Departments of Immunology and
1 Medical Biophysics, University of Toronto, Medical Sciences Building,1 King's College Circle, Toronto, Ontario M5S 1A8, Canada
Correspondence to: T. H. Watts; E-mail: tania.watts{at}utoronto.ca
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Abstract |
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Keywords: CD40 ligand, dendritic cells, endocytic pathway, GTPase
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Introduction |
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Late endosome compartments containing MHC II (MIIC) or class II-containing vesicles (CIIV) have been described in different APC (4). MIIC and CIIV represent heterogeneous multivesicular and multilaminar compartments which probably represent consecutive compartments along the late endocytic pathway. MIIC have been found in B cells, macrophages and dendritic cells (DC) and contain lysosomal markers. CIIV have been reported in murine B lymphoma cells and immature DC, and unlike MIIC are not enriched in Ii.
In B lymphocytes MHC II molecules enter endocytic compartments either directly from the trans-Golgi network (TGN) (5) or via the cell surface (6). Transport of newly synthesized MHC II through early endosomes is required for their delivery to the MIIC compartment (7,8). As antigens are transported from early to late endosomes to lysosomes, they are exposed to conditions of increasing acidity and proteolytic activity (9). Different epitopes within one antigen can vary in their processing requirements. Most epitopes for MHC II loading require harsher environments found in the later endocytic compartments; however, some can be generated in early compartments and loaded onto recycling MHC II (10,11).
Intracellular trafficking of membrane proteins requires a complex of proteins that include members of the rab family. rabs are small G proteins that are characterized by their ability to bind and to hydrolyze GTP nucleotides, which changes the protein from their active state (GTP-bound) to their inactive state (GDP-bound). rab proteins are important regulators of membrane traffic on the biosynthetic and endocytic pathways (12,13). Accumulated evidence suggests that rab GTPases recruit tethering and docking factors to establish firm contact between the membranes to fuse, after which SNAREs become involved and complete the fusion process (12). Several rab proteins have been localized to early sorting and recycling endosomal compartments (rab4, rab5, rab11, rab18, rab22 and rab25) (13). So far only rab7 (14) and rab9 (15) have been localized to late endosomes. rab9 is also expressed in the TGN and is involved in transport from late endosomes to the TGN (15). rab7 has been shown to be important for transport from early to late endosomes, and is a regulator for aggregation and fusion of late endocytic structures (1619).
To date little has been done to examine the function of rab proteins in the endocytic pathway of APC. Overexpression of dominant-negative rab6 slowed intra-Golgi transport blocking transport of newly synthesized molecules in B cells (20). rab4 has been shown to be essential in receptor mediated antigen processing in B cells (21). Also, rab5a but not rab5b or c, or rab7 were shown to be up-regulated by IFN- treatment of mononuclear cells (22).
In this study we show that the level of rab7 controls the rate of antigen processing and presentation with MHC II for a number of different antigens in B cells. Furthermore, rab7 was expressed at low levels in B cells compared to macrophages and DC. We find that rab7 protein is up-regulated in B cells upon stimulation with LPS and/or CD40 ligand (CD40L), concomitantly with up-regulation of other endocytic components. Together these results show that the level of rab7 expression influences the efficiency of antigen presentation in B cells, and that rab7 and late endosomes play an important role in antigen processing and presentation in the class II pathway.
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Methods |
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Antigen processing and presentation assays
TA3 and TA3-Rab7 cells (107/ml) were incubated with antigen (HEL, OVA or RNase; Sigma) at the doses indicated for various periods at 37°C. Cells were cooled rapidly to 4°C and washed with PBS before fixation with 1% paraformaldehyde. Following blocking with glycine and several washes, 105 cells were added to 105 T cell hybridoma cells at 37°C. After 1824 h, supernatants were incubated with 104 IL-2-dependent CTLL cells. [3H]Thymidine (Amersham Canada, Oakville, Ontario, Canada) was added to the CTLL cultures after 16 h and [3H]thymidine incorporation measured 6 h later. Results were expressed as mean + SEM. In some experiments 10 mM NH4Cl was added 3 h prior to and during incubation with antigen.
