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International Immunology, Vol. 14, No. 3, 309-318, March 2002
© 2002 Japanese Society for Immunology

Overexpression of rab7 enhances the kinetics of antigen processing and presentation with MHC class II molecules in B cells

Edward M. Bertram, Robert G. Hawley1, and Tania H. Watts

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


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
rab7 is an intracellular GTPase involved in early to late endosome fusion. By overexpressing rab7 in a B lymphoma we show that the rate of antigen presentation with MHC class II molecules is increased for four different peptide–MHC combinations, under conditions where levels of other components of the antigen-processing pathway remained constant. Resting B cells were shown to express significantly lower levels of rab7 when compared to adherent macrophages or to `immature' or `mature' dendritic cells. rab7 expression was up-regulated by stimulation of B cells with lipopolysaccharide or CD40 ligand. Other components of the endocytic pathway were also up-regulated in activated B cells, suggesting that B cell activation leads to a general enlargement of the endocytic compartment, correlating with the increased ability of activated B cells to process antigen. Taken together, our results suggest that rab7 levels regulate the rate of antigen presentation in B cells, and that rab7 and late endocytic compartments are important in MHC class II-restricted antigen presentation in B cells.

Keywords: CD40 ligand, dendritic cells, endocytic pathway, GTPase


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
MHC class II molecules present peptides on the surface of antigen-presenting cells (APC) to CD4+ T cells (1). These peptides are usually derived from exogenous proteins which are internalized into the endocytic pathway, unfolded and cleaved into small fragments during transport through increasingly acidic endocytic vesicles. Newly synthesized {alpha}ß MHC II molecules are assembled in the endoplasmic reticulum around an invariant chain (Ii) trimer, to form a nonameric complex. The Ii targets this complex to the endocytic pathway. In the late endosomal compartment, Ii is removed by proteolysis leaving a small fragment, class II-associated invariant peptide (CLIP), remaining in the binding groove of the MHC II. CLIP peptide is then exchanged for antigenic peptide before transport of the complex to the cell surface. This peptide exchange process is facilitated by HLA-DM (2). In B cells, the activity of HLA-DM is modulated by HLA-DO, another member of the MHC II family found in endosomal compartments (3).

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 (16–19).

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-{gamma} 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.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Cell lines, antibodies and reagents
The H-2dxH-2k B lymphoma TA3 (23) and the I-Ak-restricted hen egg lysozyme (HEL) (46–61)-specific T cell hybrid A2.A2 (24) were obtained from Dr L. Glimcher (Harvard University, Cambridge, MA). The I-Ak-restricted HEL(34–45)-specific T cell hybrid A6.A2 (25) and I-Ak-restricted RNase(42–56)-specific T cell hybrid TS.12 (26) was provided by Dr P. Allen (Washington University, St Louis, MO). The I-Ad-restricted ovalbumin (OVA) (323–339)-specific T hybrid DO.11.10 (27) was obtained from Dr P. Marrack and Dr J. Kappler (National Jewish Hospital, Denver, CO). The hybridomas N22, MKD6, 10-2.16 (anti-MHC II), RA3-6B2 (anti-B220), TIB-128 (anti-CD11b), N418 (anti-CD11c), GL-1 (anti-CD86), GK1.5 (anti-CD4) and 53.6.72 (anti-CD8), and the IL-2-dependent line CTLL were obtained from ATCC (Rockville, MD). All cells were grown in RPMI supplemented with 10% FCS, 2-mercaptoethanol and antibiotics. Anti-Thy1.2 antibody was obtained from Cedarlane (Hornby, Ontario, Canada). Anti-ß-actin antibody was obtained from Sigma (St Louis, MO). Anti-lamp1 and anti-transferrin receptor were obtained from BD-PharMingen (San Diego, CA). Polyclonal rabbit antibodies were made against synthetic peptides of rab7 (KQETEVELYNEFPEPIK) (14), rab5 (PKNEPQNPGANSARGR) (14) and H-2Mß (TPLPGSNYSEGWHIS) (28), covalently coupled to keyhole limpet hemocyanin (Sigma). Antibodies were purified by Protein G–Sepharose.

