The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Forquet, F.
Right arrow Articles by Davoust, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Forquet, F.
Right arrow Articles by Davoust, J.
The Journal of Immunology, 1999, 162: 3408-3416.
Copyright © 1999 by The American Association of Immunologists

Presentation of Antigens Internalized Through the B Cell Receptor Requires Newly Synthesized MHC Class II Molecules1

Frédérique Forquet2, Nicolas Barois, Patrick Machy, Jeannine Trucy, Valérie S. Zimmermann, Lee Leserman and Jean Davoust

Centre d’Immunologie de Marseille-Luminy, 13288 Marseille cedex 9, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Exogenous Ags taken up from the fluid phase can be presented by both newly synthesized and recycling MHC class II molecules. However, the presentation of Ags internalized through the B cell receptor (BCR) has not been characterized with respect to whether the class II molecules with which they become associated are newly synthesized or recycling. We show that the presentation of Ag taken up by the BCR requires protein synthesis in splenic B cells and in B lymphoma cells. Using B cells transfected with full-length I-Ak molecules or molecules truncated in cytoplasmic domains of their {alpha}- or ß-chains, we further show that when an Ag is internalized by the BCR, the cytoplasmic tails of class II molecules differentially control the presentation of antigenic peptides to specific T cells depending upon the importance of proteolytic processing in the production of that peptide. Integrity of the cytoplasmic tail of the I-Ak ß-chain is required for the presentation of the hen egg lysozyme determinant (46–61) following BCR internalization, but that dependence is not seen for the (34–45) determinant derived from the same protein. The tail of the ß-chain is also of importance for the dissociation of invariant chain fragments from class II molecules. Our results demonstrate that Ags internalized through the BCR are targeted to compartments containing newly synthesized class II molecules and that the tails of class II ß-chains control the loading of determinants produced after extensive Ag processing.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Antigens taken up in APCs via receptor-mediated endocytosis or via the fluid phase gain access to various intracellular compartments that condition their degradation and presentation. Peptides derived from the Ag can be loaded on either newly synthesized or recycling MHC class II molecules. In addition, class II peptide-loading compartments may vary depending upon cell type 1, 2, 3 and cell-specific differences in the trafficking of class II molecules. In B cells, the peptide loading of newly synthesized class II molecules requires coexpression of the invariant chain (Ii),3 H2-M, and H2-O molecules 4, 5, 6, 7 . Newly formed class II molecules are unable to bind peptides until Ii is degraded and the Ii-class II-associated Ii peptide (CLIP) fragment is dissociated from their binding groove 8 . This dissociation is catalyzed by H2-M molecules 6, 9, 10 .

The differential sensitivity of class II Ag presentation to protein synthesis inhibitors, Ii, and H2-M expression that was reported for a collection of antigenic determinants suggests the existence of multiple processing pathways 11, 12, 13, 14, 15 . In parallel with a classical pathway requiring newly synthesized class II molecules, an alternative pathway of presentation involves mature class II molecules that are expressed at the cell surface; these mature molecules recycle through endosomes, where they become available for peptide binding 16, 17 . Peptide loading of newly synthesized and mature class II molecules occurs in distinct endocytic compartments with different intracellular pH and proteolytic activities 18, 19 . Within an Ag, buried or cryptic determinants are usually presented by the classical Ii-dependent pathway involving newly synthesized class II molecules, whereas more superficial determinants can be loaded onto class II molecules recycled from the cell surface independently of Ii 18 .

In addition to the differential effects of inhibitors of protein synthesis, depending upon the Ag, APCs transfected with class II molecules lacking the cytoplasmic domain of the ß-chain have also been shown to be defective in the presentation of some but not all antigenic determinants 20, 21, 22, 23 . Truncation of the class II {alpha} and ß cytoplasmic tails interferes with endocytosis of class II molecules and with the Ii-independent presentation of some protein determinants. This finding is consistent with the involvement of class II recycling during the presentation of these peptides 24, 25 . The studies described above are derived from a diverse group of Ags that are presented by multiple cell types. In addition, the role of Ii and of class II cytoplasmic domains in Ag presentation has been analyzed using Ags internalized via pinocytosis only; no information is available on Ags targeted through the B cell receptor (BCR). Ag-specific B cells present determinants derived from cognate ligands to MHC class II-restricted CD4+ T cells at concentrations far below those required for nonspecific B cells 26 . BCR-mediated Ag processing and presentation are highly efficient as a consequence of the linked roles of the BCR with regard to the delivery of Ag to the class II peptide-loading compartment and to signaling. Signals transduced through the BCR induce changes in the intracellular localization of class II molecules and affect the Ag-presentation pathway 27, 28 . Because Ags targeted through the BCR follow an intracellular pathway that is distinct from that of Ags internalized from the fluid phase 29 , it was of interest to define the effects of inhibitors of protein synthesis and the role of class II cytoplasmic domains in the presentation of such Ags.

Our results show that when Ag is taken up via the BCR, the resulting peptides, including the so called Ii-independent peptides, are presented by newly synthesized class II molecules. The differential requirement for MHC class II cytoplasmic domains for the presentation of certain Ag-derived peptides also depends upon whether Ags are endocytosed via pinocytosis or via the BCR. The cytoplasmic tail of class II ß-chain is required for the targeting of class II molecules to compartments in which Ii-dependent antigenic determinants are loaded after BCR-mediated Ag uptake.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and cell lines

We used CBA/J mice that were transgenic for rearranged Ig heavy and light chain genes encoding a high-affinity anti-hen egg lysozyme (HEL) Ab 30, 31 . B cells from these transgenic (Tg) mice express this BCR and secrete an Ab specific for HEL. Splenic B cells were purified from these Tg mice or from CBA/J control mice after T depletion using anti-Thy-1, anti-CD4, and anti-CD8 Abs 32 plus complement; afterward, these cells were immediately used in Ag-presentation assays.

The I-A-negative B lymphoma cell line M12C3 33 was transfected with genomic DNA encoding either intact A{alpha}k and Aßk chains or those I-Ak chains with truncated cytoplasmic domains 21 . The transfectants, which were generously provided by W. Wade (Department of Microbiology, Dartmouth Medical School, Lebanon, NH), have the following MHC class II phenotypes: {alpha}wt/ßwt (I-Ak), {alpha}Cyt-12/ßwt ({alpha} cytoplasmic domain-truncated I-Ak molecule ({alpha}T)), and {alpha}wt/ßCyt-18 (ß cytoplasmic domain-truncated I-Ak molecule (ßT)). Truncation of the {alpha} or ß cytoplasmic domains was verified by blotting total lysates with Abs specific for MHC class II {alpha} or ß cytoplasmic domains (data not shown).