MIEV vector, transfections and infections
The cDNA encoding dog rab7 cDNA (14) was cloned into the MIEV retroviral vector(29). The packaging BOSC cell line was transfected with 10 µg of MIEV-Rab7 construct using calcium chloride. GP+E cells were infected with 48 h BOSC cell supernatant and EGFP+ cells sorted for high expression, using a Coulter Elite cell sorter. Sorted GP+E cells were co-cultured with TA3 cells in the presence of 8 µg/ml polybrene overnight, followed by sorting of EGFP+ TA3 cells. The packaging cell lines BOSC and GP+E were propagated in IMDM supplemented with 10% FCS.
Measurement of Ii processing from newly synthesized MHC II
Cells (2x107) were cultured in methionine-deficient medium for 60 min, washed and then pulsed with 0.5 mCi [35S]methionine (Amersham Canada) in 1 ml. After 20 min of labeling 9 volumes of prewarmed media with 1 mM cold methionine and 10% FCS were added for the chase. At each time point 2x106 cells were cooled. For immunoprecipitation, the cell pellets were lysed in 1 ml of lysis buffer with protease inhibitors (see Western blotting). Lysates were precleared with normal mouse serum and Protein ASepharose (Sigma) before the addition of skim milk (5% final). Lysates were incubated with anti-MHC II (N22) bound to Protein ASepharose, rotating overnight at 4°C. The immunoprecipitates were washed 4 times in lysis buffer and run on 12% SDSPAGE under reducing conditions. Gels were dried and exposed to autoradiography film Biomax MS (Kodak).
Western blotting
Cells pellets were lysed in 1% NP-40 in 10 mM TrisHCl, pH 7.2, 150 mM NaCl (lysis buffer) with protease inhibitors (PMSF 1 mM, EDTA 0.5 mM, pepstatin 1 µg/ml, leupeptin 10 µg/ml, aprotinin 10 µg/ml, antipain 10 µg/ml, benzamindine 17 µg/ml; Sigma). Equal amounts of Laemmli-solubilized cell lysates were loaded on 12% SDSPAGE and transferred to Immobilon-P membranes (Millipore, Bedford, MA). Blots were probe with primary antibody as indicated and detected by chemiluminescence (Amersham Pharmacia, Piscataway, NJ). Quantitation of bands was done by NIH image, version 1.61.
Immunofluorescence staining
Cells were washed in PBS before intracellular staining using the BD-PharMingen Cytofix/Cytoperm kit. Cells were stained with primary antibody followed by species-specific phycoerythrin-labeled antibody (Molecular Probes, Eugene, OR). Cells were analyzed on a FACSCalibur (Becton Dickinson, Mountain View, CA).
B cell preparations
Resting B cells were purified from total spleen cells from C57BL/6 mice (Charles River, St Constance, Quebec, Canada). Following red blood cell lysis, cells were incubated with anti-Thy1.2, anti-CD4, anti-CD8 and anti-CD11b plus complement to remove T cells and macrophages. This was followed by separation on Peroll density gradients (50/60/66/70%), with resting B cells taken at the 60/66/70% interfaces. B cells were stimulated by addition of 1 µg/ml LPS (Difco, Detroit, MI) or treatment with soluble CD40L. Soluble CD40Lmurine CD8 fusion protein was produced as a baculovirus supernatant and used as a 1:16 dilution of supernatant plus 5 µg/ml anti-CD8.
Macrophage/DC preparations
DC were generated from bone marrow by culture in 4 ng/ml recombinant granulocyte macrophage colony stimulating factor (BD-PharMingen) following the method of Inaba et al. (30). Immature DC were taken at day 6, and treated with 1 µg/ml LPS (Difco) and 4 ng/ml rIL-4 (BD-PharMingen) to generate the mature phenotype DC at day 8. Macrophages were also isolated from these preparations as the firmly adherent population that remained bound to the culture surface.
