross-Presentation in TRIF-Deficient DCs
(A) Wild-type or TRIF-deficient BM-DCs were incubated with OVA for 2 hr, fixed, and co-cultured with OT I T cells. T cell activation was depicted by ELISA.
(B) As in (A) using OT II cells.
(C) Wild-type or TRIF-deficient BM-DCs were transfected with GFP- or OVA encoding mRNA. After 3 hr, cells were fixed and co-cultured with OT I T cells.
(D) As in (A) using 10 nM SIINFEKL peptide.
(E) Wild-type or TRIF-deficient DCs were incubated with OVA for 45 min. Subsequently, the cytosolic fraction of these cells was isolated and OVA was detected by Western blot.
(F) Wild-type or TRIF-deficient BM-DCs were incubated with 200 ng/ml OVA and 9 mg/ml Cyt c. After 8 hr, cells were harvested, stained with AnnV, and analyzed by flow cytometry.
(G) Wild-type or TRIF-deficient BM-DCs were loaded with CCF4 for 1 hr and incubated with 1 mg/ml β-lactamase. After another 2 hr, β-lactamase activity was determined by flow cytometry.
(H) Wild-type or TRIF-deficient BM-DCs were transduced with lentivirus expressing GFP or GFP-Sec61β. Cells were treated with 500 ng/ml fluorochrome-labeled OVA. Subsequently, endosomes were isolated and analyzed by flow cytometry. All graphs depict representative results of at least three independent experiments. Data are represented as mean ± SEM. MFI, mean fluorescence intensity. See also Figure S7.
Immunity  , DOI: ( /j.immuni )
Copyright © 2015 Elsevier Inc. Terms and Conditions