The I-Ak-restricted, HEL-specific T cell hybridomas 3A9 and 3B11 were generously provided by L. Adorini (Roche Milano Ricerche, Milan, Italy). The 3A9 T cells recognize an Ii-dependent determinant present on the 46–61 peptide 34, 35 , and the 3B11 hybridoma recognizes the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 peptide independent of Ii 14 . The TS12 T cell hybridoma 36 that was also used in Ag-presentation assays is I-Ak-restricted but specific for the RNase A (43–56) peptide. IL-2-dependent CTLL-2 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, VA). Cell lines were cultured in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS, 20 µM 2-ME, 1 mM sodium pyruvate, and 1 mM glutamine.

Abs and reagents

The mouse hybridoma 10.2.16 producing an I-Ak-specific IgG2a mAb and the anti-mouse Fc{gamma}RII/III 2.4G2 mAb were obtained from ATCC. The IgG1 mAb HyHEL-10 recognizes an epitope on intact HEL 37 . The affinity-purified rabbit polyclonal Ab specific for the cation-independent mannose 6 phosphate receptor (M6PR) (produced by B. Hoflack, Pasteur Institute, Lille, France) was a generous gift of S. Meresse (Centre d’Immunologie de Marseille-Luminy). The rabbit polyclonal anti-Ii or anti-H-2Mß Abs were raised against synthetic peptides corresponding to sequences in the cytoplasmic domains of mouse Ii ({alpha}Cyt.Ii) or H-2Mß ({alpha}Cyt.Mß) 27, 38 . Rabbit polyclonal anti-CLIP serum ({alpha}CLIP) was produced following injection with a peptide corresponding to the mouse Ii (80–104) sequence 6 . The secondary reagents (donkey anti-mouse Igs, anti-rabbit Igs, and streptavidin) coupled to FITC or Texas Red that were suitable for flow cytometry and immunofluorescence as well as unlabeled rabbit anti-mouse IgG (RAMIG) to bind the BCR were purchased from Jackson ImmunoResearch (West Grove, PA). Human transferrin (Tf) (Sigma, St. Louis, MO) was conjugated to FITC and purified on a G25 column. HEL and RNase A were obtained from Sigma.

Liposomes

Liposomes (40 µM total lipids) were composed of 65 mol% dimyristoyl phosphatidyl choline (Sigma), 35 mol% cholesterol (Sigma), and 1 mol% phosphatidylethanolamine derivatized with the heterobifunctional cross-linking agent N-succinimidyl-3-(2-pyridyldithio)propionate, as described previously in detail 39 . These liposomes were prepared in a solution containing 10 mM carboxyfluorescein (Molecular Probes, Eugene, OR), a fluorescent marker, and 5 mg/ml HEL or RNase A in PBS. They were frozen and thawed five times and passed through polycarbonate filters (Nucleopore, Pleasanton, CA) of 80-nm pore size using an Extruder (Lipex Biomembranes, Vancouver, Canada). Liposomes were extruded 10 times until a clear solution was obtained. Unencapsulated material was removed by gel filtration over Sepharose 4B columns. Because surface expression of IgG was comparable (data not shown) between the different transfectants, we used IgG as a target for the Ag encapsulated in liposomes. Liposome-protein A (Lip-PA) were covalently coupled to N-succinimidyl-3-(2-pyridyldithio)propionate-modified (10 mol/mol) protein A from Streptococcus aureus (Pharmacia, Uppsala, Sweden). When incubated in the presence of 10 µg/ml of RAMIG, which binds to protein A, the Lip-PA preparation targets the Ag through the BCR. Liposomes were sterilized by filtration through 0.45-µm Acrodisc filters (Gelman Sciences, Ann Arbor, MI). The concentration of HEL or RNase A was determined by the known molar ratios of these proteins to the coencapsulated fluorescent marker. Liposomes of this size are known to be taken up in coated pits in lymphoid cells 32 .

Ag presentation

A total of 5 x 104 APCs were incubated with or without various doses of Ags diluted from a stock solution of free Ag or Lip-PAs containing encapsulated Ag at 5 mg/ml that were targeted with RAMIG or left untargeted. The APCs were cocultured for 24 h at 37°C with 105 T cell hybridomas. Inhibition of the protein synthesis by cycloheximide (CHX) was realized as follows: splenic B cells and I-Ak-expressing B lymphoma cells were incubated with or without 100 µM or 200 µM of CHX, respectively, for 2 h before adding the different preparations of Ags. Cells pulsed with the Ags for 4 h were washed and subsequently fixed for 30 s with 0.05% glutaraldehyde. A total of 5 x 104 fixed APCs were cocultured for 24 h at 37°C with 105 T cells. IL-2 production in culture supernatants was measured using thiazolyl blue (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma) to evaluate the growth of the IL-2-dependent CTLL-2 cell line 40 .

SDS-PAGE of surface class II complexes

A total of 15 x 106 cells were labeled by lactoperoxidase-catalyzed iodination and lysed in lysis buffer (1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and 50 mM Tris-HCl (pH 7.5)) containing protease inhibitors. Lysates were precleared with protein A-Sepharose beads (Pharmacia) and I-Ak immunoprecipitated with 10.2.16 mAb. Immunoprecipitates were extensively washed. Before electrophoresis on an SDS-polyacrylamide (12.5%) gel, the immunoprecipitated material was divided into two aliquots: the first aliquot was fully denatured at 95°C for 5 min (B); the second aliquot was incubated for 1 h at room temperature (NB) in SDS sample buffer containing 5% 2-ME to preserve the peptide-loaded, compact, MHC class II heterodimers (compact forms (CFs)) 27 .

Western blotting

Washed cells in ice-cold PBS were solubilized in 1 ml of lysis buffer and incubated for 2 h with 10.2.16 mAb bound to the protein A-Sepharose beads. Next, immunoprecipitates were washed and pellets were suspended in reducing sample buffer. Immunoprecipitates were boiled in SDS and run on 15% SDS-PAGE. After transfer onto an Immobilon-P membrane (Millipore, Bedford, MA), proteins were blotted with {alpha}Cyt.Ii or {alpha}CLIP Abs. After washing, membranes were incubated with anti-rabbit Ab conjugated to horseradish peroxidase (Jackson ImmunoResearch). Labeled proteins were detected using the enhanced chemiluminescence immunodetection kit (Amersham).

Flow cytometry and immunofluorescence

Staining for flow cytometry analysis was performed as follows: 1.5 x 105 cells/sample were incubated for 30 min on ice with fluorescent HEL-Lip-PAs in the presence or absence of RAMIG (10 µg/ml), or with 10.2.16 mAbs (1 µg/ml) in PBS containing 5% FCS and 0.02% NaN3 (FACS buffer) with 20% 2.4G2 mAb supernatant and 5% normal mouse serum to prevent binding to Fc{gamma}RII/III. After washing in FACS buffer, cells labeled with nonfluorescent 10.2.16 Ab were incubated for 30 min on ice with the FITC-coupled secondary Ab. Before analysis, cells were washed extensively in FACS buffer and fixed with 1% paraformaldehyde in PBS. Samples were analyzed using a FACScan apparatus (Becton Dickinson, San Jose, CA).

