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• W2955509991 abstract "Article4 July 2019free access Source DataTransparent process Proteasomal degradation within endocytic organelles mediates antigen cross-presentation Debrup Sengupta Debrup Sengupta Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Morven Graham Morven Graham Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Xinran Liu Xinran Liu Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Peter Cresswell Corresponding Author Peter Cresswell [email protected] orcid.org/0000-0003-0540-2351 Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Debrup Sengupta Debrup Sengupta Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Morven Graham Morven Graham Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Xinran Liu Xinran Liu Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Peter Cresswell Corresponding Author Peter Cresswell [email protected] orcid.org/0000-0003-0540-2351 Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Author Information Debrup Sengupta1, Morven Graham2, Xinran Liu2 and Peter Cresswell *,1,2 1Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA *Corresponding author. Tel: +1 203 785 5176; E-mail: [email protected] The EMBO Journal (2019)38:e99266https://doi.org/10.15252/embj.201899266 See also: M Desjardins (August 2019) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During MHC-I-restricted antigen processing, peptides generated by cytosolic proteasomes are translocated by the transporter associated with antigen processing (TAP) into the endoplasmic reticulum, where they bind to newly synthesized MHC-I molecules. Dendritic cells and other cell types can also generate MHC-I complexes with peptides derived from internalized proteins, a process called cross-presentation. Here, we show that active proteasomes within cross-presenting cell phagosomes can generate these peptides. Active proteasomes are detectable within endocytic compartments in mouse bone marrow-derived dendritic cells. In TAP-deficient mouse dendritic cells, cross-presentation is enhanced by the introduction of human β2-microglobulin, which increases surface expression of MHC-I and suggests a role for recycling MHC-I molecules. In addition, surface MHC-I can be reduced by proteasome inhibition and stabilized by MHC-I-restricted peptides. This is consistent with constitutive proteasome-dependent but TAP-independent peptide loading in the endocytic pathway. Rab-GTPase mutants that restrain phagosome maturation increase proteasome recruitment and enhance TAP-independent cross-presentation. Thus, phagosomal/endosomal binding of peptides locally generated by proteasomes allows cross-presentation to generate MHC-I-peptide complexes identical to those produced by conventional antigen processing. Synopsis How cross-presentation and Transporter associated with Antigen Processing (TAP) pathways produce the same spectrum of peptides for MHC-I loading is poorly understood. New data show that active proteasomes enter endosomes and phagosomes in dendritic cells to process luminal proteins into peptides that are loaded onto MHC-I independently of TAP function. Active proteasomes enter the lumen of endolysosomal/phagosomal compartments in dendritic cells. Human β2-microglobulin stabilizes MHC-I on the surface of TAP1-negative mouse dendritic cells, thereby facilitating TAP-independent cross-presentation. Rab-GTPase mutants that inhibit maturation of endosomes/phagosomes further enhance TAP-independent cross-presentation. TAP-independent cross-presentation and cell-surface MHC-I on TAP1−/− dendritic cells require proteasome activity. Introduction Cytotoxic T lymphocytes (CTL) that eliminate virus-infected cells or tumors do so by recognizing MHC-I molecules associated with short peptides derived from viral or tumor-specific protein antigens. Initiation of a CTL response requires priming of naïve CD8-positive T lymphocytes by professional antigen presenting cells, normally dendritic cells (DCs), by a mechanism called cross-priming or cross-presentation (Grotzke et al, 2017). This involves endocytosis of antigenic proteins or