Rab22a controls MHC ‐I intracellular trafficking and antigen cross‐presentation by dendritic cells
2016; Springer Nature; Volume: 17; Issue: 12 Linguagem: Inglês
10.15252/embr.201642358
ISSN1469-3178
AutoresIgnacio Cebrián, Cristina Croce, Néstor Guerrero, Nicolas Blanchard, Luis S. Mayorga,
Tópico(s)Immune Cell Function and Interaction
ResumoScientific Report10 October 2016free access Source DataTransparent process Rab22a controls MHC-I intracellular trafficking and antigen cross-presentation by dendritic cells Ignacio Cebrian Corresponding Author Ignacio Cebrian [email protected] orcid.org/0000-0001-6505-0875 Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina Search for more papers by this author Cristina Croce Cristina Croce Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina Search for more papers by this author Néstor A Guerrero Néstor A Guerrero Centre de Physiopathologie de Toulouse Purpan (CPTP), CNRS/INSERM/Université de Toulouse-UPS, Toulouse, France Search for more papers by this author Nicolas Blanchard Nicolas Blanchard Centre de Physiopathologie de Toulouse Purpan (CPTP), CNRS/INSERM/Université de Toulouse-UPS, Toulouse, France Search for more papers by this author Luis S Mayorga Corresponding Author Luis S Mayorga [email protected] orcid.org/0000-0002-5995-0671 Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina Search for more papers by this author Ignacio Cebrian Corresponding Author Ignacio Cebrian [email protected] orcid.org/0000-0001-6505-0875 Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina Search for more papers by this author Cristina Croce Cristina Croce Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina Search for more papers by this author Néstor A Guerrero Néstor A Guerrero Centre de Physiopathologie de Toulouse Purpan (CPTP), CNRS/INSERM/Université de Toulouse-UPS, Toulouse, France Search for more papers by this author Nicolas Blanchard Nicolas Blanchard Centre de Physiopathologie de Toulouse Purpan (CPTP), CNRS/INSERM/Université de Toulouse-UPS, Toulouse, France Search for more papers by this author Luis S Mayorga Corresponding Author Luis S Mayorga [email protected] orcid.org/0000-0002-5995-0671 Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina Search for more papers by this author Author Information Ignacio Cebrian *,1, Cristina Croce1, Néstor A Guerrero2, Nicolas Blanchard2,‡ and Luis S Mayorga *,1,‡ 1Facultad de Ciencias Médicas, Instituto de Histología y Embriología de Mendoza (IHEM)-CONICET/UNCuyo, Universidad Nacional de Cuyo, Mendoza, Argentina 2Centre de Physiopathologie de Toulouse Purpan (CPTP), CNRS/INSERM/Université de Toulouse-UPS, Toulouse, France ‡These authors contributed equally to this work *Corresponding author. Tel: +54 261 4494143; E-mail: [email protected] author. Tel: +54 261 4494143; E-mail: [email protected] EMBO Reports (2016)17:1753-1765https://doi.org/10.15252/embr.201642358 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 Cross-presentation by MHC class I molecules allows the detection of exogenous antigens by CD8+ T lymphocytes. This process is crucial to initiate cytotoxic immune responses against many pathogens (i.e., Toxoplasma gondii) and tumors. To achieve efficient cross-presentation, dendritic cells (DCs) have specialized endocytic pathways; however, the molecular effectors involved are poorly understood. In this work, we identify the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. Our results demonstrate that Rab22a is recruited to DC endosomes and phagosomes, as well as to the vacuole containing T. gondii parasites. The silencing of Rab22a expression did not affect the uptake of exogenous antigens or parasite invasion, but it drastically reduced the intracellular pool and the recycling of MHC-I molecules. The knockdown of Rab22a also hampered the cross-presentation of soluble, particulate and T. gondii-associated antigens, but not the endogenous MHC-I antigen presentation through the classical secretory pathway. Our findings provide compelling evidence that Rab22a plays a central role in the MHC-I endocytic trafficking, which is crucial for efficient cross-presentation by DCs. Synopsis Cross-presentation by MHC class I molecules allows the detection of exogenous antigens and is crucial to initiate cytotoxic immune responses against many pathogens. This report identifies the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation in dendritic cells. Endosomes, phagosomes, and Toxoplasma gondii-containing vacuoles of dendritic cells are decorated with endogenous Rab22a. Silencing of Rab22a expression reduces the intracellular pool of MHC-I, preventing phagosomal acquisition and their recycling to the plasma membrane. Rab22a knockdown impairs the cross-presentation