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 18–24 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 A–Sepharose (Sigma) before the addition of skim milk (5% final). Lysates were incubated with anti-MHC II (N22) bound to Protein A–Sepharose, rotating overnight at 4°C. The immunoprecipitates were washed 4 times in lysis buffer and run on 12% SDS–PAGE 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 Tris–HCl, 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% SDS–PAGE 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 CD40L–murine 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.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Overexpression of rab7 in a B cell lymphoma
To analyze the effects of overexpression of rab7 on antigen processing and presentation, the murine TA3 B lymphoma (H-2kxd) was infected with a retrovirus expressing the rab7 cDNA on a bicistronic transcript with EGFP. These cells (TA3-Rab7) were shown to express between 2.5- and 3.5-fold higher levels of rab7 protein (as measured in four separate experiments) when compared to the parent and control vector infected TA3 B lymphoma cells by Western blot analysis (Fig. 1AGo). Similarly TA3 cells were infected with the same vector not expressing the rab7 gene (TA3-vector control) which showed the same levels of rab7 expression as the parental TA3 cells (Fig. 1AGo). TA3-Rab7 cells were similar in size and had the same growth rate as the TA3 cells, and expressed the same levels of surface MHC II and co-stimulatory molecules CD86 and 4-1BBL as assessed by flow cytometry (data not shown). Overexpression of rab7 did not change the total levels of expression of MHC II, or late endosomal proteins lamp1, H-2M (Fig. 1B and CGo) or the early endosomal proteins rab5 (Fig. 1BGo) and transferrin receptor (Fig. 1CGo).



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  		Fig. 1. Overexpression of rab7 in TA3 B lymphomas. Western blot of cell lysates of TA3 and TA3-Rab7 B lymphomas stained with anti-Rab7 with detection by chemiluminescence (A). Western blot of TA3 and TA3-Rab7 with anti-I-Aßk (MHC II), Lamp1, H-2Mß and rab5 (B). Intracellular staining of TA3 (thin line) and TA3-Rab7 (thick line) B lymphomas stained with antibodies to rab7, H-2M, transferrin receptor (TfR), lamp1, I-Aßd and I-Aßk (C).

 
rab7 overexpression enhances the rate of antigen presentation
To examine the effect of rab7 overexpression on antigen presentation, TA3 and TA3-Rab7 were incubated with various antigens (HEL, OVA and RNase) and the cells fixed after various times. Comparison of the rate of expression of specific antigenic peptides in the context of MHC II on the cell surface between the two cell lines was made by measuring their ability to stimulate the appropriate T cell hybrids. Figure 2Go(A) shows that TA3-Rab7, when pulsed with HEL protein, was able to present the HEL(46–61):I-Ak epitope at increased rates compared to TA3. The difference in the kinetics between TA3 and TA3-Rab7 was most marked at 30 min: later time points showed that both TA3 and TA3-Rab7 had reached maximal stimulation of A2.A2 T cells. The response was dose dependent: at lower doses of HEL the overall kinetics of A2.A2 T cell activation were slowed; however, in all cases TA3-Rab7 showed an increased rate of presentation over TA3 cells (Fig. 2B and CGo). Fixed samples of both TA3 and TA3-Rab7 from each time point, when incubated with HEL(46–61) peptide, were able to stimulate A2.A2 T cells equally, confirming that the difference in the rate of T cell stimulation seen between TA3 and TA3-Rab7 was in the intracellular processing of HEL (Fig. 2DGo). TA3 and TA3-vector control showed the same kinetics of antigen processing and presentation, demonstrating that the increased kinetics of TA3-Rab7 was due to the overexpression of rab7 (data not shown).



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  		Fig. 2. Kinetics of HEL processing and presentation by TA3 and TA3-Rab7 B lymphomas. TA3 ({square}) and TA3-Rab7 ({blacksquare}) were pulsed with different HEL concentrations. At the time points shown, samples of these cells were fixed and used to stimulate HEL(46–61):I-Ak-specific A2.A2 (A–C) and HEL(34–45):I-Ak-specific A6.A2 (E–G) T cell hybrids overnight. IL-2 production, corresponding to T cell activation was assayed as described in Methods. As controls, cells were pulsed with HEL(46–61) peptide (D) and also used in an alloresponse with H-2d-reactive 2H40 T cell hybrids (H).