Internalization of the HEL encapsulated in liposomes and determination of the expression of class II molecules and Ii in different cellular compartments were performed by confocal immunofluorescence microscopy as described previously 27 . To demonstrate HEL internalization, we incubated cells with HEL-Lip-PAs in the presence of RAMIG for 4 h prior to immunofluorescence as described above. To label early endosomal compartments, FITC-coupled Tf was exposed to the cells for 20 min at 37°C before the fixation and labeling of other internal molecules. Confluent cells that had been treated with liposomes or Tf or left untreated and cultured on glass coverslips were fixed with 4% paraformaldehyde, permeabilized with 0.05% saponin in PBS, and incubated for 30 min with primary Abs. Under these conditions, the liposome-associated carboxyfluorescein is released and does not interfere with the detection of fluorescent Abs. After washing, the cells were incubated for 30 min with secondary labeled Abs and then washed. The coverslips were mounted onto glass sides with Mowiol. Confocal microscopy was performed using the TCS 4D confocal laser scanning microscope (Leica Lasertechnik, Heidelberg, Germany) interfaced with an argon/krypton ion laser.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Presentation of Ag taken up by BCR is sensitive to CHX

To determine whether the presentation of Ags targeted through the BCR is sensitive to protein synthesis inhibitors, we compared the presentation efficiency of HEL by splenic B cells that expressed or did not express a HEL-specific BCR with several HEL-specific T cell hybridomas in the presence or absence of CHX. Splenic B cells were isolated from either CBA/J control mice or Tg mice carrying genes encoding a high-affinity anti-HEL Ab 30, 31 . The presentation of the 46–61 determinant derived from extensive proteolysis of soluble HEL to the I-Ak-restricted 3A9 T cell hybridoma requires newly synthesized class II molecules and Ii expression 14 . Other determinants, such as the peripheral HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope, can associate with mature class II molecules and be presented to the 3B11 T hybridoma independent of protein synthesis 14 . We observe here that the presentation of soluble HEL targeted by the BCR in Tg splenic B cells was improved for the two epitopes studied compared with the presentation of HEL that was internalized nonspecifically (Fig. 1Go, A and C). The presentation of the synthetic HEL (46–61) peptide to the 3A9 hybridoma is comparable between Tg and control splenic B cells (Fig. 1GoB), which is consistent with the equivalent expression level of I-Ak and costimulatory molecules of normal and Tg splenic B cells (data not shown).



View larger version (19K):
[in this window]
[in a new window]
  		FIGURE 1. Sensitivity to CHX of the presentation of HEL that had been targeted through the BCR or untargeted in splenic B cells. Splenic B cells isolated from HEL-BCR Tg mice (circles) or control mice (squares) were used to stimulate the 3A9 (A) or the 3B11 (C) T cell hybridomas with various doses of HEL in the presence (open symbols) or absence (filled symbols) of CHX. As a control, splenic B cells that had been incubated with the HEL (46–61) peptide were used to stimulate the 3A9 T cells (B). All experiments have been repeated reproducibly at least three times.

 
Presentation of the protein HEL to the 3A9 T cell hybridoma was sensitive to CHX in a manner independent of the route of entry of HEL (Fig. 1GoA), whereas the presentation of the isolated HEL (46–61) synthetic peptide to the same T cell hybridoma by Tg or control B cells was not affected by CHX (Fig. 1GoB). However, presentation of the 34–45 determinant derived from intact HEL to the 3B11 T cells was sensitive to CHX in a manner that depended upon the pathway of HEL uptake. Despite the effects of CHX on overall protein synthesis, the fact that the differential effect of this drug was dependent upon the HEL-derived peptide or on its mode of entry into the APC indicates that there are no effects of CHX on class II molecules expressed at the cell surface or on other molecules required for the recognition of these Ags by the T cells. The effect of CHX on the presentation of certain epitopes consequently appears to result essentially from the inhibition of class II synthesis and transport, although effects on any protein in the Ag-presentation pathway cannot formally be ruled out. The 34–45 epitope can be presented in association with mature class II molecules recycled from the cell surface in a manner independent of protein synthesis, when the Ag is taken up from the fluid phase. Presentation of the 34–45 epitope became sensitive to CHX when HEL was targeted via the BCR (Fig. 1GoC). This result indicates that the targeting of an Ag via the BCR forces its presentation by newly synthesized class II molecules.

Binding, internalization, and presentation of liposome-encapsulated Ags in B cell lines

We extended our observations to B lymphoma cell lines to evaluate the presentation of particulate Ags targeted through the BCR by intact or truncated MHC class II molecules. The cell lines used express surface IgG, which is not specific for HEL. To evaluate the role of surface Ig in the presentation of HEL, we encapsulated HEL in liposomes that were covalently coupled to protein A. Lip-PAs were targeted to B cell surface IgG in the presence of RAMIG, an Ab which binds both protein A and surface IgG. In the present study, the Ag itself is not complexed to an Ab. The use of targeted liposomes in which the Ags are passively entrapped has the advantage of facilitating the uptake of Ag without affecting its processing, as for cross-linked Ags and immune complexes 19, 41, 42 . To analyze the liposome-targeting efficiency, we initially used FACS analysis to compare the binding of protein A-bearing, HEL containing liposomes (HEL-Lip-PAs) with B cells in the presence or absence of RAMIG (Fig. 2GoA). As expected, B cells were only able to bind the targeted liposomes. Second, we determined whether targeted HEL-Lip-PAs are internalized in B cells. The intracellular localization of HEL internalized by BCR was performed by confocal microscopy and immunofluorescence labeling of the Ag using an anti-HEL mAb. The Ag encapsulated in liposomes targeted to the BCR accumulated in large intracellular structures after 4 h (Fig. 2GoB). Intact Ag (revealed with the HyHEL-10 mAb) did not appear to colocalize with the MHC class II-enriched compartment that was induced in response to B cell activation 27 .



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 2. Targeting to the BCR of liposome-encapsulated HEL in B lymphoma cells. A, FACS analysis of the binding of HEL-Lip-PAs on I-Ak-expressing APCs after targeting through the BCR with (solid line) or without (dotted line) RAMIG. B, confocal microscopy analysis of the intracellular distribution of class II molecules labeled with 10.2.16 mAb and HEL with HyHEL-10 mAb (arrowheads) in I-Ak cells; scale bar is 6 µm. C, I-Ak-expressing B lymphoma cells were used to stimulate the 3A9 T cell hybridoma with various doses of HEL-Lip-PAs nontargeted to the BCR ({circ}) or targeted to the BCR via RAMIG (•). As a control for Fc{gamma}R binding, HEL-Lip-PAs were targeted to the BCR via RAMIG in the presence of anti-Fc{gamma}R II/III mAb ({blacksquare}). The presentation efficiency of soluble HEL is shown for comparison ({square}). Ag-presentation assays were performed on numerous occasions with irrelevant Abs in the presence of protein A-bearing liposomes. The behavior in the presence of control Abs is not different from uptake by the fluid phase (data not shown).