of soluble, particulate and T. gondii-associated antigens, but not endogenous MHC-I antigen presentation pathways. Introduction Dendritic cells (DCs) are the most potent antigen-presenting cells capable of initiating adaptive immune responses. These cells process and present antigens in the context of class I or class II molecules of the major histocompatibility complex (MHC) to trigger CD8+ or CD4+ T-cell activation, respectively. Antigens presented by MHC-I can be either endogenous or exogenous. The former antigen presentation scenario is referred to as the “classical pathway” and the latter as “cross-presentation”. Cross-presentation is pivotal to establish CD8+ cytotoxic immune responses against tumors and several pathogens, including the intracellular parasite Toxoplasma gondii 1. Compared to other professional phagocytes, DCs have developed highly specialized mechanisms of internalization to achieve an efficient cross-presentation 2. Indeed, the acidification of the phagosomal content of DCs has proved to be a very slow process due to the production of reactive oxygen species by the NADPH oxidase NOX2 3 and to an incomplete activation of the V-ATPase 4. The combination of a high pH and low levels of lysosomal proteases limits the proteolytic activity in DC phagosomes 5. This limited phagosomal proteolysis may leave intact proteins or large polypeptides for export to the cytosol, most likely via a Sec61-dependent mechanism 6, for further degradation by the proteasome. Then, the processed peptides are translocated by TAP1/2 transporters into the lumen of the ER or back into phagosomes, since these organelles recruit components from the ER–Golgi intermediate compartment by the action of the SNARE Sec22b 7. The translocated peptides are trimmed by ER aminopeptidases 8 and their analogues, such as IRAP 9, prior to loading on MHC-I molecules by the peptide loading complex 10. Finally, the MHC-I/peptide complexes are transported to the cell surface in order to trigger CD8+ T-cell activation. Although some major advances in the understanding of MHC-I intracellular trafficking during cross-presentation have recently been made, in particular those regarding the importance of Rab11a and endosomal recycling compartments (ERC) 11, the molecular effectors of the endocytic network that regulate MHC-I presentation remain poorly understood. Even less understood are the interactions that the parasitophorous vacuole (PV) of T. gondii establishes with the endocytic and exocytic pathways to intercept MHC-I molecules for efficient CD8+ T-cell activation during DC infection 12. In this study, we focused on the small GTPase Rab22a, which is a molecule that modulates central aspects of MHC-I and CD1a endocytic recycling 1314. Rab proteins mediate most intracellular membrane trafficking events by cycling between an active GTP-bound state and an inactive GDP-bound state. Working as molecular switches, different Rabs associate with specific membrane compartments giving a unique surface identity, which is required for the recruitment of molecules involved in targeting specificity 15. Rab22a has been described in early and late endosomes, but not in lysosomes in CHO cells 16. Rab22a also controls the transport of the transferrin receptor (TfR) from sorting to recycling endosomes 17, and it is associated with tubular recycling intermediates containing MHC-I molecules and transferrin 13. In the latter work, the authors have also suggested that the activation of Rab22a is required for tubule formation, while the inactive state of Rab22a is necessary for the final fusion of these tubules derived from recycling compartments with the plasma membrane 13. Moreover, Rab22a participates in the phagosome/vacuole physiology during infection and the intracellular trafficking of several pathogens, such as Anaplasma phagocytophilum 18, Neisseria meningitidis 19, Borrelia burgdorferi 20 and Mycobacterium tuberculosis 21. Nevertheless, although this Rab-GTPase plays important roles in the endocytic pathway by modulating endosomal and phagosomal functions, its role in DCs remains unknown. In this work, we demonstrate that Rab22a is widely distributed in the endocytic network of DCs and that it is early recruited to endosomes and phagosomes, as well as to the T. gondii PV, a parasite-modified compartment known to be distinct from a phagosome in infected DCs. We demonstrate that Rab22a controls MHC-I intracellular distribution, recycling and trafficking to DC phagosomes. The silencing of Rab22a expression in DCs impacts on the ability of these cells to achieve an efficient cross-presentation of soluble and particulate exogenous antigens, as well as T. gondii-derived soluble antigens. We conclude that Rab22a plays an essential