phagocytosis of virally infected cells or tumor cells followed by antigen proteolysis and binding of resulting peptides by MHC-I molecules. This is very different from the way MHC-I-peptide complexes are generated by the virally infected cells or tumor cells themselves. Here, the protein antigens are synthesized conventionally on ribosomes and peptides derived from them are generated in the cytosol by proteasomal degradation. The peptides are then translocated by TAP into the endoplasmic reticulum (ER) where those with the appropriate sequence and length bind to MHC-I molecules transiently associated with TAP via tapasin within the peptide loading complex (PLC). Trimming in the ER by dedicated aminopeptidases (ERAP-1 and -2 in humans, ERAAP in mice) may also be required for the generation of antigenic peptides of a suitable length. Ultimately, the MHC-I-peptide complexes are expressed on the plasma membrane (Blum et al, 2013). For an effective cytotoxic response, CTLs primed by cross-presenting DCs must recognize endogenous MHC-peptide complexes displayed by the infected cells or tumor. How the cross-presentation and endogenous processing pathways result in the same MHC-I complexes is poorly understood, but the predominant explanation is that exogenous antigens internalized by the DCs are translocated across endosomal or phagosomal membranes into the cytosol (Kovacsovics-Bankowski & Rock, 1995; Delamarre et al, 2003; Lu et al, 2018). Although the precise mechanism of translocation remains a matter of debate, subsequent events, including proteasomal degradation and peptide translocation by TAP, would then be similar to those involved in conventional processing of cytosolic antigens. A major difference is that TAP-mediated peptide translocation may occur in phagosomes or endosomes containing membrane recruited from the ER (Gagnon et al, 2002). MHC-I peptide binding could then occur within these compartments, either to TAP-associated MHC-I molecules also recruited from the ER (Ackerman et al, 2003, 2006; Guermonprez et al, 2003; Houde et al, 2003) or to recycling MHC-I molecules derived from the plasma membrane (Nair-Gupta et al, 2014). An alternative mechanism, commonly called the vacuolar pathway, postulates that the peptides are generated by endocytic or phagosomal proteases before binding to recycling MHC-I molecules (Song & Harding, 1996). However, this mechanism implies that the production of identical MHC-I-binding peptides by vacuolar enzymes in cross-presenting DCs and cytosolic proteasomes in the target cell is a matter of chance (Grotzke et al, 2017). Here, we present an alternative to this model for the vacuolar pathway. We propose that active proteasomes are imported into phagosomes/endolysosomes and degrade internalized antigens within them, generating the same spectrum of peptides that are produced by cytosolic proteasomes in conventional MHC-I antigen processing. This eliminates the role of coincidence in the production of the same MHC-I-peptide complexes by cross-priming DCs and the target cells ultimately recognized by mature CD8-positive CTL. Results Human β2-microglobulin rescues loss of surface MHC-I and antigen cross-presentation in BMDCs derived from TAP1−/− mice Multiple groups have shown that surface MHC-I expression is reduced on bone marrow-derived dendritic cells (BMDC) derived from TAP1−/− mice, and that these cells fail to cross-present exogenous antigens (Van Kaer et al, 1992; Kovacsovics-Bankowski & Rock, 1995; Singh & Cresswell, 2010). Two potential explanations exist for the loss of cross-presentation. In the first, peptides generated by cytosolic proteolysis are not translocated across the ER or phagosomal membrane because TAP is absent. In the second, peptides are generated in vacuolar compartments but fewer MHC-I-β2m dimers are available for binding because their assembly and transport from the ER is reduced. To distinguish these possibilities, we expressed human β2-microglobulin (hβ2m) in TAP1−/− BMDCs. The introduction of hβ2m is known to increase surface expression of H2-Kb and Db molecules on TAP1-negative mouse RMA-S cells, and Kb