 
Next, we analyzed the presentation of HEL to the 3A9 T cells depending upon the targeting of HEL-Lip-PAs in B lymphoma cells (Fig. 2GoC). The HEL encapsulated in Lip-PAs targeted to the BCR via RAMIG was more efficiently presented than soluble HEL or nontargeted HEL-Lip-PAs. Incubation with anti-Fc{gamma}R mAb did not influence the BCR targeting of encapsulated HEL or its presentation, indicating that liposomes are not internalized via the Fc{gamma}R. Thus, the presentation of liposome-encapsulated Ags is augmented by BCR-mediated uptake in B lymphoma cells, as is the uptake of free Ag in B splenocytes expressing an HEL-specific BCR.

We assessed whether CHX inhibits the presentation of Ags encapsulated in liposomes targeted through the BCR (Fig. 3Go). As seen for splenic B cells, the presentation of HEL-Lip-PAs to the 3A9 T cell hybridoma was sensitive to CHX independent of its targeting by the BCR (Fig. 3GoA). The presentation by B lymphoma cells of liposome-encapsulated HEL to the 3B11 T cell hybridoma (Fig. 3GoB) was resistant to CHX in the absence of BCR targeting. The same results were obtained with soluble Ags (data not shown). However, when liposomes are targeted via the BCR, the presentation of the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope (Fig. 3GoB) became sensitive to CHX. Liposomes are present in excess relative to the number of binding sites on the cell. The excess liposomes are not removed in a washing step. Those liposomes that are not binding are capable of being taken up by fluid phase endocytosis, albeit with reduced efficiency. This result is consistent with the data presented for the 3B11 hybridoma in Fig. 1Go. The same pattern of sensitivity to CHX depending upon BCR targeting was observed for the RNase A (43–56) epitope (Fig. 3GoC). These results indicate that, as for soluble Ags bound to the BCR, the targeting of particulate Ags through the same receptor directs their presentation to newly synthesized class II molecules. The engagement of the BCR by Ag probably transports the Ag beyond the limits of the recycling pathway.



View larger version (13K):
[in this window]
[in a new window]
  		FIGURE 3. Inhibition by CHX of the presentation of liposome encapsulated Ags targeted to the BCR in B lymphoma cells. I-Ak-expressing B lymphoma cells were used to stimulate the 3A9 (A) and the 3B11 (B) T cell hybridomas with various doses of HEL-Lip-PAs nontargeted to the BCR (squares) or targeted to the BCR via RAMIG (circles) in the presence (open symbols) or absence (filled symbols) of CHX. We also tested the ability of these cells to stimulate the TS12 T cell hybridoma in the presence of RNase A-Lip-PAs under the same conditions (C). Results are representative of three experiments.

 
Effect of class II cytoplasmic tail truncation on Ag presentation

We further analyzed whether the integrity of class II molecules affects the presentation of BCR ligands. We used B lymphoma cell lines expressing either full-length I-Ak class II molecules as described above or I-Ak class II molecules lacking the cytoplasmic domains of the {alpha}-chain ({alpha}T) or the ß-chain (ßT). FACS analysis revealed that the binding of IgG targeted-liposomes to cells expressing intact I-Ak molecules or {alpha}T and ßT class II molecules was comparable; confocal microscopy indicated that HEL-loaded liposomes were internalized in all transfectants (data not shown). Using HEL-Lip-PAs internalized by pinocytosis in the absence of RAMIG, we repeatedly observed that B cells bearing intact I-Ak class II molecules present the 46–61 peptide more efficiently than transfectants expressing I-Ak lacking either cytoplasmic domain (Fig. 4GoA). We also noticed that {alpha}T cells are slightly more potent APCs for the HEL (46–61) epitope than ßT transfectants in the absence of Ag targeting. The presentation of HEL (46–61) by full-length and {alpha}T molecules was improved by targeting the liposomes to the BCR (Fig. 4GoB). However, despite the equivalent ability of class II molecules lacking their ß cytoplasmic domain to present the synthetic HEL (46–61) peptide (Fig. 4GoC), ßT molecules were unable to present the same determinant following BCR targeting of the intact Ag, indicating that truncation of class II ß-chain affects the presentation of an epitope localized in the protein core.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 4. Influence of BCR targeting on Ag presentation in APCs expressing cytoplasmic domain-truncated I-Ak molecules. I-Ak-expressing control ({square}), {alpha}T ({circ}), and ßT ({triangleup}) B cells were used to stimulate the 3A9 (A, B, and C) or the 3B11 (D and E) T cell hybridomas with serial dilutions of nontargeted HEL-Lip-PAs (A and D), HEL-Lip-PAs targeted through the BCR via RAMIG (B and E), or HEL (46–61) peptide (C). Results are representative of five experiments for 3A9 and three experiments for 3B11.

 
In contrast to the HEL (46–61) determinant, which is uniquely presented by newly synthesized class II molecules, the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope can associate with mature I-Ak class II molecules and be presented to the 3B11 T hybridoma in the alternative pathway (Fig. 4Go, D and E). As shown previously for free Ags in murine cells 25 , we confirm here using particulate Ags that this alternative presentation pathway depends upon the integrity of the {alpha} and ß class II cytoplasmic domains. Murine B cells expressing intact I-Ak class II molecules present proteins internalized via nonspecific pinocytosis more efficiently than transfectants expressing I-Ak lacking either of the two cytoplasmic domains (Fig. 4GoD). The targeting of encapsulated HEL via the BCR improved the presentation efficiency of all transfectants and was able to restore the presentation of the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope by cytoplasmic domain truncation mutants of class II molecules to a level that was comparable with that of intact I-Ak molecules (Fig. 4GoE). Therefore, BCR-targeted Ags can reach intracellular class II molecules that are not accessible to peptides derived from fluid phase Ags.

Defects in peptide loading and intracellular distribution of cytoplasmic truncation mutants of I-Ak molecules