role in MHC-I endocytic trafficking during antigen cross-presentation by DCs. Results and Discussion Early recruitment of Rab22a to DC endosomes and phagosomes It is known that Rab22a regulates the intracellular transport of MHC-I and TfR in several cell lines 131417. Thus, it was interesting to assess the localization of this protein in the endocytic pathway of DCs. By immunofluorescence (IF), we analyzed the colocalization of endogenous Rab22a with three endocytic markers (EEA1 for early endosomes, Lamp1 for late endosomes/lysosomes, and TfR for recycling compartments) in mouse bone marrow-derived dendritic cells (BMDCs). Rab22a was present in the three compartments; however, in contrast to previous reports, the Pearson correlation coefficient (Pcc) determined for Lamp1 was markedly high (0.8412), suggesting that in DCs, Rab22a was present not only in early endosomes (Pcc, 0.4317 for EEA1) but also in late endocytic compartments (Fig 1A, upper and middle panels). Rab22a was also present in recycling compartments as indicated by a high localization with the TfR (Pcc, 0.6409; Fig 1A, lower panel). The presence of Rab22a in the recycling center was confirmed by co-staining with Rab11a, a GTPase that is specific for this compartment (Fig EV1A). In summary, our results indicate that Rab22a has a ubiquitous distribution in the endocytic and recycling compartments of DCs. Figure 1. Cellular distribution of Rab22a and its recruitment to BMDC endosomes and phagosomes Confocal microscopy analysis showing the localization of endogenous Rab22a (green) and different endocytic markers (red): early endosomal marker EEA1, late endosome/lysosomal marker Lamp1, and the recycling compartment marker TfR in BMDCs at steady state. The means ± SEM of the Pearson correlation coefficients (Pcc) from Rab22a and EEA1 (0.4317 ± 0.05101), Lamp1 (0.8412 ± 0.03562), and TfR (0.6409 ± 0.02999) were estimated from 15 images analyzed for each marker. Scale bars: 5 μm. Colocalization of endogenous Rab22a (red) and EEA1 (magenta) around endosomes containing fluorescent soluble OVA (OVA-FITC, green) after 30 min of internalization by BMDCs. More than 60% of the endosomes were positive for Rab22a and EEA1. The indicated boxes are shown at higher magnification in the insets. Scale bars: 5 μm. IF detection of endogenous Rab22a (green) and Lamp1 (red) after 1 h of phagocytosis of 3-μm latex beads (LB) in BMDCs. Around 50% of the phagosomes were double positive for Rab22a and Lamp1. Asterisks indicate the LB. Scale bars: 5 μm. BMDCs were incubated with 3-μm magnetic beads for 15 min at 37°C and chased for 0 or 45 min. The panel shows immunoblotting of purified phagosomes, and the total cell lysates (TCL) analyzed for Lamp1 and Rab22a. A total protein amount of 10 μg and 50 μg was loaded for purified phagosomes and TCL, respectively. The blot is representative of three independent experiments. Data information: In (A–C), the nuclear marker DAPI (blue) and DIC images are shown in the left panels. Overlays are shown in the right panels. Data are representative of three independent experiments. Source data are available online for this figure. Source Data for Figure 1 [embr201642358-sup-0002-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Subcellular localization of Rab22a in BMDC and JAWS-II DC Confocal microscopy analysis showing endogenous Rab22a (red) co-localizing with endogenous Rab11a (green) at the recycling center in BMDCs at steady state. The nuclear marker DAPI (blue) and DIC images are shown on the left panel. Scale bars: 5 μm. JAWS-II DC was incubated with 3-μm magnetic beads for 15 min at 37°C and chased for 0 and 45 min. Immunoblotting of purified phagosomes was analyzed for Lamp1 and Rab22a. Ten micrograms of protein was loaded on each lane for purified phagosomes. JAWS-II DC was incubated with 3-μm LB for 15 min at 37°C and chased for 0, 45, or 165 min. Rab22a staining on isolated phagosomes was measured by FACS at the indicated time periods. Data are representative of three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint The recruitment of Rab22a to compartments containing soluble and particulate exogenous antigens was then explored. We observed that many early endosomes, as labeled with the EEA1 marker, were positive for Rab22a and internalized soluble fluorescent ovalbumin (OVA) after 30 min of uptake (Fig 1B). When BMDCs were fed during 1 h with 3-μm latex beads (LB), a clear recruitment of Rab22a and Lamp1 was observed around LB phagosomes (Fig 1C). To confirm these observations, the presence of Rab22a in purified BMDC phagosomes was assessed by Western blot. As expected, an increase in Lamp1 over time was observed, whereas Rab22a was recruited as soon as 15 min after internalization