is well expressed on the surface when introduced into TAP-negative human T2 cells (Anderson et al, 1993). We predicted that the expression of hβ2m in TAP1−/− BMDCs would enhance surface Kb expression, and that cross-presentation would be restored if TAP is only necessary to increase the MHC-I available for vacuolar loading. As expected, TAP1−/− DCs exhibited low cell surface Kb, measured by flow cytometry, that was partially restored by the introduction of hβ2m (Fig 1A). Cross-presentation by BMDCs of phagocytosed OVA associated with latex beads was assessed by measuring the stimulation of IL-2 release by the OVA-specific Kb-restricted hybridoma B3Z. Virtually, no cross-presentation was observed in TAP1−/− BMDCs transduced with a control vector (Fig 1B) but it was partially restored by a vector expressing hβ2m (Fig 1B). This is consistent with the hypothesis that the lack of cross-presentation by TAP1−/− BMDCs is at least partially a consequence of a reduction of available MHC-I-β2m dimers compared to wild-type cells. Figure 1. Expression of hβ2m partially rescues surface Kb expression and antigen cross-presentation in TAP1−/− BMDC, but does not rescue endogenous antigen presentation Cell surface H2-Kb levels of wild type and TAP1−/− BMDC transduced with control vector or a vector expressing hβ2m were analyzed. Cross-presentation of OVA by wild type and TAP1−/− BMDC expressing hβ2m was compared to control BMDC, by measuring IL-2 production by a Kb-SIINFEKL-specific T-cell hybridoma (B3Z) cultured with paraformaldehyde-fixed BMDC that were incubated with varying numbers of OVA-coated latex beads for 6 h, prior to fixation. The effect of hβ2m on the presentation of peptides derived from endogenous antigen was assessed by measuring IL-2 levels in the culture supernatant of the B3Z hybridoma incubated with fixed VV-OVA-infected WT and TAP1−/− BMDC transduced with control vector or hβ2m. Data Information: In (B) and (C), a representative experiment of three independent experiments is shown. The mean ± SD of assay triplicates are plotted. *P < 0.05, **P < 0.01, and ****P < 0.001 (Student's t-test). Download figure Download PowerPoint As a control, we asked whether the expression of hβ2m impacted conventional antigen presentation. To test this, TAP1−/− BMDC with or without hβ2m were infected with vaccinia virus-encoding OVA (VV-OVA; Ackerman et al, 2006) and their ability to mediate direct presentation of OVA was assessed. In contrast to cross-presentation (Fig 1B), endogenous antigen presentation by TAP1−/− BMDC was not rescued by the introduction of hβ2m (Fig 1C), consistent with the known requirement of TAP for delivery of OVA-derived peptides for binding to assembling Kb-β2m dimers (Androlewicz et al, 1993; Procko & Gaudet, 2009). Rab-GTPase mutants that restrict phagosomal degradation enhance TAP-independent cross-presentation Cross-presentation via the vacuolar pathway is thought to depend on the generation of the appropriate peptides by phagosomal or lysosomal proteases (Song & Harding, 1996; Campbell et al, 2000; Shen et al, 2004), while cross-presentation by the cytosolic pathway is believed to be enhanced by a reduction in lysosomal proteolysis that allows intact proteins or large fragments of them to be translocated into the cytosol for proteasomal degradation. Vacuolar proteolysis is restrained in cross-presenting DCs by an increase in lysosomal pH relative to non-cross-presenting cells, such as macrophages (Savina et al, 2006; Samie & Cresswell, 2015). If the TAP-independent cross-presentation observed in Fig 1 is a consequence of conventional phagosomal proteolysis, one would predict that introducing Rab-GTPase mutants that restrain the maturation of phagosomes to phagolysosomes would inhibit it. We therefore independently expressed three different Rab-GTPase mutants, Rab5ACA, Rab22ACA, and Rab7ADN, each of which has been shown to impair phagosomal maturation and degradation in distinct cell types (Duclos et al, 2000; Harrison et al, 2003; Roberts et al, 2006), together with hβ2m, in TAP1−/− bone marrow-derived cells to assess their impact on cross-presentation