To complement our Ag presentation results with MHC class II surface expression and peptide-loading ability in the different B cell transfectants, we performed FACS analysis, immunoprecipitation of surface-iodinated class II molecules, and Western blotting of the MHC class II-associated Ii. Comparable surface expression of I-Ak class II molecules was detected on the different transfectants (Fig. 5GoA). However, a marked reduction in the proportion of endogenous I-Ak CFs was revealed with surface iodination in {alpha}T B cells as was a slight reduction in ßT B cells (Fig. 5GoB). These CFs, which are resistant to denaturation in SDS detergent at 25°C, reflect the presence of class II heterodimers loaded with peptides derived from intrinsic proteins or serum components. 35S pulse-chase labeling indicated that the truncation of either cytoplasmic domain delayed the appearance of peptide-loaded MHC class II CFs (data not shown). Whereas the proportion of endogenous compact class II molecules is higher at the surface of ßT than {alpha}T B cells, HEL (46–61) presentation efficiency is higher for {alpha}T than for ßT B cells. This discrepancy could be explained by a difference in the access of truncated class II molecules to peptide loading compartments or by a difference in the conformational stability of these molecules. Identification of the intermediate Ii fragments bound to class II molecules is instrumental to follow class II transport and peptide loading in B cells 27 . In this study, we monitored the pattern of the full-length Ii (p31) and Ii (p12) degradation products associated with class II molecules to follow class II maturation (Fig. 5GoC). Class II molecules are not prominently associated with p12 Ii fragments in B cells expressing either intact or {alpha}T class II molecules, which can present BCR ligands (Fig. 5GoC). Truncation of the I-Ak ß cytoplasmic domain strongly increased the level of association of the CLIP-containing p12 Ii fragments. To demonstrate the specificity of the different bands observed on the gels corresponding to I-Ak-associated proteins, we used the 10.2.16 mAb for immunoprecipitation of the non-I-Ak-expressing parental M12C3 line as a negative control. None of the above bands were observed (data not shown). A slightly higher m.w. band to p31, which we have not identified, coprecipitates with class II molecules on these gels.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5. MHC class II characterization in cells expressing intact or cytoplasmic domain-truncated I-Ak molecules. A, class II surface expression of cells with truncated class II cytoplasmic domains was assessed by FACS using the anti-I-Ak 10.2.16 mAb (open histograms) and was controlled using the secondary reagent (filled histograms). B, surface class II molecules were visualized after membrane 125I labeling followed by class II immunoprecipitation with the 10.2.16 mAb; class II dimers are partially present as CFs in non-boiled SDS extracts (NB lanes) and are fully dissociated in boiled SDS extracts (B lanes). C, the expression of Ii (p31) or Ii fragment (p12) associated with class II molecules was analyzed on Western blots after immunoprecipitation of the complexes with the 10.2.16 mAb. The proteins were resolved on SDS-PAGE, transferred onto an Immobilon-P membrane, blotted with {alpha}Cyt.Ii or {alpha}CLIP Abs, and revealed with the enhanced chemiluminescence detection kit.

 
To localize class II molecules and Ii fragments in B lymphoma cells expressing full-length or truncated class II molecules, we performed double fluorescence confocal microscopy using the 10.2.16 mAb directed against I-Ak and the rabbit {alpha}Cyt.Ii Ab. In cells expressing full-length I-Ak or {alpha}T molecules (Fig. 6Go, I-Ak and {alpha}T lanes), Ab to these molecules was mainly localized at the cell surface, whereas Ab to Ii showed a reticulated intracellular staining consistent with an endoplasmic reticulum expression pattern. In these cells, MHC class II molecules and Ii are not detected in endolysosomal compartments at the steady state (Fig. 7Go and data not shown). In ßT B cells, class II molecules are expressed at the cell surface but are also concentrated with Ii in intracellular compartments (Fig. 6Go, ßT lane). To determine in which intracellular compartments class II and Ii accumulate in ßT cells, we performed double immunofluorescence with different markers specific for various endocytic compartments: Tf is internalized in early endosomes, M6PR accumulates in late endosomes, and H-2M is present in lysosome-related compartments 43 . With such markers, we observed (Fig. 7Go) that class II and Ii molecules, which colocalize only slightly with internalized Tf, accumulate preferentially in late endosomal compartments labeled with the anti-M6PR Ab and do not colocalize with H-2M. This intracellular accumulation of class II/Ii complexes in late endosomes, which correlated with the accumulation of Ii p12 fragments (Fig. 5Go) and with an impairment of HEL (46–61) presentation (Fig. 4Go), suggests that the ß cytoplasmic domain of class II molecules is involved in the transport of I-Ak molecules to compartments in which Ii is terminally degraded and in which class II molecules can be loaded with epitopes derived from protein cores. The class II ß cytoplasmic domain allows class II molecules to quit late endosomes to reach deep lysosome-related compartments, which also contain H-2M.



View larger version (73K):
[in this window]
[in a new window]
  		FIGURE 6. Localization of MHC class II molecules and Ii in B cell lines expressing intact or truncated class II molecules. The intracellular distribution of class II molecules and Ii was determined by confocal microscopy analysis in B cell lines. Class II molecules were labeled with the 10.2.16 mAb in I-Ak, {alpha}T, and ßT B cells (A, B, and C, respectively). Ii was labeled with {alpha}Cyt.Ii Ab in the same cells (D, E, and F, respectively). Arrows indicate the colocalization of class II and Ii molecules in intracellular compartments of ßT B cells (C and F). Scale bar is 10 µm.

 


View larger version (76K):
[in this window]
[in a new window]
  		FIGURE 7. Intracellular localization of MHC class II molecules and Ii in B cell lines expressing intact or ß-truncated class II molecules. The distribution of class II molecules and Ii in different endolysosomal compartments was determined by double immunofluorescence and confocal microscopy analysis in transfected B cell lines. Class II molecules (in red) were labeled with the 10.2.16 mAb in I-Ak cells (A–C) and ßT B cells (D–F). Ii was labeled (in red) with {alpha}Cyt.Ii Ab in ßT B cells only (G–I). Early endosomal compartments (in green) were detected with internalized FITC-Tf (A, D, and G). Late endosomal compartments (in green) were labeled with an anti-M6PR Ab (B, E, and H). Lysosome-related compartments (in green) were stained with an {alpha}Cyt.Mß Ab (C, F, and I). Arrows either indicate an accumulation of class II and Ii molecules (in red) in vesicles distinct from those labeled with Tf or H-2M or indicate the colocalization (in yellow) of class II molecules and M6PR (E) or of Ii molecules and M6PR (G) in ßT B cells. Scale bar is 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Presentation of peripheral protein determinants such as the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope is resistant to protein synthesis inhibitors and independent of Ii and H2-M expression when the Ags are internalized via the fluid phase 13, 18 . These epitopes are thought to be presented by mature class II molecules that recycle from the cell surface. Presentation of the epitopes derived from protein cores such as the HEL (46–61) determinant is sensitive to protein synthesis inhibitors and depends upon Ii and H2-M expression. Such antigenic determinants are only presented by newly synthesized class II molecules 18 . Using splenic B cells from mice that express or do not express anti-HEL surface Igs, we showed that Ags targeted through the BCR are presented at an Ag concentration that is 1000-fold lower than Ags endocytosed via the fluid phase, and that practically all of the advantage conferred by BCR targeting for the presentation of either of these HEL epitopes is sensitive to CHX (Fig. 1Go). We extended these observations to B lymphoma cell lines in which surrogate Ag-specific presentation could be achieved by encapsulating Ag in liposomes and targeting it with Ab directed at the non-HEL-specific cell surface Ig expressed by the lines (Figs. 2Go and 3Go). Presentation of the Ags encapsulated in liposomes and targeted to surface IgG is also markedly improved compared with nontargeted Ags, but to a lesser extent (50- to 100-fold) than in splenic B cells. Using this system, we found that the presentation of peripheral determinants, such as the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and RNase A (43–56) epitopes, also became dependent upon protein synthesis. This experimental system permitted us to use cells transfected with genes encoding truncated class II molecules and to analyze the role of class II {alpha} and ß cytoplasmic domains in the presentation of different antigenic determinants following fluid phase or BCR-mediated uptake of Ag by transfected B lymphoma cells.