and remained in the phagosomes for up to 60 min (Fig 1D). Similar results were obtained with the JAWS-II DC line, where Rab22a was detected in purified phagosomes at 15 min after internalization and decreased at 60 min (Fig EV1B). These observations were confirmed through the quantification of Rab22a levels in isolated phagosomes by flow cytometry (Fig EV1C). Altogether, these experiments demonstrate that Rab22a is distributed widely in the endocytic pathway of DCs and that it is early and efficiently recruited to endosomes and phagosomes containing internalized material. The localization of Rab22a in other cell types is mainly restricted to early, late, and recycling endosomes, but it is excluded from other compartments with higher proteolytic capacity, such as lysosomes 16. Intriguingly, the location of Rab22a in DCs seems to be slightly different. Indeed, this GTPase was found to be more abundant in recycling endosomes and late endosomes/lysosomes. It can be hypothesized that this difference in Rab22a location, with a wider distribution along the endocytic pathway of DCs, is important for the interception of exogenous antigens after their internalization. The intracellular trafficking of MHC-I molecules is a Rab22a-dependent process To assess the role of Rab22a in the endocytic pathway of DCs, we generated stable knockdown JAWS-II DCs (Rab22a KD cells) by means of lentivirus-delivered short hairpin RNAs (shRNA). As shown in the immunoblot of Fig 2A, the expression of Rab22a was lowered to 40% as compared to DCs transduced with the scramble shRNA used as control (Scramble cells). This level of KD efficiency was confirmed by RNA extraction and qPCR (Fig 2B). Despite this modest KD efficacy, we observed a drastic effect on the intracellular distribution of MHC-I molecules, as shown by IF detection and confocal microscopy. In particular, we noticed the disappearance of the perinuclear MHC-I pool corresponding to the recycling center that markedly colocalized with Rab22a in Scramble DCs (Fig 2C). In contrast, the plasma membrane-associated labeling did not seem to be affected (Fig 2C). This alteration in MHC-I distribution was then assessed by flow cytometry. MHC-I molecules exposed on the plasma membrane were labeled with a FITC-conjugated anti-H-2Kb-specific antibody in non-permeabilized cells, while the internal pool was labeled after cell fixation and permeabilization with saponin. As depicted in Figs 2D and EV2A, the amount of MHC-I expressed on the cell surface did not exhibit significant differences between KD and control cells. In contrast, the total amount of MHC-I (cell surface + intracellular pool) was strongly decreased in the Rab22a KD DCs. To confirm these results, we performed this set of experiments also with primary BMDCs. We silenced the expression of Rab22a and we observed, by confocal microscopy (Fig EV2B) and by flow cytometry (Fig EV2C and D), a clear disappearance of the MHC-I intracellular pool, while the amount of MHC-I present at the cell surface remained unchanged, as compared to Scramble BMDCs. Therefore, it can be assumed that the reduction in the expression of Rab22a causes an important decrease in the intracellular pool of MHC-I molecules in both, JAWS-II DCs and BMDCs. Figure 2. Rab22a controls the intracellular trafficking of MHC-I molecules in DCs Immunoblotting and densitometry quantification of Rab22a in JAWS-II DCs infected with lentiviruses encoding a random sequence (Scramble) and a shRNA specific for silencing Rab22a (Rab22a KD). Data are representative of at least three independent experiments. RNA extraction and qPCR quantification of Rab22a in the same cells analyzed in (A). Data show mean ± SEM of triplicate values and are representative of two independent experiments. **P = 0.0047. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD JAWS-II DCs. Nuclei stained with DAPI and DIC images are shown on the left. Overlay is shown in the right panels. Scale bars: 5 μm. Data are representative of at least 30 images analyzed for each experimental condition from three independent experiments. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (total) Scramble and Rab22a KD JAWS-II cells. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. P = 0.1082 (ns) and ***P = 0.0003. MHC-I staining on isolated phagosomes was measured by FACS at the indicated time periods after 3-μm LB internalization by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. ***P < 0.001 at 3 h and *P < 0.05 at 5 h between both DC types. MHC-I molecules recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. *P < 0.05 at 10 min and **P < 0.01 at 20 and 40 min. The transferrin (Tfn) recycling ability was measured by FACS at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data represent mean ± SEM of three independent experiments. P > 0.05 (ns) at 10, 20, and 40 min. Data information: In (B and D), the two-tailed Student's unpaired t-test was performed. In (E–G), a two-way ANOVA and the Bonferroni post-test were performed. Source data are available online for this figure. Source Data for Figure 2 [embr201642358-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Rab22a expression controls the intracellular pool of MHC-I molecules in DCs Representative FACS profile of intact (left panel) and saponin-permeabilized (right panel) Scramble and Rab22a KD JAWS-II DCs after H-2Kb staining. IF labeling and confocal microscopy analysis showing the distribution of MHC-I molecules (H-2Kb, green) and Rab22a (red) in Scramble and Rab22a KD BMDCs at steady state. DIC images are shown in the left panels. Overlay is shown in the right panels. Scale bars: 5 μm. Representative FACS profiles of intact (left panel) and saponin-permeabilized (right panel) Scramble and Rab22a KD BMDCs after H-2Kb staining. FACS analysis of MHC-I labeled in intact (cell surface) and permeabilized (intracellular) Scramble and Rab22a KD BMDCs. Data represent mean ± SEM of triplicate values. P = 0.6283 (ns) and **P = 0.0046. The two-tailed Student's unpaired t-test was performed. Representative dot plot obtained from flow cytometry analysis after the isolation of 3-μm LB phagosomes. The population corresponding to individual phagosomes (red circle) is easily distinguished from the cellular debris generated after sample processing. More than ten thousand phagosomes from each condition were analyzed in each experiment. Representative FACS profiles of H-2Kb staining on isolated phagosomes at the indicated time periods after 3-μm LB internalization by Scramble and Rab22a KD JAWS-II DCs. MHC-I staining on isolated phagosomes was measured by FACS at 3 h post-internalization of 3-μm LB by Scramble and Rab22a KD BMDCs. Data represent mean ± SEM of triplicate values. **P = 0.0070. The two-tailed Student's unpaired t-test was performed. Representative FACS profiles of MHC-I molecules (H-2Kb FITC) recycling at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Representative FACS profiles of TfR (Alexa 488-transferrin) recycling at the indicated time periods by Scramble and Rab22a KD JAWS-II DCs. Data information: For (A, E, F, H, and I), data are representative of three independent experiments. For (B–D and G), data are representative of two independent experiments. Download figure Download PowerPoint The remarkable changes observed in the intracellular pool of MHC-I molecules prompted us to investigate whether Rab22a was controlling the acquisition of MHC-I molecules to DC phagosomes, a process already described for Rab11a KD DCs 11. To address this issue, we isolated LB-containing phagosomes from Scramble and Rab22a KD JAWS-II DCs at 15 min, 1, 3, and 5 h post-internalization and we analyzed the presence of MHC-I by flow cytometry (Fig EV2E). A striking impairment in the phagosomal recruitment of MHC-I molecules was observed in Rab22a KD DCs, as compared to Scramble DCs (Figs 2E and EV2F). Interestingly, results showed the existence of two different kinetics for both cell types: while in Scramble DCs, phagosomal MHC-I labeling decreased after 3 h post-internalization, in Rab22a KD DCs, the staining was lower but sustained over time (Fig 2E), indicating that Rab22a may also be playing an important role during MHC-I molecules export from the phagosome to the cell surface. To confirm the defect of MHC-I phagosomal acquisition after the silencing of Rab22a, we isolated phagosomes at 3 h post-internalization from primary transduced BMDCs. Again, the flow cytometric analysis demonstrated a significant reduction in MHC-I molecules in Rab22a KD BMDC phagosomes, as compared to Scramble cells (Fig EV2G). Our results demonstrate that Rab22a has a crucial role in the regulation of the MHC-I phagosomal recruitment by DCs. Although previous studies carried out in other cell types have demonstrated that Rab22a controls the recycling of MHC-I molecules, data regarding the role of this GTPase in the fast recycling of TfR are controversial 1317. Therefore, these aspects of the endocytic trafficking were assessed herein. We evaluated the MHC-I and TfR recycling capacities of Scramble and Rab22a KD JAWS-II DCs by flow cytometry. Thus, the decrease in the mean fluorescent intensities (MFI) of FITC-conjugated H-2Kb, and Alexa 488-conjugated transferrin were measured in order to assess the efficiency of molecular recycling to the plasma membrane after binding, internalization, and cell surface acid stripping. Interestingly, in Rab22a KD DCs, we observed a significant recycling capacity inhibition for MHC-I (Figs 