after differentiation into DCs. Constitutive expression of Rab5ACA and Rab22ACA affected BMDC differentiation so they were placed under the control of a doxycycline-inducible (Tet-ON)-based promoter (Appendix Fig S1A). Rab7ADN did not affect differentiation and was expressed under a constitutive promoter (Appendix Fig S1B). The expression of the Rab mutants does not alter the cell surface MHC-I levels or the differentiation of BMDCs (Appendix Fig S1C–E). To verify that the Rab mutants affect phagosomal degradation, we adapted a previously described assay (Savina et al, 2010). BMDCs expressing the Rab mutants were incubated with latex beads covalently conjugated with Alexa 647-labeled OVA. Following a 1-h pulse to allow phagocytosis, the cells were extracted with detergent (1% Triton X-100 plus 0.1% SDS) at different time points. Phagosomal degradation of the OVA was assessed using flow cytometry to measure the reduction in Alexa 647 fluorescence of the released beads. Consistent with previous reports, the rate of phagosomal degradation was reduced compared to control cells in BMDC expressing Rab5ACA, Rab22ACA, or Rab7ACA (Fig EV1A and B). Click here to expand this figure. Figure EV1. Expression of Rab5ACA, Rab22ACA, and Rab7ADN restricts the degradation of phagocytosed antigen Phagocytosed latex beads covalently conjugated with Alexa 647-OVA were extracted from BMDC expressing GFP or GFP-Rab5ACA or GFP-Rab22ACA or Rab7ADN at 1-h intervals, and analyzed for the loss of Alexa 647 fluorescence by flow cytometry. The mean Alexa 647 fluorescence on the beads normalized to the fluorescence at the 1-h time point is plotted (n = 3). Data representation: In (A), a representative of three independent experiments is shown. In (B), the mean (±SEM) is plotted and analyzed by non-linear one-phase decay curve. The t½ obtained from the above analysis was compared. *P < 0.05. **P < 0.01, and ***P < 0.005 (one-way ANOVA). Download figure Download PowerPoint To investigate the role of phagosomal degradation in TAP-independent cross-presentation, we co-expressed the individual Rab mutants with hβ2m in BMDCs derived from wild-type and TAP1−/− mice and analyzed cell surface Kb expression and cross-presentation efficiency. Kb levels on TAP1−/− BMDCs co-expressing hβ2m plus the Rab mutants were not significantly different from TAP1−/− BMDC expressing hβ2m alone (Fig 2A–C). However, simultaneous expression of the Rab mutants and hβ2m further enhanced the cross-presentation efficiency of both TAP1−/− BMDC and wild-type BMDC (Fig 2D–F). The expression of hβ2m was confirmed by flow cytometry (Appendix Fig S2). Figure 2. Expression of Rab5ACA, Rab22ACA, and Rab7ADN, respectively, increases hβ2m-enhanced TAP-independent cross-presentation A–C. Surface H2-Kb levels of wild-type and TAP1−/− BMDC co-expressing Rab mutants and hβ2m were analyzed by flow cytometry. D–F. Cross-presentation of OVA by wild-type and TAP1−/− BMDC co-expressing Rab mutants and hβ2m was analyzed, and IL-2 release is shown (D–F). Data information: In (D–F), representative means (±SD) of at the least three independent experiments per Rab mutants setup in triplicate are plotted. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, and “ns” is not significant (Student's t-test). Download figure Download PowerPoint The above data suggest that reduced phagosomal maturation combined with the availability of a stable pool of cell surface Kb molecules may be the key requirements for efficient cross-presentation in the absence of a functional TAP transporter. TAP-dependent cross-presentation is thought to require limited endocytic proteolysis followed by antigen translocation into the cytosol and subsequent proteasomal degradation. However, the above experiments show that TAP-independent cross-presentation is enhanced in the presence of Rab-GTPase mutants that restrict lysosomal degradation. Clearly, a protease must be required to produce the epitope, raising the possibility that proteasomes may remain a critical protease even in TAP-independent antigen cross-presentation. TAP-independent cell surface stability of MHC-I and antigen