Role of MHC class II tails in the presentation of fluid phase Ags

Previous studies performed in human fibroblasts and B cells 24 or in transfected rat blood leukemia (RBL) cells 25 showed that Ag presentation was differentially affected by class II cytoplasmic domain truncations, depending upon the nature of the Ag 19 or the determinant presented within a protein when Ags were taken up from the fluid phase. Class II cytoplasmic domains are essential to promote the internalization of surface class II molecules in these cells 24 . In agreement with these results, we observed that the alternative presentation pathway of the HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 epitope was equally affected by the truncation of {alpha} or ß class II cytoplasmic domains in murine B cells, even when this Ag was encapsulated in nontargeted liposomes (Fig. 4Go, D and E).

However, we observed that the presentation of the 46–61 epitope derived from intact HEL was also sensitive to the truncation of class II cytoplasmic tails of {alpha}- and ß-chains, with a stronger inhibition observed in truncated ß-chain transfectants (Fig. 4GoA). Our results suggest that the ßT cytoplasmic domain is important in directing class II molecules to compartments involved in the processing of core epitopes such as HEL (46–61) peptide. In contrast to this observation, presentation of the HEL (46–61) epitope to the same T cell hybridoma used here was not affected by truncation of the cytoplasmic tail of the MHC class II ß-chain transfected with Ii into RBL cells 25 . This difference could have several explanations. First, Ag taken up by fluid phase endocytosis by cells transfected with wild-type class II molecules was presented at least an order of magnitude more efficiently by the mouse lymphoma used here than by the rat RBL cells used in that study. Second, RBL cells retain wild-type class II molecules in intracellular compartments, whereas our murine B lymphoma transfectants express wild-type class II molecules mostly at the cell surface (Fig. 6Go). In APCs in which newly synthesized class II molecules accumulate intracellularly before being exported to the surface, truncation of the ß cytoplasmic tail could be of minor importance. Such class II molecules may reside long enough in late endosome-related compartments to meet core epitopes. In murine B cells, truncation of the ß cytoplasmic domain induced an intracellular accumulation of class II and Ii molecules in compartments in which the M6PR colocalized (Fig. 7Go). These cells had a reduced ability to present the 46–61 peptide derived from HEL, suggesting that the access of ßT class II molecules to compartments in which Ii is degraded and core epitopes are produced is regulated differentially in RBL cells and in B lymphoma cells. In addition, the nature of the compartments in which class II molecules accumulate constitutively or as a consequence of ß-chain truncation could be different in both cells.

Influence of BCR targeting on Ag presentation by truncated class II molecules

Our understanding of the role of class II cytoplasmic domains in different Ag-presentation pathways is based essentially on studies performed with Ags internalized from the fluid phase. However, the capture of Ags by B cells is mostly mediated through BCR recognition. When HEL was targeted by the BCR, we observed a strong augmentation in the presentation efficiency of the HEL-derived (46–61) epitope, compared with HEL internalized via the fluid phase (Fig. 4Go). Ag targeting to the BCR rescued the presentation of HEL by {alpha}T class II molecules to a level that was equivalent to that seen for cells expressing intact I-Ak class II molecules for Ag taken up by fluid phase endocytosis. Nevertheless, improved presentation of HEL (46–61) epitope by ßT class II molecules was not observed under the same BCR-targeting conditions. The data obtained using the same Ag, APC, and T cell hybridoma recognizing the HEL-derived peptide 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 indicate that before the molecules pass into a compartment that is dependent upon the I-Ak ß-chain, they have access to compartments which do not require the presence of the I-A ß cytoplasmic domain.

Whereas the cytoplasmic domain of the class II ß-chain is involved in the transport of newly synthesized class II molecules into deep endosomal compartments, the {alpha} cytoplasmic domain is not necessary for intracellular class II transport. This finding is supported by immunofluorescence analysis of class II distribution in the {alpha}T cells, which is similar to that seen in the wild-type molecule. The {alpha} cytoplasmic domain does not influence the Ii-p12 release from class II molecules, because an equal quantity of p12 remains associated with {alpha}T and full-length I-Ak molecules. The reduced Ag presentation seen in {alpha}T cells with Ags internalized via the fluid phase could result from a conformational instability of these truncated {alpha}T molecules to bind peptides; this instability is partially overcome by a concentration of Ags following BCR internalization in specific compartments, although the precise cause of the reduced presentation remains unknown. BCR-mediated uptake directs Ags for presentation in a manner that is dependent upon protein synthesis, as shown by the sensitivity to CHX of HEL presentation to T hybridomas, regardless of the peptide presented (Figs. 1Go and 3Go). As well as acting on routes of Ag internalization, BCR engagement could also control Ag presentation by acting on the maturation pathway of newly synthesized class II molecules 27 .

Relationships between MHC class II molecule transport and Ag-processing compartments

In ßT B cells, the degradation pattern of Ii shows that Ii-p12 fragments are associated with accumulated intracellular class II molecules (Fig. 5Go). ß-chain truncation consequently blocks the access of class II molecules to compartments in which class II/p12 fragment complexes are processed into Ii-p10 fragments and class II/CLIP complexes and in which the HEL (46–61) peptide is generated and/or loaded onto class II molecules. The di-leucine motifs present in the cytoplasmic domain of the class II ß-chain have been shown to regulate Ag presentation by acting on the intracellular transport of class II molecules 25 ; these motifs act in conjunction with the two di-leucine motifs present in the cytoplasmic tail of Ii, which are essential for the transport of class II-Ii complexes to endosomal vesicles 44 . Moreover, the di-leucine motif of the ß-chain is required for Ii-independent presentation following fluid phase Ag uptake 25 . Our results show that the cytoplasmic domain of class II ß-chains controls the access of newly synthesized class II molecules to compartments, probably lysosome-related, in which class II molecules achieve their maturation and in which the Ag internalized by the BCR is terminally processed.

Our results are consistent with the model illustrated in Fig. 8Go. Mature class II molecules recycling through endosomal compartments (compartment 3) in a protein synthesis-independent but I-A{alpha}- and ß-chain-dependent manner can bind peptides such as HEL 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 when Ags are internalized via pinocytosis. This peripheral compartment seems inaccessible to Ags endocytosed through the BCR. BCR ligands either are directly targeted to specialized endosomal compartments (compartment 1) or pass through early endosomal compartments (compartment 3) and are only released from immune complexes in late endosomal compartments (compartment 1). These late endosomal compartments contain newly synthesized class II molecules that are delivered in a manner not requiring the integrity of their {alpha} and ß cytoplasmic domains. These compartments permit mild Ag degradation or are in continuity with compartments having this capacity 45 . Class II ß cytoplasmic tails control access to deeper lysosome-related compartments (compartment 2) and the presentation of peptides requiring extensive Ag degradation. In addition to the effect of BCR engagement on the intracellular transport of newly synthesized class II molecules 27 , our results show that BCR engagement also targets Ags into compartments that are only accessible to these newly formed class II molecules. The dual role of BCR in Ag internalization and in cell signaling thus conveys cognate BCR ligands to newly synthesized class II molecules to transform B cells into very efficient APCs.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 8. MHC class II peptide-loading compartments accessible to Ags internalized through the BCR. {alpha}ßIi, class II/Ii complexes; {alpha}ßp, class II molecules loaded with antigenic peptides. Compartment 1, late endosomal compartment permitting mild Ag degradation where epitopes such as the HEL (34–45) associate with class II molecules; compartment 2, lysosome-related compartment with extensive Ag degradation where epitopes such as HEL (46–61) associate with class II molecules; compartment 3, early endosomal compartments where class II molecules recycle.