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Results |
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The HEL(4661):I-Ak epitope has been previously characterized as being processed in late endocytic compartments. In contrast, the RNase(4256):I-Ak epitope has been characterized as being Ii independent, and able to be processed and loaded onto recycling MHC II in early endosomes (11,31,32). Figure 3(A) shows that overexpression of rab7 also increased the rate of presentation of RNase(4256):I-Ak. As seen for HEL protein, TA3-Rab7 when pulsed with intact RNase were able to process and present the RNase(4256): I-AK epitope to TS.12 T cell hybrids at increased rates compared to TA3.
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Addition of NH4Cl to cells inhibits the degradation of antigen by increasing the pH in late endocytic compartments, thereby preventing production of epitopes that require a more acidic pH for processing (33). In view of our finding that rab7 enhanced the rate of processing of RNase(4256): I-Ak, it was important to confirm the previous findings on the pH dependence of antigen processing in the TA3 cells. Figure 4 shows that NH4Cl treatment of TA3 significantly prevented production of the HEL(4661):I-Ak epitope (Fig. 4A
). The same was true in TA3 cells overexpressing rab7 (Figure 4B
). NH4Cl treatment of TA3 cells did not prevent presentation of RNase(4256):I-Ak (Fig. 4C
). However, NH4Cl treatment of TA3-Rab7 showed inhibition of processing of RNase only at the earliest time points, the same time points that showed enhancement of presentation by rab7 overexpression (Fig. 4C
). Thus a subfraction of RNase processing appears to take place in more acidic compartments and can be enhanced by rab7 overexpression. In contrast, the NH4Cl-independent component of RNase processing, thought to take place in early endosomes, may take place more slowly, as seen by lack of inhibition at the later time points. As expected, NH4Cl treatment did not inhibit presentation of HEL(4661) peptides by TA3 to A2.A2 T cells (Fig. 4E
).
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To test whether increased rates of transport of newly formed MHC IIßIi complexes through the endosomal pathway are influenced by overexpression of rab7, TA3 and TA3-Rab7 were pulsed with [35S]methionine, and at various time points during the chase MHC II proteins were immunoprecipitated and amounts of MHC II-associated Ii determined by SDSPAGE (Fig. 5
). Again, there was no detectable difference between TA3 or TA3-Rab7 in the amount of Ii or its major cleavage product (the p10 fragment) at any time point looked at. Similarly, the level of [35S]methionine-labeled SDS-stable MHC II dimers following various times of chase did not vary between TA3 and TA3-Rab7 (data not shown).
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rab7 levels differ in different APC types and are regulated in B cells by activation
The finding that increasing the expression of wild-type rab7 in a B cell line by only a few fold had a substantial effect on the kinetics of antigen presentation prompted us to examine the levels of endogenous rab7 in normal APC. Significantly higher levels of rab7 were found in adherent macrophages compared to B and T cells (Fig 6A). As a control the levels of ß-actin in these cell types were shown to be similar. Given that overexpression of rab7 in B cells could enhance antigen presentation, we speculated that B cells might up-regulate rab7 during activation. Indeed stimulation of B cells with either CD40L, LPS or a combination of the two up-regulated rab7 protein by 24 h, with maximal induction by 48 h after activation (Fig. 6B
). In addition, other components of both the early and late endocytic pathway, including lamp1, H-2Mß and rab5, were also increased upon B cell activation, under conditions where actin levels remained constant. Thus there appears to be a general increase in the expression of components of the endocytic pathway upon B cell activation. rab7 levels were also measured in DC cultured from mouse bone marrow precursors using granulocyte macrophage colony stimulating factor. At day 6 of culture, cells were treated with LPS and IL-4 to induce maturation. `Mature' DC are characterized by high cell surface MHC II, high CD86 and low uptake of FITCdextran, whereas the `immature' DC population expresses lower levels of both MHC II and CD86 at the cell surface and shows high uptake of FITCdextran. Intracellular staining of the surface MHC II high and surface MHC II low populations of DC with anti-Rab7 antibody showed similar high levels of rab7 expression in the two populations and this was similar to that found in adherent macrophages from the same cultures (data not shown).
