A Role for the Endoplasmic Reticulum Protein Retrotranslocation Machinery during Crosspresentation by Dendritic Cells  Anne L. Ackerman, Alessandra Giodini, Peter Cresswell  Immunity  Volume 25, Issue 4, Pages 607-617 (October 2006) DOI: 10.1016/j.immuni.2006.08.017 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Exogenous ICP47 (1-35) Abrogates the Presentation of Exogenous and Cytosolic Ags (A) KG1.Kb cells were incubated for 16 hr with 10 mg/ml OVA (bold lines) or BSA (thin lines) in the presence of the indicated concentrations of ICP47 (1-35) or control ICP47 (35-1) peptide. The generation of Kb-SIINFEKL complexes was assessed by flow cytometry with the 25D1.16 mAb specific for the complex. (B) The effect of ICP47 (1-35) on the crosspresentation of OVA by KG-1.Kb was examined by means of IL-2 secretion, measured by ELISA, by B3Z hybridoma cells, which also recognize the Kb-SIINFEKL complex. The graphs show the mean values (± SD) from three independent experiments. (C) Surface expression of Kb and HLA-DR was measured by flow cytometry with the mAbs Y3 and L243, respectively, on KG-1.Kb cells incubated for 12 hr in 100 μM ICP47 (1-35) (solid line) or ICP47 (35-1) (dotted line). The dashed line indicates the isotype control. (D) KG1.Kb (left) and PeCr2.Kb (right) cells were infected with VSV-OVA in the presence of ICP47 peptides. ICP47 (35-1)-treated (solid lines) and ICP47 (1-35)-treated (dotted lines) cells were assessed for generation of the Kb-SIINFEKL epitope after 12 hr by flow cytometry with the 25D1.16 mAb. The dashed line indicates the isotype control. (E) Presentation of cytosolic OVA by KG1.Kb and PeCr2.Kb was quantitated over the course of VSV-OVA infection by 25D.16 staining. Values are expressed as a percent of the maximum signal seen in untreated cells. (F) Inhibition of endogenous OVA presentation was confirmed with the B3Z hybridoma with VSV-OVA-infected KG1.Kb cells fixed at 0, 8, 12, and 16 hr postinfection. IL-2 released was quantitated by ELISA. Graphs show the mean values (± SD) from three independent experiments. ICP47 (35-1)-treated and ICP47 (1-35)-treated samples are shown as light gray and dark gray bars. All results are representative of at least three experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Exogenous ICP47 (1-35) Downregulates Surface MHC Class I in Crosspresenting Cells (A–C) Immature DCs (A), macrophages, primary B cells, or the PeCr2 B cell line (B), or KG-1 DLCs (C) were incubated with the indicated concentrations of ICP47 (1-35) (thick lines) or the control ICP47 (35-1) peptide (thin lines) for 16 hr at 37°C then analyzed by flow cytometry for MHC class I surface expression. The dashed lines show isotype control staining. (D) Quantitation of MHC class I surface expression by DCs (squares), macrophages (circles), and B cells (triangles) incubated with different concentrations of ICP47 (1-35) (open symbols) or the control ICP47 (35-1) peptide (closed symbols). The values represent the means (± SD) of triplicate samples. All results are representative of at least three experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 ER Recruitment to Phagosomes and Inhibition of TAP Exogenous ICP47 (1-35) (A) KG-1 cells were incubated for 15 min with microspheres coupled to a glycosylation acceptor peptide (solid line) or a control peptide (gray line). Glycosylation of the microspheres was determined by flow cytometry after detergent lysis of cells, staining with Con A-Alexa 647. The dotted line shows Con A binding to glycosylation acceptor microspheres isolated after phagocytosis by KG-1 cells pretreated with tunicamycin. (B) Phagosomes purified from KG-1 DLCs incubated for 15 min with latex beads and ICP47 (1-35) (closed circles) or ICP47 (35-1) (open squares) were further incubated with an 125I-labeled glycosylation acceptor peptide for the indicated times. (C and D) TAP-translocated glycosylated peptides were recovered with Con A-Sepharose beads and counted in a γ counter. TAP translocation by (C) purified microsomes from the KG-1 cells and (D) the cells after SLO permeabilization was examined as in (B). Bound peptide recovered in the presence of 5 U/ml apyrase (gray line) served as a negative control. All results are representative of at least three independent experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 ExoA Reverses ICP47 (1-35)-Mediated TAP Inhibition (A) Cell lysates from KG-1 cells incubated with 30 μg/ml ExoA for 2 hr were subjected to immunoprecipitation (IP) with ExoA Ab (ExoA) or control serum (Cont) followed by SDS-PAGE under nonreducing (−DTT) or reducing (+DTT) conditions and the samples immunoblotted with biotinylated ExoA Ab. Recombinant ExoA (rExoA) was included as a positive control (lane 4). (B) Microsomes from KG-1 cells incubated with or without ExoA (30 μg/ml) for 2 hr were incubated with recombinant biotinylated p97 or GST (as a control) for 1 hr at 4°C. Pelleted microsomes were solubilized in digitonin containing the crosslinker DSP. As a control (lane 6), microsomes containing ExoA were mixed with microsomes incubated with biotinylated p97 during solubilization and crosslinking. p97 was isolated with streptavidin-agarose, and potential crosslinked components were detected by immunoblotting with Abs to ExoA, MHC class I heavy chain, and the ribosomal subunit L22. Samples in the two right lanes of each blot are recombinant ExoA (rExoA) and control microsomal lysate. (C) TAP-mediated peptide translocation was assessed with purified phagosomes. Addition of ICP47 (1-35) (closed circles) significantly blocked peptide transport compared to control cells (open circles). The addition of ExoA alone (open squares) had no significant effect on peptide translocation, but significantly reversed ICP47 (1-35)-mediated inhibition of TAP function (closed squares). The graphs show the mean (± SD) of three independent experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 ExoA Inhibits Crosspresentation but Not Constitutive Presentation of a Cytosolic Ag (A) KG1.Kb cells were incubated with 10 μg/ml ExoA (squares) or BSA (circles) for 18 hr in the presence of varying concentrations of exogenous OVA. Inhibition of OVA crosspresentation was assessed by coculture of these cells with the Kb-SIINFEKL-specific T hybridoma, B3Z. IL-2 production was quantitated by ELISA; the values indicate the mean (± SD) of triplicate samples. (B) KG1.Kb cells were incubated with either ExoA or BSA in the presence of varying concentrations of SIINFEKL peptide. The ability of these cells to stimulate B3Z-mediated IL-2 production was assessed by ELISA. The values indicate the mean (±SD) of triplicate samples. (C and D) Endogenous OVA presentation is unaffected by treatment with ExoA. The generation of the Kb-SIINFEKL epitope by KG1.Kb cells expressing endogenous OVA (C) or (D) by KG1.Kb cells infected with rVV-OVA (solid lines) or control rVV (dashed gray lines), was examined by flow cytometric staining with the 25D1.16 mAb after treatment with either ExoA or BSA. In (C), treated samples are indicated by small dashes, whereas untreated samples are shown as a solid line. The large dashes indicate control staining of OVA-expressing KG-1 cells lacking Kb expression. All results are representative of at least three independent experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 In Vitro Reconstitution of Exogenous Antigen Retrotranslocation (A) Purified luciferase-loaded phagosomes were incubated for 30 min in the absence (column 1) or presence (column 2) of cytosol. In column 3, apyrase-conjugated beads were added to deplete ATP and then removed by centrifugation. Columns 4–6 show control phagosomes, isolated from a mixture of KG-1 cells that had internalized latex beads alone and KG-1 cells that had internalized fluid phase luciferase, incubated as in columns 1–3. Relative luciferase activity transported out of phagosomes during the reaction, measured with a luciferin substrate by luminometry, was normalized to the total luciferase released upon detergent lysis of phagosomes. (B) Purified luciferase-loaded phagosomes were incubated with increasing concentrations of recombinant WT p97 (light gray columns) or DN p97 (dark gray columns) and luciferase export was measured as in (A). The columns on the right show export with cytosol (black column) or without cytosol (white column). Values are indicated as a percentage of those obtained with cytosol. (C) Purified luciferase-loaded phagosomes were incubated without cytosol (white column), with cytosol alone (black column), or with cytosol plus the indicated concentrations of WT p97 (light gray columns) or DN p97 (dark gray columns), and luciferase export was measured as in (A). Values are indicated as a percentage of those obtained with cytosol alone. All graphs show the mean values (± SD) derived from four or more independent experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 7 DN p97 Inhibits Crosspresentation but Not Constitutive Endogenous OVA Presentation (A) 6 hr after transient transfection with vectors expressing EGFP alone (right) or a combination of EGFP and either WT (left) or DN (middle) p97, KG1.Kb cells were incubated for 24 hr in the presence of exogenous OVA (bold lines) or BSA (thin lines). Inhibition of OVA crosspresentation was assessed by flow cytometry with the 25D1.16 mAb. Transfected cells were differentiated from untransfected cells by gating on EGFP expression. (B) 30 hr after transient transfection of KG1.Kb.OVA cells with the indicated EGFP coexpressing vectors, the generation of the Kb-SIINFEKL epitope was examined by flow cytometry as in (A). EGFP-positive samples are shown as small dashes, while EGFP-negative samples are shown as a solid line. The large dashes indicate control staining of OVA-expressing KG-1 cells lacking Kb expression. All results are representative of at least three independent experiments. Immunity 2006 25, 607-617DOI: (10.1016/j.immuni.2006.08.017) Copyright © 2006 Elsevier Inc. Terms and Conditions