 


   Footnotes
 
1 This work was supported by the Institut National de la Santé et de la Recherche Médicale, by the Centre National de la Recherche Scientifique, and by grants from both the Association pour la Recherche sur le Cancer and the Ligue Nationale contre le Cancer. N.B. was the recipient of a predoctoral SIDACTION fellowship from the Fondation pour la Recherche Médicale. Back

2 Address correspondence and reprint requests to Dr. Frédérique Forquet, Centre d’Immunologie de Marseille-Luminy, Parc Scientifique de Luminy, Case 906, 13 288 Marseille, France. E-mail address: Back

3 Abbreviations used in this paper: Ii, invariant chain; BCR, B cell receptor; CLIP, class II-associated Ii peptide; HEL, hen egg lysozyme; RAMIG, rabbit anti-mouse IgG; {alpha}T, {alpha} cytoplasmic domain-truncated I-Ak molecule; ßT, ß cytoplasmic domain-truncated I-Ak molecule; Tg, transgenic; M6PR, mannose 6 phosphate receptor; CHX, cycloheximide; Lip-PA, liposome-protein A; Tf, transferrin; CF, compact form; RBL, rat blood leukemia. Back

Received for publication August 12, 1998. Accepted for publication December 14, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Qiu, Y., X. Xu, A. Wandinger-Ness, D. P. Dalke, S. K. Pierce. 1994. Separation of subcellular compartments containing distinct functional forms of MHC class II. J. Cell Biol. 125:595.[Abstract/Free Full Text]
  2. West, M. A., J. M. Lucocq, C. Watts. 1994. Antigen processing and class II MHC peptide-loading compartments in human B-lymphoblastoid cells. Nature 369:147.[Medline]
  3. Amigorena, S., J. R. Drake, P. Webster, I. Mellman. 1994. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B lymphocytes. Nature 369:113.[Medline]
  4. German, R. N., F. Castellino, R. Han, C. Reis e Sousa, P. Romagnoli, S. Sadegh-Nasseri, G.-M. Zhong. 1996. Processing and presentation of endocytically acquired protein antigens by MHC class II and class I molecules. Immunol. Rev. 151:5.[Medline]
  5. Viville, S., J. Neefjes, V. Lotteau, A. Dierich, M. Lemeur, H. Ploegh, C. Benoist, D. Mathis. 1993. Mice lacking the MHC class II-associated invariant chain. Cell 72:635.[Medline]
  6. Miyazaki, T., P. Wolf, S. Tourne, C. Waltzinger, A. Dierich, N. Barois, H. Ploegh, C. Benoist, D. Mathis. 1996. Mice lacking H2-M complexes, enigmatic elements of the MHC class II peptide-loading pathway. Cell 84:531.[Medline]
  7. Liljedahl, M., O. Winqvist, C. D. Surh, P. Wong, K. Ngo, L. Teyton, P. A. Peterson, A. Brunmark, A. Y. Rudensky, W.-P. Fung-Leung, L. Karlsson. 1998. Altered antigen presentation in mice lacking H2-O. Immunity 8:233.[Medline]
  8. Roche, P. A., P. Cresswell. 1991. Proteolysis of the class II-associated invariant chain generates a peptide binding site in intracellular HLA-DR molecules. Proc. Natl. Acad. Sci. USA 88:3150.[Abstract/Free Full Text]
  9. Denzin, L. K., P. Cresswell. 1995. HLA-DM induces CLIP dissociation from MHC class II {alpha}ß dimers and facilitates peptide loading. Cell 82:155.[Medline]
  10. Vogt, A. B., H. Kropshofer, G. Moldenhauer, G. J. Hämmerling. 1996. Kinetic analysis of peptide loading onto HLA-DR molecules mediated by HLA-DM. Proc. Natl. Acad. Sci. USA 93:9724.[Abstract/Free Full Text]
  11. St.Pierre, Y., T. H. Watts. 1990. MHC class II-restricted presentation of native protein antigen by B cells is inhibitable by cycloheximide and brefeldin A. J. Immunol. 145:812.[Abstract]
  12. Kakiuchi, T., M. Watanabe, N. Hozumi, H. Nariuchi. 1990. Differential sensitivity of specific and nonspecific antigen-presentation by B cells to a protein synthesis inhibitor. J. Immunol. 145:1653.[Abstract]
  13. Nadimi, F., J. Moreno, F. Momburg, A. Heuser, S. Fuchs, L. Adorini, G. J. Hämmerling. 1991. Antigen presentation of hen egg-white lysozyme but not of ribonuclease A is augmented by the major histocompatibility complex class II-associated invariant chain. Eur. J. Immunol. 21:1255.[Medline]
  14. Momburg, F., S. Fuchs, J. Drexler, R. Busch, M. Post, G. J. Hämmerling, L. Adorini. 1993. Epitope-specific enhancement of antigen presentation by invariant chain. J. Exp. Med. 178:1453.[Abstract/Free Full Text]
  15. Pinet, V., M. S. Malnati, E. O. Long. 1994. Two processing pathways for the MHC class II-restricted presentation of exogenous influenza virus antigen. J. Immunol. 152:4852.[Abstract]
  16. Harding, C. V., R. W. Roof, E. R. Unanue. 1989. Turnover of Ia-peptide complexes is facilitated in viable antigen-presenting cells: biosynthetic turnover of Ia vs. peptide exchange. Proc. Natl. Acad. Sci. USA 86:4230.[Abstract/Free Full Text]
  17. Salamero, J., M. Humbert, P. Cosson, J. Davoust. 1990. Mouse B lymphocyte-specific endocytosis and recycling of MHC class II molecules. EMBO J. 9:3489.[Medline]
  18. Griffin, J. P., R. Chu, C. V. Harding. 1997. Early endosomes and a late endocytic compartment generate different peptide-class II MHC complexes via distinct processing mechanisms. J. Immunol. 158:1523.[Abstract]
  19. Pinet, V. M., E. O. Long. 1998. Peptide loading onto recycling HLA-DR molecules occurs in early endosomes. Eur. J. Immunol. 28:799.[Medline]
  20. Nabavi, N., Z. Ghogawala, A. Myer, I. J. Griffith, W. F. Wade, Z. Z. Chen, D. J. McKean, L. H. Glimcher. 1989. Antigen presentation abrogated in cells expressing truncated Ia molecules. J. Immunol. 142:1444.[Abstract]
  21. Wade, W. F., Z. Z. Chen, R. Maki, S. McKercher, E. Palmer, J. C. Cambier, J. H. Freed. 1989. Altered I-A protein-mediated transmembrane signaling in B cells that express truncated I-Ak protein. Proc. Natl. Acad. Sci. USA 86:6297.[Abstract/Free Full Text]
  22. Smiley, S. T., T. M. Laufer, D. Lo, L. H. Glimcher, M. J. Grusby. 1995. Transgenic mice expressing MHC class II molecules with truncated {alpha}ß cytoplasmic domains reveal signaling-independent defects in antigen presentation. Int. Immunol. 7:665.[Abstract/Free Full Text]