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Discussion |
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Activated B cells have previously been shown to present MHC IIantigen complexes to T cells at a faster rate than resting B cells (35,36). This has been attributed to several factors including increased rates of fluid phase pinocytosis (35) and up-regulation of co-stimulatory activity (3739). Signals through the antigen receptor, CD40 or by LPS can up-regulate co-stimulatory activities and convert resting B cells into effective APC for naive T cells (38,39). In our studies, T cell hybridomas were used as a read-out for antigen presentation. Because these T cell hybridomas are relatively insensitive to co-stimulation, they can be used to analyze the rate of MHC IIpeptide complex formation on the cell surface relatively independently of other co-stimulatory interactions. Our data support the additional concept that activated B cells are more efficient in antigen presentation at least in part due to increases in rab7 and other components of the endocytic pathway. The effects of rab7 overexpression may be to allow activated B cells to transport antigen and/or newly synthesized MHC II from early endosomes into the MIIC (late endocytic) compartment at increased rates compared to resting B cells and/or to increase the overall size of the MIIC compartment, thereby increasing the efficiency of the antigen loading process.
In contrast to the results with B cells, we found that both `immature' and `mature' bone marrow-derived DC have similar levels of rab7 expression and these levels are comparable to those seen in adherent macrophage populations (data not shown). Thus our data add to the accumulating evidence for differences between myeloid and lymphoid antigen presenting cells.
Macrophages have increased rates of fluid phase pinocytosis (macropinocytosis) compared to B cells and can present antigen with MHC II at faster rates (35,4042). Optimal T cell activation can occur within 60 min for adherent macrophages incubated with antigen compared with 68 h for B cells. Our results show that adherent macrophages contain considerably more rab7 than B cells. We have not fully assessed the activation state of the macrophages used here, as the adherence step used to separate macrophages and DC may well alter their level of activation. However, these macrophages do not express detectable MHC II, arguing that they are not fully activated for antigen presentation function. Furthermore, their rab7 levels did not change after addition of LPS (data not shown). Regardless of their activation state, the high levels of rab7 in adherent macrophages are consistent with the observation that macrophages have increased rates of antigen processing compared to B cells. It is also recognized that there are many other factors that effect the presentation of particular MHC IIantigen complexes in B cells and macrophages. For example, B cells but not macrophages express a chaperone HLA-DO that modulates the ability of HLA-DM to enhance peptide loading (3).
As mentioned above, rab7 could enhance the rate of antigen presentation by enhancing the rate of antigen delivery to the late endosome or by enhancing the rate of delivery of newly synthesized MHC II to this compartment, or by a combination of these effects. The fact that both MHC IIIi as well as proteins taken up from outside the cell have been shown to reach the MIIC via an early endosome intermediate (1,7) makes it likely that rab7 is acting on both these processes. Overexpression of rab7 has also been shown to increase the size of the late endosome/lysosome compartment (19) and thus may have a general effect on the efficiency of processes that take place in the late endosome. However, in our studies we were unable to detect an overall difference in the degradation of labeled proteins or in the rate of processing of the MHC IIIi complex (Fig. 5). The rate of transport of horseradish peroxidase through the endosome was also similar in TA3 and TA3-Rab7, and MHC II complexes arrive at the surface at a similar rate in both cell types (data not shown). As discussed above, the failure to detect these changes biochemically may reflect the differences in sensitivity of the antigen presentation assay versus these bulk biochemical assays. Indeed, previous studies with overexpression of rab7 in BHK and HeLa cells, showed no detectable increase in the rate of degradation of radiolabeled proteins in cells overexpressing wild-type rab7 when compared to control cells (16,43). In contrast, overexpression of dominant-negative rab7 was able to decrease transport from early to late endosomes in those studies. Recently, others have used constitutively active forms of rab7 to analyze rab function (19). Again, while dominant-negative forms impaired protein degradation, dominant active and wild-type forms of rab7 did not appear to change the level of protein degradation in the cells (19). Our attempts to overexpress a dominant-negative form of rab7 in TA3 cells were unsuccessful, as constitutive expression of a dominant-negative form of rab7 is lethal to the cells (18). Nevertheless, the overexpression studies imply that rab7 is limiting for antigen presentation in B cells and also imply that rab7 and the late endosome are key players in MHC class II-restricted presentation.