cross-presentation are proteasome-dependent Restricting peptide delivery to the ER, for example, by inhibiting TAP, results in a reduction in MHC-I cell surface expression. We hypothesized that if the residual surface MHC-I observed in TAP1−/− BMDCs depends on peptides generated by proteasomes, then a proteasome inhibitor would also reduce cell surface expression. We tested this prediction by measuring surface Kb levels on TAP1−/− BMDCs in response to varying doses of epoxomicin and found that after 6 h treatment there was a concentration-dependent reduction of Kb expression (Fig 3A and B). To confirm that this was a result of loss of peptide generation, we repeated the experiment in the presence of exogenously added SIINFEKL peptide and found that this rescued the loss of Kb surface expression (Fig 3A and B). We repeated this analysis using TAP1−/− BMDC transduced with hβ2m and again observed that surface Kb expression was reduced by proteasome inhibition and that this was reversed by adding SIINFEKL peptide (Fig 3C and D). These findings suggest that peptide generation by proteasomes is required for maintaining the stability of surface MHC-I molecules in TAP1−/− BMDCs. Figure 3. Proteasomal activity is required for stable surface Kb expression in TAP1−/− dendritic cells and antigen cross-presentation A–D. TAP1−/− BMDC transduced with vector control (A) or hβ2m (C) were incubated with varying concentrations of epoxomicin for 6 h in the absence or presence of exogenously added Kb-binding SIINFEKL peptide and analyzed for surface expression of Kb by flow cytometry. Representative plots are shown (A, C). The impact of epoxomicin on cell surface Kb was analyzed by plotting Kb surface expression on cells incubated with epoxomicin, normalized to that on cells incubated without epoxomicin (n = 3) (B, D). E. The effect of epoxomicin on cross-presentation of OVA-coated beads by TAP1−/− BMDC expressing hβ2m was determined by incubating the BMDC with OVA-coated latex beads in the presence of varying doses of epoxomicin and 10 μM gB peptide. After 6 h, the cells were fixed and incubated with B3Z cells and IL-2 production was measured. Data information: In (B, D), the means (±SEM) of three independent experiments are plotted. Data were analyzed by performing a non-linear regression analysis. In (E), a representative experiment of three independent experiments is shown. The mean (±SD) of assay triplicates is plotted. Data were analyzed by performing a linear regression analysis. Download figure Download PowerPoint Conceivably, the reduction in Kb cell surface expression by the inhibition of proteasomes could play a role in the inhibition of cross-presentation in TAP1−/− BMDCs independently of a reduction in the generation of the SIINFEKL epitope derived from OVA. To dissect the potential roles of the two parameters, we used the peptide stabilization strategy to uncouple proteasome-dependent epitope generation from down-regulation of cell surface MHC-I. We used a different Kb-binding peptide, an epitope derived from HSV-glycoprotein B (gB; Singh & Cresswell, 2010), to prevent the down-regulation of Kb in response to epoxomicin (Fig EV2). We then measured the impact of varying doses of epoxomicin on OVA cross-presentation by TAP1−/− BMDC expressing or not expressing hβ2m in the presence of the HSV-gB peptide. Down-regulation of Kb was indeed inhibited, but the increased cross-presentation induced by hβ2m remained sensitive to proteasome inhibition (Figs 3E and EV2). Click here to expand this figure. Figure EV2. Rescue of epoxomicin-mediated down-regulation of surface Kb by HSV-gB peptide A–D. Cell surface Kb expression of TAP1−/− BMDC transduced with control vector (A) or hβ2m expressing vector (C), incubated with varying doses of epoxomicin in the presence and absence of 10 μM gB peptide, was analyzed by flow cytometry. (B, D) Mean (±SEM) surface Kb levels normalized to untreated cells were plotted (n = 2). Download figure Download PowerPoint Reconstitution of TAP-independent antigen cross-presentation in non-immune cells The proteasome-dependent but TAP-independent antigen cross-presentation pathway we observed could be a specialized mechanism evolved in mouse BMDC or result from loss of TAP as a PLC structural component rather than a peptide delivery function. To examine this question, we reconstituted the pathway in a non-immune human cell line and combined this with an alternative means of inhibiting TAP function. We previously showed that the non-hematopoietic 293T cell line can efficiently mediate cross-presentation of OVA immune complexes when supplied with human FcRγIIb and Kb (Giodini et al, 2009). We expressed two different viral TAP inhibitors in these 293T-FcR-Kb cells. One, US6, is encoded by human cytomegalovirus (HCMV), and the second, ICP47, is encoded by herpes simplex virus-1 (HSV-1). US6 and ICP47 inhibit human TAP function via distinct mechanisms, on the luminal and cytosolic sides of the ER, respectively (Lehner et al, 1997; Neumann et al, 1997). TAP inhibition was confirmed by measuring the resulting decrease in surface H2-Kb levels by flow cytometry (Fig EV3A). The impact of the viral inhibitors on cross-presentation was assessed by comparing 293T-FcR-Kb cells expressing the viral genes to cells expressing LacZ as a control. The cross-presentation efficiency of the cells expressing the US6 or ICP47 was reduced by approximately 80% compared to cells expressing LacZ (Figs 4A and B, and EV3C), even though surface Kb levels remained substantial, suggesting that the availability of stable pool of cell surface Kb may not be sufficient to drive maximum TAP-independence. We hypothesized that reduced phagosomal degradation in the cells expressing the viral TAP inhibitors could also be required, and used the Rab mutants to inhibit phagosomal maturation as described above for the BMDCs. Indeed, co-expression of US6 or ICP47 with any of the Rab mutants (Fig EV3A and B) that delayed phagosomal maturation reversed the ability of the viral TAP inhibitors to inhibit cross-presentation (Figs 4A and B, and EV3C); 293T-FcR-Kb cells expressing US6 or ICP47 plus the respective Rab mutants cross-presented OVA at an efficiency close to 80% of the control cells expressing LacZ plus the Rab mutants (Fig 4A and B). The residual 20% reduction in cross-presentation may be attributable to the reduction of surface Kb molecules caused by the viral proteins (Fig EV3A). Click here to expand this figure. Figure EV3. Expression of Rab mutants in 293T-FcR-Kb and impact of co-expression of US6 or ICP47 with Rab mutants of the cell surface H2-Kb and cross-presentation H2-Kb on the cell surface of 293T-FcR-Kb co-expressing US6 or ICP47 along with Rab mutants was measured by flow cytometry. Transient expression of Rab5ACA, Rab22ACA, and Rab7ADN in 293T-FcR-Kb was evaluated by immunoblotting with anti-Myc. Representative plots of at the least three independent experiments of 293T-FcR-Kb cells co-expressing US6 or ICP47 with LacZ or Rab mutants were incubated with opsonized OVA-coated latex beads for 12 h. The effect of US6 or ICP47 along with the Rab mutants on cross-presentation was assessed by paraformaldehyde fixation and measuring IL-2 production by added B3Z cells. The assay was set up in triplicates, and the mean (±SD) of the triplicates is plotted. Source data are available online for this figure. Download figure Download PowerPoint Figure 4. Reconstitution of TAP-independent cross-presentation in 293T-FcR-Kb cells by expressing Rab5ACA, Rab22ACA, and Rab7ADN A, B. 293T-FcR-Kb cells co-expressing empty vector or Rab mutants (Rab5ACA, Rab22ACA, and Rab7ADN) individually, with either LacZ as a control or US6 (A) or ICP47 (B), were analyzed for cross-presentation of OVA using B3Z cells. The effects of US6 and ICP47 on cross-presentation were analyzed by plotting the mean percentage of IL-2 release by cells co-expressing US6 (A) or ICP47 (B) with control vector or Rab mutants compared to cells co-expressing LacZ with control vector or Rab mutants, respectively. C–E. 293T-FcR-Kb cells expressing Rab5ACA (C), Rab22ACA (D), and Rab7ADN (E) were incubated with opsonized OVA-coated latex beads in the presence of varying doses of epoxomicin. After 6 h, the cells were fixed and incubated with B3Z cells, and IL-2 production was measured. Data information: In (A) and (B), means (±SEM) of three independent experiments for each Rab mutants are plotted. **P < 0.01 (Student's t-test). Representative experiments of three independent experiments are shown for each Rab mutants (C–E). The means (±SD) of assay triplicates are plotted. Data were analyzed by performing a linear regression analysis. Download figure Download PowerPoint Overall, the data above suggest that a major fraction of cross-presentation by 293T-FcR-Kb cells expressing the Rab mutants is TAP-independent, and, as for the TAP1−/− BMDCs, we further found that this enhancement of cross-presentation remained sensitive to proteasome inhibition (Fig 4C–E). The requirement for both reduced phagosomal degradation and a stable pool of cell surface Kb molecules for proteasome-dependent cross-presentation cannot be explained by current models. Proteasomes are imported into the endolysosomal/phagosomal lumen We postulated that the entry of active proteasomes that generate the SIINFEKL epitope into the endolysosomal lumen could explain the proteasome dependence we observe in cells lacking TAP activity. To approach this question, we first used immunoelectron microscopy to examine endolysosomal compartments in BMDCs for the presence of proteasomes. Endolysosomal membranes were labeled with anti-LAMP1 antibody (5 nm gold particles, white arrowhead), and proteasome distribution was first analyzed using a rabbit antibody specific for the immunoproteasome subunit LMP2 (Appendix Fig S3A), detected using 15 nm gold particles (black arrowhead). In addition to cytosolic labeling, we also observed LMP2 subunits localized to LAMP1-positive vacuoles in BMDC (Fig 5A and B). As a specificity control, we compared the labeling of INF-γ-treated MEF cells derived from wild-type and LMP2 knockout mice. The labeling of LMP2 knockout MEF cells was much lower than wild-type MEF cells (Fig 5A and B, Appendix Fig S3B), establishing a background level of labeling by the LMP2 antibody. To further verify proteasome localization within the endolysosomal compartment, we performed immunoelectron microscopy using LAMP1 antibody together with antibodies raised against three different subunits of constitutive proteasomes (15 nm gold particles, black arrowhead), namely α5 and β5, which are components of the outer and inner rings of the 20S particle, respectively, and S2, located in the 19S cap of the 26S particle. In addition to cytosolic labeling, all three components were located in membrane-bound compartments labeled with LAMP1 (Fig 5C and D, Appendix Fig S3C). Quantitative analysis revealed that approximately 15% of the proteasomes, defined by labeling with antibodies against three distinct proteasome subunits, were within the LAMP1-positive membrane compartment (Fig 5E). Figure 5. Proteasomes access the endolysosomal and phagosomal lumen A. EM micrographs of double immuno-gold labeling using antibodies against immunoproteasome subunit LMP2 and the endolysosomal membrane marker LAMP1 in BMDC, WT MEF, and LMP2 KO MEF. Large gold particles (15 nm, black arrowhead) label LMP2 and small gold particles label LAMP1 (5 nm, white arrowhead; Scale bars = 500 nm). The insets are the magnification of region of interest containing LAMP1-positive vacuole (Scale bars = 100 nm), marked by the black rectangle. B. The distribution of LMP2-positive signals (n = 22 images) assessed by plotting the number of gold particles labeling LMP2 within the LAMP1-positive membrane compartment versus those outside the LAMP1-positive organelles. C. Immuno-gold labeling with antibodies against constitutive proteasome subunits, α5, β5, and 19 S2 (15 nm, black arrowhead), co-labeled with a" @default.
• W2955509991 created "2019-07-12" @default.
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• W2955509991 date "2019-07-04" @default.
• W2955509991 modified "2023-10-14" @default.
• W2955509991 title "Proteasomal degradation within endocytic organelles mediates antigen cross‐presentation" @default.
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