2F and EV2H), but not for TfR (Figs 2G and EV2I), as compared to Scramble DCs. Taken together, these results support the hypothesis that Rab22a is crucial to stabilize the intracellular pool of MHC-I molecules at the ERC, where it colocalizes with Rab11a and TfR. Moreover, we observed that Rab22a is necessary for the normal recruitment of MHC-I molecules to DC phagosomes. In this sense, it is worth mentioning that the acquisition kinetics of MHC-I to Scramble DC phagosomes is in accordance with the phagosomal recruitment and subsequent disappearance of Rab22a. The role of Rab22a in MHC-I molecules recycling has been already described by others 1314; however, the modulatory capacity of this small GTPase has never been addressed in immune cells. In our study, a severe defect of MHC-I molecules recycling by Rab22a KD DCs was observed. Interestingly, the fast recycling, as evaluated by the use of fluorescent-labeled transferrin, is not altered in these cells. Rab22a is required for antigen cross-presentation by DC Since Rab22a impacted on the intracellular trafficking of MHC-I molecules to DC phagosomes, we analyzed the effect of Rab22a silencing on antigen cross-presentation. Scramble and Rab22a KD JAWS-II DCs were fed with soluble OVA or OVA-coated beads during 5 h. Then, DCs were extensively washed, fixed, and incubated with the β-galactosidase-inducible B3Z T-cell hybrid, which is specific for the OVA peptide (SIINFEKL) in association with H-2Kb MHC-I molecules. A strong inhibition of the cross-presentation capacity for soluble OVA and OVA-coated LB in Rab22a KD DCs was observed, as compared to Scramble DCs (Fig 3A and B, respectively). The synthetic peptide SIINFEKL, which does not need further processing in intracellular compartments, was presented equally well in both DC types (Fig 3C), demonstrating that the impairment of cross-presentation in Rab22a KD DCs was due to a defect in endosomal and phagosomal uptake and/or antigen processing. Figure 3. Rab22a controls cross-presentation by DCs without affecting antigen internalization, phagosomal degradation, or endogenous MHC-I presentation A–C. The cross-presentation ability after incubation with (A) soluble OVA, (B) OVA/BSA-coated beads, and (C) the SIINFEKL control peptide at the indicated concentrations by Scramble and Rab22a KD JAWS-II DCs was evaluated with the B3Z hybridoma. Data represent mean ± SEM of triplicate values and are representative of three independent experiments. (A) ***P = 0.0001 and (B) P = 0.1432 (ns); **P = 0.0044. The two-tailed Student's unpaired t-test was performed. D, E. Evaluation of endocytosis and phagocytosis in Scramble and Rab22a KD JAWS-II DCs. (D) The endocytosis of fluorescent OVA after 1 h of internalization and (E) the phagocytosis of 3-μm fluorescent LB at different times of internalization were assessed by FACS analysis. The antigen internalization was conducted at 37°C for effective uptake and at 4°C as negative control. In (D), data represent mean ± SEM of triplicate values and are representative of three independent experiments. F. The kinetics of OVA degradation, as percentage of proteases inhibitors, in isolated phagosomes at the indicated time periods post-internalization from Scramble and Rab22a KD JAWS-II DCs was assessed by FACS analysis. Data represent mean ± SEM of three independent experiments. G. Immunoblotting of Rab22a and Actin in BMDCs infected with lentiviruses encoding a random sequence (Scramble) and two shRNA specific for silencing Rab22a (Rab22a KD #1 and #2). H, I. The cross-presentation capacity after the incubation with (H) soluble and (I) the SIINFEKL control peptide at the indicated concentrations by Scramble, Rab22a KD #1, and Rab22a KD #2 BMDCs was evaluated as described before for JAWS-II DCs. Data represent mean ± SEM of triplicate values and are representative of two independent experiments. ***P = 0.0001. The two-tailed Student's unpaired t-test was performed. J. Soluble OVA was electroporated into the cytosol of Scramble and Rab22a KD JAWS-II DCs, and T-cell activation was determined 2 h later with the B3Z hybridoma. To control endogenous MHC-I antigen presentation specificity, DCs were also treated with brefeldin A (BFA). The use of this drug markedly reduced CD8+ T-cell response to similar levels obtained by DCs without any antigen (∅). Data represent mean ± SEM of triplicate values and are representative of three independent experiments. K. Immunoblotting showing the amount of OVA incorporated by Scramble (a) and Rab22a KD (b) JAWS-II DCs after electroporation and BFA treatment. Source data are available online for this figure. Source Data for Figure 3 [embr201642358-sup-0004-SDataFig3.pdf] Downloa
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