Overexpression of wild-type rab7 in TA3 B cells led to enhanced kinetics of antigen presentation for four different MHC IIantigen combinations. The effects of increased rab7 expression were most striking for OVA(323339):I-Ad, which was very poorly presented by TA3 cells in the absence of rab7 overexpression. The level of rab7 obtained in transfected TA3 cells approximates the levels of rab7 found in activated B cells (Fig. 6B) and thus the data obtained with TA3 B lymphoma cells lend support to the idea that rab7 accounts at least in part for the enhanced rate of antigen presentation in activated normal B cells. The greater effect of rab7 overexpression on Ad-restricted presentation of OVA323339 could reflect allele specific differences in processing. Alternatively, the larger size of the OVA protein compared to HEL or RNase appears to result in a longer processing time and this longer time may result in greater sensitivity to modulation by rab7.
Previous results have shown that both HEL(4661):I-Ak (10,11,44) and OVA(323339):I-Ad (45) epitopes are loaded onto MHC II in late endocytic compartments. Thus rab7, by enhancing early to late endosome fusion, may well be acting by facilitating entry of HEL and OVA into the late endosome compartment. The RNase(4256):I-Ak epitope has been shown to be generated in early endocytic compartments, and is HLA-DM independent (11) and Ii independent (32,46), suggesting that delivery of this epitope to the MIIC might be irrelevant to its processing. In TA3 B lymphomas we found that presentation of this epitope was resistant to inhibition by NH4Cl treatment, confirming that a large fraction of processing of RNase can take place independently of a highly acidic environment. However, a fraction of RNase did show NH4Cl sensitivity at early time points, the same time points where effects of rab7 overexpression are observed. Thus while the bulk of RNase appears to be processed in an NH4Cl-independent manner, likely in early endosomes, at earlier time points one can detect a small NH4Cl-dependent processing step whose kinetics of presentation can be enhanced by rab7. The early endosome, NH4Cl-independent processing of RNase is not detected until 2 h, likely because peptide loading is slower in the early endosomes than in late endosomes. The RNase epitope may be destroyed at longer times in the late endosome, hence the switch from NH4Cl-dependent to -independent processing over time. These data are consistent with the loading of MHC II with peptide being more efficient in the later endocytic compartments of B cells. This may be due to the higher levels of HLA-DM and/or more efficient release of Ii and its processing intermediates in this compartment.
In conclusion, our studies show that rab7 is expressed at low levels in resting B cells when compared to myeloid APC. However, rab7 can be up-regulated in B cells by CD40L or LPS and this enhanced rab7 expression correlates with an increased rate of MHC II-restricted antigen presentation in B cells. In support of this, overexpression of rab7 in a B cell line results in enhanced antigen presentation for every antigenMHC II combination examined. These results highlight the importance of rab7 and the late endosome compartment in the antigen-processing and/or MHC II-loading process.
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Acknowledgments |
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Abbreviations |
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APC antigen-presenting cell |
CD40L CD40 ligand |
CIIV class II-containing vesicle |
CLIP class II-associated invariant peptide |
DC dendritic cell |
HEL hen egg lysozyme |
Ii invariant chain |
MIIC MHC class II compartment |
OVA ovalbumin |
TGN trans-Golgi network |
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Notes |
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Transmitting editor: H. Ploegh
Received 17 August 2001, accepted 3 December 2001.
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References |
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