 
Similarly, TA3-Rab7 cells were able to process and present the HEL(34–45):I-Ak epitope at increased rates compared to the TA3 parental line (Fig. 2EGo), although the kinetic effects for this epitope were less pronounced than for the HEL(46–61):I-Ak epitope. This response was also seen to be dose dependent (Fig. 2F and GGo). At all time points shown, TA3 and TA3-Rab7 were equally proficient in the stimulation of the alloreactive T cell hybridoma 2H40 (Fig. 2HGo).

The HEL(46–61):I-Ak epitope has been previously characterized as being processed in late endocytic compartments. In contrast, the RNase(42–56):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 3Go(A) shows that overexpression of rab7 also increased the rate of presentation of RNase(42–56):I-Ak. As seen for HEL protein, TA3-Rab7 when pulsed with intact RNase were able to process and present the RNase(42–56): I-AK epitope to TS.12 T cell hybrids at increased rates compared to TA3.



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  		Fig. 3. Kinetics of RNase and OVA processing and presentation by and TA3-Rab7 B lymphomas. TA3 ({square}) and TA3-Rab7 ({blacksquare}) were pulsed with either RNase or OVA. At the time points shown samples of these cells were fixed and used to stimulate RNase(42–56):I-Ak-specific TS.12 (A) or OVA(323–339):I-Ad-specific DO.11.10 (B) T cell hybrids overnight. IL-2 production, corresponding to T cell activation was assayed as described in Methods.

 
Figure 3Go(B) shows that the effect of rab7 overexpression on antigen presentation by TA3 cells was not specific to I-Ak molecules. TA3 cells incubated with native OVA process and present OVA(323–339):I-Ad quite poorly to DO.11.10 T cell hybrids. It can be seen that even by 360 min very few OVA(323–339):I-Ad epitopes have been produced. In contrast, TA3-Rab7 pulsed with OVA were substantially more efficient at OVA presentation at all time points where significant presentation was detected.

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(42–56): I-Ak, it was important to confirm the previous findings on the pH dependence of antigen processing in the TA3 cells. Figure 4Go shows that NH4Cl treatment of TA3 significantly prevented production of the HEL(46–61):I-Ak epitope (Fig. 4AGo). The same was true in TA3 cells overexpressing rab7 (Figure 4BGo). NH4Cl treatment of TA3 cells did not prevent presentation of RNase(42–56):I-Ak (Fig. 4CGo). 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. 4CGo). 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(46–61) peptides by TA3 to A2.A2 T cells (Fig. 4EGo).



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  		Fig. 4. Effect of NH4Cl treatment on the kinetics of antigen processing and presentation of TA3 B lymphomas. TA3 and TA3-Rab7 cells were cultured alone ({square}) or with 10 mM NH4Cl ({blacksquare}) and pulsed with either HEL or RNase. At the time points shown samples of these cells were fixed and used to stimulate HEL(46–61):I-Ak-specific A2.A2 (A and B) and RNase(42–56):I-Ak-specific TS.12 (C and D) T cell hybrids overnight. IL-2 production, corresponding to T cell activation was assayed as described in Methods. As a control, cells were pulsed with HEL(46–61) peptide at each time point (E) and used to stimulate A2.A2 T cell hybrids.

 
Enhanced expression of rab7 could influence the kinetics of appearance of antigen–MHC II complexes on the cell surface by increasing the rate of antigen delivery to late endocytic compartments, thereby increasing the rate of generation of antigenic peptide fragments. Additionally, increased rab7 could increase the rate of delivery of the MHC II–Ii precursor to the MIIC. However, TA3 and TA3-Rab7 were shown to degrade [125I]OVA at similar rates (data not shown). We also measured the rate of movement of horseradish peroxidase through the endocytic pathway by subcellular fractionation of TA3 and TA3-Rab7 cells, and also failed to see an overall difference in traffic through the endocytic pathway (data not shown).

To test whether increased rates of transport of newly formed MHC II–{alpha}ß–Ii 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 SDS–PAGE (Fig. 5Go). 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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  		Fig. 5. Rates of Ii processing by TA3 and TA3-Rab7 B lymphomas. Cells were pulsed with [35S]methionine for 20 min and chased for the times indicated before immunoprecipitation with anti-MHC II antibody. Samples were boiled in reducing conditions before loading on 12% SDS–PAGE.

 
To measure the rate of appearance of newly synthesized MHC II at the cell surface, we used [35S]methionine-labeled cells followed by cell surface biotinylation during the chase followed by sequential double immunoprecipitation (anti-MHC II, followed by streptavidin). Again we failed to see a difference between TA3 and TA3-Rab7 cells (data not shown). The inability to see a clear difference in the overall rates of total protein degradation and movement through the endocytic pathway or in traffic back to the cell surface between TA3 and TA3-Rab7 is likely due to the insensitivity and lack of specificity of bulk biochemical assays compared to the sensitivity of the functional T cell assays. T cells are sensitive to peptide–MHC II complexes at the cell surface at <200 copies/cell (34). Thus the change in absolute number of MHC–peptide complexes at the surface which gives rise to a change in kinetics of the T cell functional assay may be too small to be discriminated by the other approaches used here.

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 6AGo). 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. 6BGo). 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 FITC–dextran, whereas the `immature' DC population expresses lower levels of both MHC II and CD86 at the cell surface and shows high uptake of FITC–dextran. 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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  		Fig. 6. rab7 expression differs in different APC and is up-regulated in activated B cells. Western blot of cell lysates of different APC and T cells. Blots were stained with either anti-Rab7 antibody or anti-ß-actin antibody and detected by chemiluminescence (A). Western blot of B cells that were stimulated with either CD40L, LPS or a combination of both for the times indicated. Blots were stained with anti-Rab7 and anti-ß-actin antibody (C). Western blots of B cells that were stimulated with CD40L and LPS. Blots were stained with anti-Lamp1, anti-H-2Mß, anti-Rab5 and anti-ß-actin antibody.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
rab7 is important for transport of proteins from early to late endosomal compartments in eukaryotic cells (16–19). To date, its role in APC has not been examined. In this study we found that overexpression of rab7 alone could increase the kinetics of antigen presentation in a B cell lymphoma line for several different MHC II–peptide combinations, in particular the poorly processed OVA(323–339) epitope presented with I-Ad. Interestingly, we found that the levels of rab7 are strikingly different between myeloid APC and B cells. Furthermore, the level of rab7 in B cells was found to be significantly up-regulated by agents known to stimulate B cell activation (CD40L and LPS). These data, combined with the observation that resting B cells show slower kinetics of antigen presentation compared to activated B cells (35,36), suggest that rab7 expression is limiting in resting B cells and that up-regulation of rab7 together with other components of the endocytic pathway contributes to enhanced antigen presentation by activated versus resting B cells. Although in normal B cells increases in rab7 upon activation were accompanied by increases in the levels of other components of the endocytic pathway, in the TA3 B lymphoma overexpression of rab7 did not change the levels of other components of the MHC II-processing pathway and the endocytic pathway (Fig. 1BGo). Thus it appears that rab7 levels alone can influence the rate of antigen processing and presentation in a B cell line.

Activated B cells have previously been shown to present MHC II–antigen 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 (37–39). 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 II–peptide 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 (35,40–42). Optimal T cell activation can occur within 60 min for adherent macrophages incubated with antigen compared with 6–8 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 II–antigen 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 II–Ii 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 II–Ii complex (Fig. 5Go). 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 II–antigen combinations. The effects of increased rab7 expression were most striking for OVA(323–339):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. 6BGo) 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 OVA323–339 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(46–61):I-Ak (10,11,44) and OVA(323–339):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(42–56):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 antigen–MHC 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.


   Acknowledgments
 
We thank M. Zerial for dog rab7 cDNA, M. D. Goldstein, B. Ghumman, W. Dawicki and I. C. Boulton for technical assistance, and J. L. Cannons for reading this manuscript. This work was supported by a grant from the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to T. H. W.)


   Abbreviations
 
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

   Notes
 
1 Present address: Hematopoiesis Department, Holland Laboratory, American Red Cross, Rockville, MD 20855, USA Back

Transmitting editor: H. Ploegh

Received 17 August 2001, accepted 3 December 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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