  23. Smiley, S. T., A. Y. Rudensky, L. H. Glimcher, M. J. Grusby. 1996. Truncation of the class II ß-chain cytoplasmic domain influences the level of class II invariant chain-derived peptide complexes. Proc. Natl. Acad. Sci. USA 93:241.[Abstract/Free Full Text]
  24. Pinet, V., M. Vergelli, R. Martin, O. Bakke, E. O. Long. 1995. Antigen presentation mediated by recycling of surface HLA-DR molecules. Nature 375:603.[Medline]
  25. Zhong, G. M., P. Romagnoli, R. N. German. 1997. Related leucine-based cytoplasmic targeting signals in invariant chain and major histocompatibility complex class II molecules control endocytic presentation of distinct determinants in a single protein. J. Exp. Med. 185:429.[Abstract/Free Full Text]
  26. Lanzavecchia, A.. 1996. Mechanisms of antigen uptake and presentation. Curr. Opin. Immunol. 8:348.[Medline]
  27. Barois, N., F. Forquet, J. Davoust. 1997. Selective modulation of the major histocompatibility complex class II antigen presentation pathway following B cell receptor ligation and protein kinase C activation. J. Biol. Chem. 272:3641.[Abstract/Free Full Text]
  28. Siemasko, K., B. J. Eisfelder, E. Williamson, S. Kabak, M. R. Clark. 1998. Signals from the B lymphocyte antigen receptor regulate MHC class II containing late endosomes. J. Immunol. 160:5203.[Abstract/Free Full Text]
  29. Rouas-Freiss, N., F. Housseau, J.-M. Bidart, C. Bonnerot, S. Amigorena, J.-G. Guillet, D. Bellet. 1993. Deficient antigen processing of a protein quaternary structure can be overcome by receptor-mediated uptake. Eur. J. Immunol. 23:3335.[Medline]
  30. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
  31. Grivel, J. C., P. Ferrier, N. Renard, G. Jolly, T. Jarry, L. Leserman. 1993. Rapid induction of anti-idiotypic responses to unmodified monoclonal antibodies from syngeneic mice following primary immunization. J. Immunol. Methods 158:173.[Medline]
  32. Machy, P., J. Barbet, L. D. Leserman. 1982. Differential endocytosis of T and B lymphocyte surface molecules evaluated with antibody-bearing fluorescent liposomes containing methotrexate. Proc. Natl. Acad. Sci. USA 79:4148.[Abstract/Free Full Text]
  33. Glimcher, L. H., D. J. McKean, E. Choi, J. G. Seidman. 1985. Complex regulation of class II gene expression: analysis with class II mutant cell lines. J. Immunol. 135:3542.[Abstract]
  34. Adorini, L., E. Appella, G. Doria, Z. A. Nagy. 1988. Mechanisms influencing the immunodominance of T cell determinants. J. Exp. Med. 168:2091.[Abstract/Free Full Text]
  35. Adorini, L., A. Sette, S. Buus, H. M. Grey, M. Darsley, P. V. Lehmann, G. Doria, Z. A. Nagy, E. Appella. 1988. Interaction of an immunodominant epitope with Ia molecules in T-cell activation. Proc. Natl. Acad. Sci. USA 85:5181.[Abstract/Free Full Text]
  36. Lorenz, R. G., A. N. Tyler, P. M. Allen. 1988. T cell recognition of bovine ribonuclease: self/non-self discrimination at the level of binding to the I-Ak molecule. J. Immunol. 141:4124.[Abstract]
  37. Smith-Gill, S. J., T. B. Lavoie, C. R. Mainhart. 1984. Antigenic regions defined by monoclonal antibodies correspond to structural domains of avian lysozyme. J. Immunol. 133:384.[Abstract]
  38. Barois, N., F. Forquet, J. Davoust. 1998. Actin microfilaments control the MHC class II antigen presentation pathway in B cells. J. Cell Sci. 111:1791.[Abstract]
  39. Leserman, L., J. Barbet, F. Kourilsky, J. N. Weinstein. 1980. Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A. Nature 288:602.[Medline]
  40. Mosmann, T.. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55.[Medline]
  41. Watts, C., A. Lanzavecchia. 1993. Suppressive effect of antibody on processing of T cell epitopes. J. Exp. Med. 178:1459.[Abstract/Free Full Text]
  42. Simitsek, P. D., D. G. Campbell, A. Lanzavecchia, N. Fairweather, C. Watts. 1995. Modulation of antigen processing by bound antibodies can boost or suppress class II major histocompatibility complex presentation of different T cell determinants. J. Exp. Med. 181:1957.[Abstract/Free Full Text]
  43. Mellman, I.. 1996. Endocytosis and molecular sorting. Annu. Rev. Cell Biol. 12:575.[Medline]
  44. Pieters, J., O. Bakke, B. Dobberstein. 1993. The MHC class II-associated invariant chain contains two endosomal targeting signals within its cytoplasmic tail. J. Cell Sci. 106:831.[Abstract]
  45. Geuze, H. J.. 1998. The role of endosomes and lysosomes in MHC class II functioning. Immunol. Today 19:282.[Medline]



This article has been cited by other articles:


Home page
 Int ImmunolHome page
C. Chalouni, J. Banchereau, A. B. Vogt, V. Pascual, and J. Davoust
Human germinal center B cells differ from naive and memory B cells by their aggregated MHC class II-rich compartments lacking HLA-DO
Int. Immunol., April 1, 2003; 15(4): 457 - 466.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
C. M. Snyder, X. Zhang, and L. J. Wysocki
Negligible Class II MHC Presentation of B Cell Receptor-Derived Peptides by High Density Resting B Cells
J. Immunol., April 15, 2002; 168(8): 3865 - 3873.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Gugasyan, C. Velazquez, I. Vidavsky, B. M. Deck, K. van der Drift, M. L. Gross, and E. R. Unanue
Independent Selection by I-Ak Molecules of Two Epitopes Found in Tandem in an Extended Polypeptide Antigen
J. Immunol., September 15, 2000; 165(6): 3206 - 3213.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Forquet, F.
Right arrow Articles by Davoust, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Forquet, F.
Right arrow Articles by Davoust, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS