Download Compartments Target the Antigen to Distinct Intracellular B Cell

B Cell Receptors and Complement Receptors
Target the Antigen to Distinct Intracellular
Compartments
This information is current as
of June 18, 2017.
Laure A. Perrin-Cocon, Christian L. Villiers, Jean Salamero,
Françoise Gabert and Patrice N. Marche
J Immunol 2004; 172:3564-3572; ;
doi: 10.4049/jimmunol.172.6.3564
http://www.jimmunol.org/content/172/6/3564
Subscription
Permissions
Email Alerts
This article cites 65 articles, 34 of which you can access for free at:
http://www.jimmunol.org/content/172/6/3564.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2004 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
The Journal of Immunology
B Cell Receptors and Complement Receptors Target the
Antigen to Distinct Intracellular Compartments1
Laure A. Perrin-Cocon,* Christian L. Villiers,2* Jean Salamero,† Françoise Gabert,* and
Patrice N. Marche*
T
he efficiency of Ag presentation depends not only on the
efficiency of Ag internalization but also on the intracellular transport of Ags toward compartments equipped for
processing and loading of antigenic peptides on MHC class II molecules (MHC-II).3 It was previously shown that B cell receptor
(BCR)-mediated uptake rapidly targets Ags toward multilamellar
or multivesicular compartments (1, 2), rich in neosynthesized class
II molecules and specialized for efficient peptide loading, which
were named MHC-II loading compartments (MIIC) (3– 6). Neosynthesized class II molecules are targeted from the secretory
trans-Golgi network to early endosomes before reaching MIIC
where they accumulate (7, 8). Therefore, in B cells, class II mol*Laboratoire d’Immunochimie, Département de Réponse et Dynamique Cellulaires,
Commissariat à l’Energie Atomique, Institut National de la Santé et de la Recherche
Médicale, Unité 548, Université Joseph Fourier, Grenoble, France; and †Laboratoire
de Compartimentation et Dynamique Cellulaire, Unité Mixte de Recherche 144, Centre National de la Recherche Scientifique, Institut Curie, Paris, France
Received for publication September 6, 2002. Accepted for publication January
6, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by specific grants from the Délégation Générale de
l’Armement (DSP 99-34-038) and the Ministère de l’Éducation Nationale, de la Recherche, et de la Technologie (Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires) and by institutional grants from Institut
National de la Santé et de la Recherche Médicale and Commissariat à l’Energie
Atomique. L.A.P.-C. was initially supported by a fellowship from the Ministère de
l’Éducation Nationale, de la Recherche, et de la Technologie, and then by the Ligue
Nationale Contre le Cancer.
2
Address correspondence and reprint requests to Dr. Christian L. Villiers, Laboratoire d’Immunochimie, Département de Réponse et Dynamique Cellulaires, Commissariat à l’Energie Atomique, 17 rue des Martyrs, F-38054 Grenoble, cedex 9, France.
E-mail address: [email protected]
3
Abbreviations used in this paper: MHC-II, MHC class II molecule; BCR, B cell
receptor; MIIC, MHC-II loading compartment; Ii, invariant chain; HEL, hen egg
lysozyme; TeNT, tetanus neurotoxin; CR, complement receptor; TfR, transferrin receptor; Lamp-2, lysosomal-associated membrane protein-2; PNS, postnuclear supernatant; MF, magnetic fraction; NP-40, Nonidet P-40; CIIV, class II-containing vesicle; DAF, decay-accelerating factor; MCP, membrane cofactor protein.
Copyright © 2004 by The American Association of Immunologists, Inc.
ecules are present throughout the endocytic pathway (9). The addressing of neosynthesized class II molecules is monitored by their
association with the invariant chain (Ii), which chaperons the
newly formed complex to the endocytic pathway (10 –12). Once in
the endocytic compartments, the invariant chain is degraded by
proteases into class II-associated invariant chain peptide, which is
exchanged with an antigenic peptide under HLA-DM (DM) catalysis, a protein highly enriched in MIIC (13–15). MHC-II-peptide
complexes are then exported to the plasma membrane. Surface
class II molecules can be reinternalized into early endosomes (16),
where they may exchange their peptide and then recycle to the
plasma membrane (17). Some peptides rapidly generated in the
endocytic pathway are presented by these recycling class II molecules (18).
The BCR is internalized via clathrin-coated pits and delivered to
early endosomes. Signals in the cytoplasmic tails of surface Ig and
side-chains Ig␣ and Ig␤ control the internalization and addressing
of this receptor to the appropriate compartment. The mutation of
one tyrosine (Y587) in the cytoplasmic tail of surface IgM does not
affect the efficiency of Ag capture but alters Ag presentation to T
lymphocytes (19, 20), highlighting the importance of the addressing role of the receptors. Furthermore, Ig␣ and Ig␤ control the
transport to late endosomes, leading to efficient Ag presentation
(21). Analysis of the addressing motifs of these chains has revealed
that Ig␣ targets the Ag to compartments containing neosynthesized
class II molecules, resulting in presentation of numerous epitopes,
whereas Ig␤ targets the Ag toward recycling class II molecules,
resulting in presentation of a restricted number of epitopes (22).
Because the internalization capacities of these receptors are quite
similar, the targeting of the Ag thus appeared to be crucial for their
presentation.
Evidence from in vivo and in vitro experiments demonstrate that
the complement component C3 plays a critical role in the specific
immune response. For instance, mice deficient in C3 show severely impaired Ab responses to T cell-dependent Ags (23). Under
0022-1767/04/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells.
This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in
which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by
B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the
intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas
both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to
compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly
targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein
content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive
less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are
distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation. The Journal of Immunology, 2004, 172: 3564 –3572.
The Journal of Immunology
Materials and Methods
Materials
4.2 (HLA DR3/DR1101) is B cell clone specific for TeNT (generous gift
from Dr. A. Lanzavecchia (Basel Institute, Basel, Switzerland)); SWEIG
(HLA DR1101) is an EBV-transformed B cell line and was obtained from
American Type Culture Collection (Manassas, VA). TDR11-IV2 is a T cell
clone specific for the peptide p30 (947–967) of TeNT, obtained from a
homozygous HLA DR1101 donor as described (28). All were grown in
RPMI 1640 medium with Glutamax I/10% heat-inactivated FCS (all from
Life Technologies, Cergy Pontoise, France)
Antibodies. Rabbit polyclonal antiserum against Rab7 was generously
provided by Dr. P. Chavrier (Institut Curie, Paris, France) (40); mAb
against cytoplasmic tails of MHC-II ␣-chain (DA6-147) and Ii (Pin1) have
been previously described (41, 42). L243 Ab was purified from HB55
hybridoma (American Type Culture Collection). 5C1 is an anti-HLA-DM␣
Ab (43). HRP-conjugated polyclonal anti-rabbit IgG was from Jackson
ImmunoResearch Laboratories (West Grove, PA), and HRP-conjugated
polyclonal anti-mouse IgG was from Sigma-Aldrich (St. Quentin-Fallavier,
France). mAb against transferrin receptor (TfR; clone DF1513) was purchased from Sigma-Aldrich; mouse anti-lysosomal-associated membrane
protein-2 (Lamp-2) (CD107b) was from BD PharMingen (San Diego, CA);
Texas Red-labeled donkey anti-mouse IgG and donkey anti-rabbit IgG
were from Jackson ImmunoResearch Laboratories.
TeNT was from Institut Mérieux (Marcy L’Etoile, France). Human C3
was purified as described (44). TeNT-C3b complexes were prepared as
described (25).
T cell proliferation assay
4.2 or SWEIG cells were incubated for 300 min at 37°C with TeNT or
TeNT-C3b, washed, and further incubated for 0 – 40 h at 37°C. After fixation (0.15% paraformaldehyde for 10 min), 5 ⫻ 103 cells were incubated
in 96-well flat-bottom microtiter plates with the T cell clone TDR11-IV2
(2 ⫻ 104 cells). After 20 h, supernatants (100 ␮l) were assayed for the
presence of IL-2 by incubation with the IL-2-dependent CTLL-2 cell line
(1 ⫻ 104 cells/well) for 20 h. Then, 1 ␮Ci of [3H]thymidine (Applied
Biosystems, Courtaboeuf, France) was added to the culture for 6 h, and
proliferation was measured by counting [3H]thymidine incorporation into
the cells.
Microbeads coupled to receptor ligand
Microbeads of 10-nm diameter (iron concentration, 6 mg/ml) were prepared as described previously (45).
TeNT or C3b (240 ␮g/ml) were covalently linked to microbeads in 0.1
M phosphate buffer (pH 7.5), by addition of 0.025% (w/w) glutaraldehyde.
Excess glutaraldehyde was inactivated by ethanolamine (0.1 M; pH 7.5).
Seventy-five to 90% of the protein was covalently bound to microbeads.
TeNT and C3b binding and internalization
TeNT and C3b were labeled with Alexa 488 (see Immunofluorescence and
confocal microscopy) or iodinated using the Iodogen method previously
described (46), and covalently linked to microbeads as described above. To
analyze the binding of C3b microbeads to cells, B lymphoblastoids were
washed and incubated in 20 mM NaCl/8 mM Na2HPO4/1.5 mM KH2PO4/
240 mM sucrose/1% OVA for 1 h on ice with various amounts of microbeads bearing fluorescent C3b. When mentioned, incubation was conducted in presence of nonlabeled C3b microbeads in excess. After three
washes in the same buffer, cells were analyzed by flow cytometry.
To compare internalization via BCR and CR, iodinated TeNT or C3b
microbeads (3.6 ␮g of protein) were incubated with 107 4.2 B cells at 4°C
for 1 h. Cells were washed three times and further incubated at 37°C for 30
min. The amount of radioactivity bound to the cells was quantified before
(total binding) and after (amount internalized) incubation.
Radiolabeling and biotinylation of cells
Cells (5 ⫻ 108) in Met/Cys-free DMEM were pulse-labeled with 2 mCi
35
S-Promix (Amersham Biosciences, Les Ulis, France) for 20 min at 37°C.
After washing in cold PBS (pH 7.4), cells were incubated at 15 ⫻ 106
cells/ml with 0.5 mg/ml N-hydroxysulfosuccinimide ester-long chain biotin (Pierce Chemicals, Rockford, IL) for 30 min on ice, under intermittent
shaking. Then, cells were washed three times with DMEM before use.
Electron microscopy
Cells were incubated with TeNT or C3b microbeads at 4°C and internalized at 37°C for 10 or 60 min, as described for magnetic fractionation.
Cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH
7.2) for 1 h at room temperature. Pelleted cells were postfixed in 1% OsO4
for 1 h at 4°C and included in Epon. Ultrathin sections were stained with
5% uranyl acetate and lead citrate, and examined with a JEOL (Peabody,
MA) 1200 EX II transmission electron microscope at 80 kV.
Subcellular fractionation on Percoll gradient
Centrifugation using self-forming Percoll density gradient was performed
in 13-ml Quick-Seal tubes (Beckman Instruments, Gagny, France), successively filled with the following: 1-ml cushion of 60% (w/v) sucrose, 11
ml of 17% (v/v) Percoll (Amersham Biosciences), and 1 ml of the fraction
to analyze. All solutions were in homogenization buffer. Sealed tubes were
centrifuged (20,000 ⫻ g; 90 min; 4°C) in a Ti 70.1 rotor (Beckman Instruments). Gradients were collected from the bottom of the tube in
0.65-ml fractions. To determine the position of organelles in the gradient,
4.2 B cells were incubated at 37°C for 30 min with 125I-labeled ferritransferrin prepared as described (29) and fractionated on Percoll gradient,
and several markers were quantified in the fractions. Early endosomes were
localized using 125I-labeled transferrin labeling. Lysosomes were characterized by their high ␤-galactosaminidase and cathepsin B activity, which
were determined as described (29), using paranitro-phenyl-N-acetylgalactosaminide (5 mM in 10% DMSO) and benzyloxycarbonyl-Arg-Arg-2naphtylamide (20 mM in DMSO), respectively, as substrate.
Immunofluorescence and confocal microscopy
TeNT or C3b were labeled with Alexa 488-protein labeling kit (Molecular
Probes, Leiden, The Netherlands) or with Cy3 (Amersham Pharmacia Biotech, Piscataway, NJ) as recommended by the manufacturer. Fluorescent
proteins were fixed on microbeads as described above.
For TeNT internalization, cells were washed in RPMI 1640 and incubated (108 cells/ml) with TeNT microbeads for 1 h on ice. After two
washes, cells were further incubated at 37°C for the indicated chase time
minus 10 min. Cells were then diluted to 5 ⫻ 106/ml and incubated for 10
min at 37°C on poly-L-lysine-coated lamellas. For C3b internalization,
cells were washed three times in RPMI 1640, resuspended at 2 ⫻ 106
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
activation, at a site of inflammation, for example, C3 is cleaved
into C3b, which can covalently bind to proximal Ags, leading to
the formation of C3b-Ag complexes (24). C3b acts as a natural
adjuvant of the secondary immune response. When C3b is coupled
to hen egg lysozyme (HEL) (HEL-C3b), 200 times less HEL is
required to immunize mice and elicit a B cell response compared
with HEL alone (25), and humoral immunity to HEL persists much
longer than when immunization is performed with CFA (26).
Moreover, a recombinant fusion protein containing three copies of
C3d coupled to HEL is highly immunogenic, inducing a maximal
Ab response at doses a thousand times lower than native HEL Ag
(27). In vitro, the covalent binding of C3b to Ags such as tetanus
neurotoxin (TeNT) enhances their presentation to specific T cells
(28). One part of this effect can be explained by increased internalization of C3b-TeNT complexes mediated by complement receptors (CR) (29). Moreover, C3b induces some modifications of
processing of the Ag linked (30), resulting in increased formation
of highly stable MHC-II-peptide complexes (31). Thus, the binding of C3 to Ags modifies not only their capture but also their
processing and presentation to T cells, resulting in enhanced specific immune response in vivo.
The mechanism of action of C3 is poorly understood; nevertheless, CR have been largely implicated. Knockout mice for CR1 and
CR2 show impaired primary and secondary T-dependent humoral
responses (32, 33), similarly to C3-deficient mice. Saturation of
CR by injection of soluble CR2 or Abs against CR1/2 strongly
inhibits Ab response to immunizations (34 –37). In vitro, coligation of CR2 and BCR results in enhanced release of intracellular
Ca2⫹ (38) and lowers the threshold for B cell activation (27, 39),
leading to significant augmentation of B cell responses.
Therefore, BCR and CR both sustain efficient Ag presentation.
To investigate the role of C3 in the intracellular traffic of Ags, we
analyzed the intracellular targeting induced by C3 CR and compared it with the trafficking induced after BCR internalization.
3565
3566
BCR AND CR INTRACELLULAR TARGETING OF Ags
NP-40/50 mM Tris (pH 7.4)). Proteins were eluted by incubation at 90°C
for 10 min, in denaturing solution (4 M urea/1% SDS/0.1 M Tris (pH 8.8))
and separated by SDS-PAGE before electrotransfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Class II molecules were
detected by immunoblotting with DA6.147 Ab. Biotinylated proteins were
detected using streptavidin-HRP (Pierce Chemicals). 35S-Labeled molecules were quantified with a PhosphorImager (Amersham Biosciences).
Immunoblotting
FIGURE 1. BCR and CR induce efficient Ag presentation to T cells.
Nonspecific (SWEIG) or TeNT-specific (4.2) B cells were incubated at
37°C with TeNT or TeNT-C3b for 300 min, washed, and chased for 0 – 40
h. Cells were cocultured with TDR11-IV2 cells for 20 h. T cell stimulation,
resulting from the presentation of TeNT by B cells, was measured by
[3H]thymidine incorporation as described in Materials and Methods.
Reduced and denatured samples were analyzed on a 12% SDS-PAGE.
Proteins were electrotransferred (100 V for 1 h) to Immobilon-P membranes (Millipore) in 10 mM 3-[cyclohexylamino]propane-1-sulfonic acid
(pH 11)/10% methanol. Blots were saturated with 5% fat-free milk powder
in PBS/0.1% Tween 20. Washing and incubations with primary and secondary Abs were conducted in PBS/0.1% Tween 20. Detection was performed using HRP-conjugated secondary Abs with the ECL kit (Amersham Biosciences). Densitometry of the film was performed after scanning
using ImageMaster software (Amersham Biosciences).
Results
BCR and CR internalization both result in efficient Ag
presentation
The efficiency of presentation of the TeNT immunodominant
epitope p30 by B lymphocytes was analyzed following three different internalization pathways: pinocytosis, and BCR- and CRmediated endocytosis. Presentation of the epitope p30 was assessed by the stimulation of the specific T cell clone TDR11-IV2.
Due to their expression of the TeNT-specific IgG, 4.2 B cells presented the immunodominant p30 epitope much more efficiently
Magnetic fractionation
After washing, B cells (108 cells/ml) were incubated for 1 h on ice with
TeNT or C3b microbeads (final iron concentration, 1 mg/ml). Cells were
washed twice with 50 ml of cold DMEM and further incubated at 37°C
(5 ⫻ 106 cells/ml) for the indicated time. Cells were rapidly cooled to 4°C,
washed once with 50 ml of PBS, and resuspended in homogenization buffer
(250 mM sucrose/1 mM HEPES/1 mM EDTA (pH 7.2)). Cellular plasma
membranes were disrupted by a cell disrupter (one shot, 0.75-kW model;
Constant Systems, Warwick, U.K.) with a pressure of 350 bars. Nuclei and
intact cells were removed by centrifugation (750 ⫻ g; 10 min; 4°C), and
the postnuclear supernatant (PNS) was kept for purification of intracellular
compartments.
Separation of intracellular compartments containing microbeads was
performed by magnetic fractionation: a column filled with 1.5 g of slightly
compacted steel wire (Spontex, Nanterre, France) was placed in a 0.6-T
U-shaped permanent magnet (MACS; Miltenyi Biotec, Bergisch Gladbach,
Germany) and equilibrated with homogenization buffer. The PNS was
pumped into the column at a flow rate of 1 ml/min. After washing with 10
vol of homogenization buffer, the column was removed from the magnetic
field, and the retained material or magnetic fraction (MF) was eluted in 4
ml of the same buffer. To remove extravesicular proteins, MF was treated
by 0.1 ␮g/ml proteinase K (Roche Diagnostics, Meylan, France) for 2 min
on ice. The reaction was stopped by the addition of 100 ␮l of protease
inhibitor mixture (250 mM Pefabloc; 250 mM iodoacetamide; 50 ␮g/ml
pepstatin A). Vesicles were washed and concentrated by ultracentrifugation
(100,000 ⫻ g; 40 min; 4°C) in a SW60 rotor (Beckman Instruments).
Immunoprecipitations
MF and PNS were solubilized in 200 ␮l of lysis buffer (300 mM NaCl/
0.5% Nonidet P-40 (NP-40)/50 mM Tris (pH 7.4)). After five cycles of
freezing/thawing, insoluble material was pelleted by ultracentrifugation
(400,000 ⫻ g; 15 min; 4°C). Supernatants were precleared with protein
A-Sepharose 4B (Amersham Pharmacia Biotech) for 1 h at 4°C. Half of the
preparation was incubated for 1 h at 4°C with L243 mAb bound to protein
A-Sepharose for immunoprecipitation of class II molecules. The second
part was incubated for 1 h at 4°C with streptavidin-Sepharose (Zymed, San
Francisco, CA) to retain total biotinylated proteins. Immunoprecipitates
were washed twice with high salt buffer (500 mM NaCl/0.5% NP-40/50
mM Tris (pH 7.4)) and twice with low salt buffer (150 mM NaCl/0.5%
FIGURE 2. Characterization of C3b binding. 4.2 B lymphocytes were
incubated at 4°C for 1 h with different concentrations of fluorescent C3b
linked to microbeads. A, Cells were washed before measurement of the
fluorescence bound to the cells. B, Analysis of the specificity of C3b binding to B cells was performed by addition of unlabeled C3b (120 ng) during
incubation with 20 ng of fluorescent C3b microbeads.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
cells/ml, and allowed to adhere on poly-L-lysine-coated lamellas for 10 min
at 37°C. Lamellas were washed three times with cold C3b buffer (20 mM
NaCl/8 mM Na2HPO4/1.5 mM KH2PO4/240 mM sucrose/1% OVA) and
incubated with Alexa 488-C3b microbeads, for 1 h on ice. After two
washes, cells were incubated in the same buffer at 37°C, for the indicated
chase time.
For cointernalization, cells were incubated in C3b buffer in presence of
Alexa 488-TeNT microbeads and Cy3-C3b microbeads for 1 h on ice. Cells
were washed and further incubated in the same buffer at 37°C, and allowed
to adhere on lamellas at 37°C for the last 10 min of incubation. Cells were
then processed for immunofluorescence staining as described previously
(47). The coverslips were mounted and viewed under a confocal microscope (TCS4D; Leica Lasertechnic, Heidelberg, Germany).
The Journal of Immunology
3567
FIGURE 3. Internalization of TeNT and C3b microbeads. 4.2 B cells were incubated at 4°C with TeNT
(A) or C3b microbeads (B). Cells were washed before
internalization at 37°C and prepared for transmission
electron microscopy on ultrathin sections as described
in Materials and Methods. Arrows indicate the
microbeads.
Internalization by BCR and CR
To allow specific binding of C3b or TeNT ligands, 4.2 B cells were
first incubated for 1 h on ice. Then, the specificity of the binding
was checked: the binding of C3b was mediated by a specific receptor, because it was dose dependant, reached saturation above 20
ng (Fig. 2A), and could be inhibited by 70% by competition with
6-fold excess unlabeled C3b (B). TeNT binding was specific to
BCR expression on 4.2 B cells and could be displaced by unlabeled TeNT (29, 48). Similar results were obtained with TeNT
linked to microbeads (data not shown). After incubation with the
cells at 4°C, ligand in excess was removed before internalization at
37°C for 0 –120 min. Internalization of TeNT and C3b microbeads
was analyzed by electron microscopy: after incubation at 37°C,
electron-dense microbeads were localized into intracellular vesicles on ultrathin sections of cells (Fig. 3). Because B cells do not
possess a strong capability of endocytosis, few microbead-positive
intracellular vesicles could be observed on each section. Further-
more, TeNT and C3b microbeads were internalized with similar
efficiency as shown in Table I. Intracellular endocytic compartments were separated on Percoll gradient according to their density
and localized as follows: early endosomes were labeled by internalization of 125I-labeled ferri-transferrin and localized in fractions
13–15; lysosomes were in fractions 4 –7, as characterized by their
high ␤-galactosidase and cathepsin B activity (Fig. 4A). In agreement with previous results (45), microbeads did not interfere with
the distribution of TeNT in light fractions (enriched in endosomes)
and dense fractions (enriched in lysosomes) whether or not it was
linked to the protein (Fig. 4B). Analysis by SDS-PAGE of fraction
content indicated that 80 –90% of 125I-labeled ligands remained
complexed with microbeads (data not shown). This allowed us to
follow the intracellular targeting of the ligands. Without internalization (chase, 0 min), ligands were essentially associated with the
plasma membrane (fractions 18 –21) (Fig. 4, B and C). Upon incubation at 37°C, radiolabeled TeNT was recovered in light endosomes and progressively accumulated in dense fractions (4 –7)
containing lysosomes (Fig. 4B), whereas C3b was very rapidly
localized in dense fractions, and its distribution did not vary significantly during the chase time (C). These results may be explained by differences either in the targeting of the ligands or in the
kinetics of their traffic in the same endocytic pathway.
Table I.
Internalization of TeNT and C3b in 4.2 B cellsa
6
Binding (ng/10 cells)
Internalization (%)
TeNT
C3b
11.1
72
11.3
89
a
4.2 B lymphocytes were incubated with 125I-labeled C3b or 125I-labeled TeNT
microbeads (3.6 ␮g of protein for 107 cells) for 1 h at 4°C, washed, and incubated at
37°C for 30 min to allow internalization. Internalization is expressed as percentage of
bound ligand. Results from one representative experiment are shown.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
than did nonspecific B cells (SWEIG), which internalized TeNT by
pinocytosis (Fig. 1). When TeNT was opsonized by C3b, its presentation by nonspecific B cells was almost as efficient as that
mediated by 4.2 cells. In these conditions, SWEIG cells could
internalize TeNT-C3b complexes by CR with an efficiency comparable with that of BCR, as previously shown using another B cell
line (28). Furthermore, p30 epitope presentation is slightly enhanced when TeNT-C3b complexes are presented by TeNT-specific B cells compared with free TeNT. When 4.2 and SWEIG cells
were compared, few differences in the kinetics of presentation
were observed: whereas 4.2 cells induced T cell proliferation just
after Ag loading (0-min chase), SWEIG cells needed ⬎300 min to
present antigenic peptide after TeNT-C3b complex internalization
(Fig. 1). This could be due either to differences in the capacity of
these cell lines to process Ags or to differences in the kinetics of
the intracellular pathways mediated by BCR and CR. To compare
the intracellular traffic induced by these two receptors, we next
followed the internalization of TeNT and C3b in the same B cell
line, 4.2.
3568
BCR AND CR INTRACELLULAR TARGETING OF Ags
BCR and CR ligands are internalized through distinct endocytic
pathways
Comparison of vesicular content
FIGURE 4. Subcellular distribution of TeNT and C3b microbeads. A,
125
I-labeled ferri-transferrin was internalized by 4.2 B cells for 20 min at
37°C. Cells were homogenized and fractionated on Percoll gradient. Fractions were analyzed for their content in 125I-labeled ferri-transferrin,
␤-galactosaminidase, and cathepsin B activity. B—D, 4.2 B cells were
incubated at 4°C (1 h) with 125I-labeled TeNT (B), 125I-labeled TeNT microbeads (B and C), or 125I-labeled C3b microbeads (D) and chased at 37°C
for 0, 10, 30, 60, or 120 min. Cells were homogenized and fractionated on
Percoll gradient. Fractions were collected and the amount of iodinated
ligand in each fraction was counted. B, Comparison of the percentage of
125
I-labeled TeNT found in light fractions enriched in endosomes (fractions
13–15) and dense fractions enriched in lysosomes (fractions 4 –7) after 30
min of chase, when TeNT is linked or not to microbeads. C and D, 125I
distribution on Percoll gradients. Data are expressed as percentage of total
radioactivity recovered in the gradient.
To characterize each endocytic pathway, compartments targeted
by BCR and CR were isolated by magnetic fractionation, after
internalization of TeNT or C3b microbeads, respectively. Their
content in specific proteins, involved in vesicular transport (Rab)
or in Ag presentation, was analyzed by Western blot. As shown in
Fig. 6A, after 120 min of chase, MHC-II, Ii, and HLA-DM, as well
as the late endosome marker Rab7, were detected in isolated compartments whether microbeads were internalized by BCR or CR.
Nevertheless, the kinetic evolution of the protein content in both
pathways was very different. When internalization was mediated
by BCR, compartments containing microbeads progressively acquired Rab7, with a maximum between 60 and 120 min of chase
(Fig. 6B). However, in compartments reached after internalization
by CR, a constant but low level of Rab7 was observed, which
corresponded to the level found after 10-min internalization of
BCR. In both cases, the early endosome and plasma membrane
marker Rab5, was found at the very beginning of the chase and
decreased after longer incubations (data not shown). Therefore,
unlike BCR, CR does not target its ligand to classical late endosomes during the first 2 h after its internalization.
Because both receptors lead to efficient Ag presentation, the
molecules involved in Ag processing were determined in these
different compartments. In compartments accessed after BCR internalization, the amounts of MHC-II, p31 Ii, and HLA-DM increased upon incubation at 37°C to reach a maximum at 120 min
and decreased later on (Fig. 6, C–E). In conclusion, after 60 min of
chase, BCR ligand is found in late endocytic compartments,
equipped to load antigenic peptides on class II molecules. Vesicles
purified after CR internalization also contained MHC-II, Ii, and
HLA-DM, but in constant amounts during the first 120 min of
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
The intracellular pathways of CR and BCR ligands were analyzed
by confocal microscopy. TeNT and C3b were labeled with Alexa
488 and fixed on microbeads. After 10-min internalization, TeNT
microbeads were observed in peripheral and pericentriolar vesicles, positive for TfR (Fig. 5A). After 60 min of chase, TeNT
partly colocalized with MHC-II and Lamp-2. BCR intracellular
targeting has been extensively studied, and in agreement with previous results from different laboratories (1, 2, 49, 50), we found
that, in 4.2 B cells, BCR is first delivered to TfR-positive endosomes before reaching late endocytic compartments containing
MHC-II and Lamp-2 that have been characterized as MIIC.
The intracellular traffic of C3b is quite different. C3b microbeads remained mostly in peripheral vesicles and never colocalized with TfR (Fig. 5B). After 60 min of chase, C3b partly
colocalized with MHC-II but not with the lysosomal Lamp-2 (Fig.
5B). Observations in immunoelectron microscopy, confirmed that,
after 60 min of chase, C3b microbeads were not found in Lamp2-positive compartments (data not shown). Furthermore, when
TeNT (labeled with Alexa 488) and C3b (labeled with Cy3) were
simultaneously internalized, the proteins were localized in distinct
compartments after 10 or 60 min incubation at 37°C (Fig. 5C and
data not shown). After 5 h of chase, although the remaining fluorescent signals were very weak, some colocalization of TeNT and
C3b could be observed, suggesting that at least part of the ligands
may finally converge.
These results demonstrate that BCR and CR ligands are targeted
to different early intracellular compartments. C3b do not access the
MIIC presentation compartment at least during the first hour after
internalization.
The Journal of Immunology
3569
FIGURE 5. Localization of internalized TeNT and
C3b microbeads by confocal microscopy. A and B, 4.2
cells were incubated at 4°C with Alexa 488-TeNT (A)
or Alexa 488-C3b microbeads (B) (in green) and
chased for 0, 10, or 60 min at 37°C, as indicated beside images. Cells were processed for immunofluorescence intracellular staining for TfR, MHC-II, and
Lamp-2 (in red). C, 4.2 cells were incubated at 4°C
with Alexa 488-TeNT microbeads and Cy3-C3b microbeads and chased for 10 min. The merged images
of green and red fluorescence channels are shown.
Origin of MHC-II in both endocytic pathways
Because MHC-II found in endocytic compartments may come either from the plasma membrane after reinternalization or from the
Golgi apparatus after neosynthesis, we have analyzed the origin of
these molecules in compartments reached by BCR and CR ligands.
Neosynthesized molecules were metabolically labeled, and cell
surface molecules were biotinylated before incubation in the presence of TeNT or C3b microbeads. After receptor internalization
and 120 min of chase, compartments containing TeNT or C3b
microbeads were magnetically isolated. Mature MHC-II were immunoprecipitated using L243 Ab. The proportion of neosynthesized and molecules reinternalized from plasma membrane was
determined on the same protein blot, by radioactivity and biotin
detection, respectively. Controls performed on the PNS showed
that the overall amount of MHC-II immunoprecipitated was equivalent whether internalization was mediated by BCR or CR (data
not shown). In isolated vesicles, the amount of biotinylated
MHC-II did not significantly differ in BCR and CR compartments,
whereas the amount of neosynthesized MHC-II in CR compartments was significantly reduced (⫺28.5 ⫾ 2.5%) compared with
BCR compartments (Fig. 7).
Discussion
Using biochemical analysis and microscopic observations, we
have shown that the intracellular traffic induced by the internalization of BCR and CR are independent. BCR endocytosis is mediated by clathrin-coated pits (51), leading to colocalization with
TfR in early peripheral sorting endosomes and pericentriolar recycling endosomes. After 1 h of chase, the Ag concentrates in
pericentriolar compartments, accumulating MHC-II, HLA-DM,
and Ii, labeled by late endosomal markers Rab7 and Lamp-2.
These compartments have the characteristics of MIIC. In contrast,
endocytosed C3b is retained in peripheral vesicles devoid of TfR
and Lamp-2. These compartments contain MHC-II, Ii, and
HLA-DM at a constant level for 2 h. They are rapidly accessed by
endocytosed C3b, but they are distinct from BCR-targeted early
endosomes by the lack of TfR and their apparent high density on
Percoll gradient. The absence of intracellular colocalization of
BCR and CR ligands at early stage of the endocytosis process
indicates that the receptors follow distinct internalization pathways. Their ligands are found in compartments differentiated by
their composition and their intracellular evolution, C3b remaining
confined in specific compartments for at least 2 h. BCR and CR
ligands may converge later on. Therefore, C3b opsonization of
Ags not only enhances their capture by nonspecific cells, using CR
that are widely expressed, but also modifies their targeting within
B lymphocytes.
These results raise the question of the targeting of C3b-Ag complexes in specific B cells. As shown previously, cross-linking of
BCR and CR by such complexes induces intracellular signaling
leading to B cell activation (39), and compared with resting cells,
these activated cells may also have modified intracellular traffic. In
conclusion, more investigation will be necessary to analyze the
effect of C3b-Ag complexes on the intracellular traffic in specific
B cells to discriminate between consequences of cell activation
and modification of intracellular pathway due to receptor internalization, because both may modify Ag processing and presentation.
Although internalization via BCR and CR results in very different trafficking within the cell, we have shown, in agreement
with previous results (28), that both pathways result in efficient Ag
presentation of an immunodominant epitope, although with different kinetics. C3b compartments are equipped to load antigenic
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
chase and increased only after 240 min of chase (Fig. 6, C–E).
Therefore, Ag processing in these distinct compartments may be
quite different.
3570
BCR AND CR INTRACELLULAR TARGETING OF Ags
FIGURE 6. Evolution of the content of BCR or CR compartments along
the endocytic pathway. 4.2 cells were incubated at 4°C with TeNT or C3b
microbeads. After the indicated chase time, cells were homogenized, and
compartments were isolated by magnetic fractionation. Compartment proteins were separated by SDS-PAGE and analyzed by Western blotting for
the following: Rab7, MHC-II, Ii, and HLA-DM. A, Western blots on BCR
or CR compartments isolated after 120 min of chase. B—E, Kinetic evolution of protein content, detected by Western blot, in BCR and CR
compartments.
peptides, because they contain neosynthesized and membrane
MHC-II, HLA-DM, and HLA-DO (data not shown). Furthermore,
we found comparable amounts of MHC-II in C3b compartments
isolated after 1 h as in TeNT-containing compartments reached
after 1 h of BCR internalization (Fig. 6C) that were characterized
as MIIC. The presence of HLA-DM in C3b compartments after 10
min of internalization is in agreement with the finding of HLA-DM
at the cell surface and in endosomes of B lymphocytes (52), and
not only in lysosomes as described initially. Moreover, we have
also detected this molecule by Western blot in Percoll gradient
light fractions enriched in endosomes prepared from our B lymphocytes (data not shown). The generation of antigenic peptides by
limited proteolysis of Ags is supported by the presence of active
proteases throughout the endocytic pathway even in early endosomes, where peptide loading on recycling MHC-II takes place
(18) and may be influenced by HLA-DM (53, 54). Therefore, Ag
processing and the resulting loading of antigenic peptides onto
MHC-II could occur in C3b compartments. The delay in Ag presentation observed when TeNT is linked to C3b (Fig. 1) may result
either from differences in the capacity of 4.2 and SWEIG cells to
present Ags or from the retention of C3b-TeNT complexes in the
compartments that we describe, either because these compartments
do not allow Ag presentation despite their content in class II and
HLA-DM molecules or because the processing in these compartments is slower than that occurring in MIIC. In all cases, the particular trafficking induced by CR modify the conditions of processing of the opsonized Ag.
Two types of peptide-loading compartments have been described and named MIIC and class II-containing vesicles (CIIV).
MIIC are late endocytic compartments, containing Lamp-2, where
neosynthesized MHC-II and HLA-DM molecules accumulate (3,
55). CIIV were first isolated from B lymphocytes by subcellular
fractionation (4). The absence of lysosomal markers such as
Lamp-2 distinguish them from MIIC. They are connected to endocytic compartments, because internalized material can reach
them, and they contain stable MHC-II-peptide complexes and few
HLA-DM molecules. They are a site of terminal degradation of Ii
and probably of peptide loading (56). More recently, Turley et al.
(57) have described, in dendritic cells, nonlysosomal peripheral
vesicles involved in the transport of these complexes from MIIC to
the plasma membrane that corresponded to CIIV. Ags were first
targeted to MIIC where processing and peptide loading took place,
and then MHC-II-peptide complexes were delivered to CIIV to be
transported to the membrane. Compartments reached by C3b share
some features with CIIV: they are nonlysosomal peripheral vesicles enriched in MHC-II, containing HLA-DM molecules, and devoid of Lamp-2 and TfR. Therefore, either C3b is retained in specialized atypical endosomal compartments devoid of TfR, or C3b
is directly targeted to CIIV export vesicles where peptide loading
can occur as shown previously (56). In conclusion, C3b is internalized independently of BCR and remains for several hours in
compartments that differ from MIIC by their physical properties
and their protein content. This will have some repercussions on Ag
processing according to proteases and other enzymatic activities
located in these compartments. The repertoire of peptides that will
be selected for presentation may also be affected by the difference
in the ratio between reinternalized and neosynthesized MHC-II.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 7. Quantification of recycling and neosynthesized MHC-II in
BCR and CR compartments. 4.2 cells were radiolabeled with [35S]methionine/cysteine (for 20 min) and surface biotinylated, before incubation at
4°C with TeNT or C3b microbeads. Microbeads were chased for 120 min
at 37°C, and cells were homogenized to prepare the PNS. BCR or CR
compartments were isolated by magnetic fractionation and homogenized in
lysis buffer. MHC-II were immunoprecipitated from MF or PNS with L243
Ab, and proteins were analyzed by SDS-PAGE. Biotinylated MHC-II (recycling MHC-II) were detected by streptavidin-HRP in chemiluminescence. Radiolabeled MHC-II (neosynthesized MHC-II) were detected by
exposure in a phosphor imager. The amount of MHC-II in MF is normalized by the amount of MHC-II immunoprecipitated from PNS and expressed as a percentage compared with BCR compartments (biotin, 100 ⫽
200,000 arbitrary units; 35S, 100 ⫽ 29,000 arbitrary units).
The Journal of Immunology
Acknowledgments
We are grateful to Prof. J. Trowsdale for providing HLA-DM 5C1 mAb
and Dr. C. Roucard for expert analysis of HLA-DM and HLA-DO distribution. We thank I. Pignot-Paintrand (Département de Réponse et Dynamique Cellulaires/Institut Federatif de Recherche 27, Institut National de
la Santé et de la Recherche Médicale, Commissariat à l’Energie Atomique,
Grenoble, France) and Y. Bailly (Strasbourg, France) for electron microscopy observations, and Maighread Gallagher for correction of the
manuscript.
References
1. Cheng, P. C., C. R. Steele, L. Gu, W. Song, and S. K. Pierce. 1999. MHC class
II antigen processing in B cells: accelerated intracellular targeting of antigens.
J. Immunol. 162:7171.
2. Drake, J. R., T. A. Lewis, K. B. Condon, R. N. Mitchell, and P. Webster. 1999.
Involvement of MIIC-like late endosomes in B cell receptor-mediated antigen
processing in murine B cells. J. Immunol. 162:1150.
3. Neefjes, J. J., V. Stollorz, P. J. Peters, H. J. Geuze, and H. L. Ploegh. 1990. The
biosynthetic pathway of MHC class II but not class I molecules intersects the
endocytic route. Cell 61:171.
4. Amigorena, S., J. R. Drake, P. Webster, and I. Mellman. 1994. Transient accumulation of new class II MHC molecules in a novel endocytic compartment in B
lymphocytes. Nature 369:113.
5. West, M. A., J. M. Lucocq, and C. Watts. 1994. Antigen processing and class II
MHC peptide-loading compartments in human B-lymphoblastoid cells. Nature
369:147.
6. Tulp, A., D. Verwoerd, B. Dobberstein, H. Ploegh, and J. Pieters. 1994. Isolation
and characterization of the intracellular MHC class II compartment. Nature
369:120.
7. Pond, L., and C. Watts. 1999. Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells. J. Biol. Chem. 274:18049.
8. Brachet, V., G. Pehau-Arnaudet, C. Desaymard, G. Raposo, and S. Amigorena.
1999. Early endosomes are required for major histocompatibility complex class
II transport to peptide-loading compartments. Mol. Biol. Cell 10:2891.
9. Castellino, F., and R. N. Germain. 1995. Extensive trafficking of MHC class
II-invariant chain complexes in the endocytic pathway and appearance of peptideloaded class II in multiple compartments. Immunity 2:73.
10. Lotteau, V., L. Teyton, A. Peleraux, T. Nilsson, L. Karlsson, S. L. Schmid,
V. Quaranta, and P. A. Peterson. 1990. Intracellular transport of class II MHC
molecules directed by invariant chain. Nature 348:600.
11. Odorizzi, C. G., I. S. Trowbridge, L. Xue, C. R. Hopkins, C. D. Davis, and
J. F. Collawn. 1994. Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment. J. Cell Biol. 126:317.
12. Pieters, J., O. Bakke, and B. Dobberstein. 1993. The MHC class II-associated
invariant chain contains two endosomal targeting signals within its cytoplasmic
tail. J. Cell Sci. 106:831.
13. Sherman, M. A., D. A. Weber, and P. E. Jensen. 1995. DM enhances peptide
binding to class II MHC by release of invariant chain-derived peptide. Immunity
3:197.
14. Denzin, L., and P. Cresswell. 1995. HLA-DM induces CLIP dissociation from
MHC class II ␣␤ dimers and facilitates peptide loading. Cell 82:155.
15. Sloan, V. S., P. Cameron, G. Porter, M. Gammon, M. Amaya, E. Mellins, and
D. M. Zaller. 1995. Mediation by HLA-DM of dissociation of peptides from
HLA-DR. Nature 375:802.
16. Salamero, J., M. Humbert, P. Cosson, and J. Davoust. 1990. Mouse B lymphocyte
specific endocytosis and recycling of MHC class II molecules. EMBO J. 9:3489.
17. Pinet, V., M. S. Malnati, and E. O. Long. 1994. Two processing pathways for the
MHC class II-restricted presentation of exogenous influenza virus antigen. J. Immunol. 152:4852.
18. Pinet, V. M., and E. O. Long. 1998. Peptide loading onto recycling HLA-DR
molecules occurs in early endosomes. Eur. J. Immunol. 28:799.
19. Mitchell, R. N., K. A. Barnes, S. A. Grupp, M. Sanchez, Z. Misulovin,
M. C. Nussenzweig, and A. K. Abbas. 1995. Intracellular targeting of antigens
internalized by membrane immunoglobulin in B lymphocytes. J. Exp. Med.
181:1705.
20. Shaw, A. C., R. N. Mitchell, Y. K. Weaver, J. Campos-Torres, A. K. Abbas, and
P. Leder. 1990. Mutations of immunoglobulin transmembrane and cytoplasmic
domains: effects on intracellular signaling and antigen presentation. Cell 63:381.
21. Siemasko, K., B. J. Eisfelder, C. Stebbins, S. Kabak, A. J. Sant, W. Song, and
M. R. Clark. 1999. Ig␣ and Ig␤ are required for efficient trafficking to late endosomes and to enhance antigen presentation. J. Immunol. 162:6518.
22. Bonnerot, C., D. Lankar, D. Hanau, D. Spehner, J. Davoust, J. Salamero, and
W. H. Fridman. 1995. Role of B cell receptor Ig␣ and Ig␤ subunits in MHC class
II-restricted antigen presentation. Immunity 3:335.
23. Fischer, M. B., M. Ma, S. Goerg, X. Zhou, J. Xia, O. Finco, S. Han, G. Kelsoe,
R. G. Howard, T. L. Rothstein, et al. 1996. Regulation of the B cell response to
T-dependent antigens by classical pathway complement. J. Immunol. 157:549.
24. Anh-Tuan, N., A. Falus, G. Füst, K. Merétey, and S. R. Hollan. 1984. Appearance
of covalently bound antigen in immune complexes formed during the activation
of complement. J. Immunol. Methods 75:257.
25. Villiers, M. B., C. L. Villiers, A. M. Laharie, and P. N. Marche. 1999. Amplification of the antibody response by C3b complexed to antigen through an ester
link. J. Immunol. 162:3647.
26. Villiers, M. B., C. L. Villiers, A. M. Laharie, and P. N. Marche. 1999. Different
stimulating effects of complement C3b and complete Freund’s adjuvant on antibody response. Immunopharmacology 42:151.
27. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, and D. T. Fearon.
1996. C3d of complement as a molecular adjuvant: bridging innate and acquired
immunity. Science 271:348.
28. Jacquier-Sarlin, M. R., F. M. Gabert, M. B. Villiers, and M. G. Colomb. 1995.
Modulation of antigen processing and presentation by covalently linked complement C3b fragment. Immunology 84:164.
29. Villiers, M. B., C. L. Villiers, M. R. Jacquier Sarlin, F. M. Gabert, A. M. Journet,
and M. G. Colomb. 1996. Covalent binding of C3b to tetanus toxin: influence on
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
Indeed, C3 induces some differences in antigenic peptides that can
be eluted from MHC-II (V. A. Serra, unpublished observations).
Similarly, as in early endosomes (18), peptides of high affinity may
be loaded on recycling MHC-II and could form highly stable
MHC-II-peptide complexes.
There are several candidates for the receptors inducing the targeting of C3b to CIIV-like compartments; among them are the
following: decay-accelerating factor (DAF; CD55), membrane cofactor protein (MCP; CD46), and CR1 (CD35). DAF is a GPIanchored receptor mainly localized at the plasma membrane in
lipid rafts (58). Like other GPI receptors, it may be internalized
into caveolae vesicles devoid of TfR. MCP is a transmembrane
receptor that can induce efficient Ag presentation by MHC-II (59,
60), indicating that this receptor gains access to some peptideloading compartments, but its intracellular traffic has not been
characterized. Furthermore, DAF and MCP are strongly expressed
on B lymphocytes. CR1 binds C3b and its internalization is not
mediated by clathrin-coated pits (61), but this receptor is very
weakly expressed on our cells and may be excluded as a single
candidate for intracellular targeting of C3b. However, it has been
established that CR1 could cooperate with another receptor: CR2
(CD21). CR2 has high affinity for C3dg and to a lesser degree for
iC3b, two fragments generated by cleavage of C3b, and it was
shown to induce very efficient presentation of C3d-HEL complexes (62). CR2/CR1 complexes induce intracellular signaling
and up-regulation of costimulatory molecules (63); these two receptors could also cooperate to enhance endocytosis of C3b and its
fragments (64), but their endocytic pathway has not been explored.
In conclusion, CR1 and DAF could account for the peculiar intracellular pathway of C3b, and MCP and CR2 could account for
efficient Ag presentation. Blocking agents specific to each receptor
could not individually inhibit C3b internalization, whereas their
use in combination is much more efficient (data not shown),
strongly suggesting that these receptors are not mutually exclusive.
The formation of complexes between Ags and C3b is a physiological process occurring naturally in vivo when complement system is activated, by immune complexes, for example. In vivo experiments on immunization have shown that Ag opsonization by
C3b improves the immune response, inducing a long-lasting Ab
response to low doses of Ag, indicating that this complement protein has an adjuvant effect (25, 26). In this study, we show that CR
and BCR are first targeted to distinct compartments, likely able to
induce modifications of the peptides generated, and then presented
to T cells. In contrast, a smaller fragment of C3, C3d rapidly targets Ags toward MIIC, via internalization by CD21/CD19 complexes (62, 65), illustrating the multiple roles of C3 that can be
relayed by several receptors. Taken together, these results indicate
that binding of C3b to Ags is an intrinsic and important step of the
immune response. The modification of Ag targeting induced by
C3b provides new explanation for the understanding of the adjuvant effect of C3b on humoral immunity.
3571
3572
30.
31.
32.
33.
34.
35.
36.
37.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49. Aluvihare, V. R., A. A. Khamlichi, G. T. Williams, L. Adorini, and
M. S. Neuberger. 1997. Acceleration of intracellular targeting of antigen by the
B-cell antigen receptor: importance depends on the nature of the antigen-antibody
interaction. EMBO J. 16:3553.
50. Cassard, S., J. Salamero, D. Hanau, D. Spehner, J. Davoust, W. H. Fridman, and
C. Bonnerot. 1998. A tyrosine-based signal present in Ig␣ mediates B cell receptor constitutive internalization. J. Immunol. 160:1767.
51. Salisbury, J. L., J. S. Condeelis, and P. Satir. 1980. Role of coated vesicles,
microfilaments, and calmodulin in receptor-mediated endocytosis by cultured B
lymphoblastoid cells. J. Cell Biol. 87:132.
52. Arndt, S. O., A. B. Vogt, S. Markovic-Plese, R. Martin, G. Moldenhauer,
A. Wolpl, Y. Sun, D. Schadendorf, G. J. Hammerling, and H. Kropshofer. 2000.
Functional HLA-DM on the surface of B cells and immature dendritic cells.
EMBO J. 19:1241.
53. Pathak, S. S., J. D. Lich, and J. S. Blum. 2001. Cutting edge: editing of recycling
class II:peptide complexes by HLA-DM. J. Immunol. 167:632.
54. Albert, L. J., L. K. Denzin, B. Ghumman, N. Bangia, P. Cresswell, and
T. H. Watts. 1998. Quantitative defect in staphylococcal enterotoxin A binding
and presentation by HLA-DM-deficient T2.Ak cells corrected by transfection of
HLA-DM genes. Cell. Immunol. 183:42.
55. Peters, P. J., J. J. Neefjes, V. Oorschot, H. L. Ploegh, and H. J. Geuze. 1991.
Segregation of MHC class II molecules from MHC class I molecules in the Golgi
complex for transport to lysosomal compartments. Nature 349:669.
56. Amigorena, S., P. Webster, J. Drake, J. Newcomb, P. Cresswell, and I. Mellman.
1995. Invariant chain cleavage and peptide loading in major histocompatibility
complex class II vesicles. J. Exp. Med. 181:1729.
57. Turley, S. J., K. Inaba, W. S. Garrett, M. Ebersold, J. Unternaehrer,
R. M. Steinman, and I. Mellman. 2000. Transport of peptide-MHC class II complexes in developing dendritic cells. Science 288:522.
58. Parolini, I., M. Sargiacomo, M. P. Lisanti, and C. Peschle. 1996. Signal transduction and glycophosphatidylinositol-linked proteins (lyn, lck, CD4, CD45, G
proteins, and CD55) selectively localize in Triton-insoluble plasma membrane
domains of human leukemic cell lines and normal granulocytes. Blood 87:3783.
59. Rivailler, P., M. C. Trescol-Biemont, C. Gimenez, C. Rabourdin-Combe, and
B. Horvat. 1998. Enhanced MHC class II-restricted presentation of measles virus
(MV) hemagglutinin in transgenic mice expressing human MV receptor CD46.
Eur. J. Immunol. 28:1301.
60. Gerlier, D., M. C. Trescol-Biemont, G. Varior-Krishnan, D. Naniche, I. FugierVivier, and C. Rabourdin-Combe. 1994. Efficient major histocompatibility complex class II-restricted presentation of measles virus relies on hemagglutininmediated targeting to its cellular receptor human CD46 expressed by murine B
cells. J. Exp. Med. 179:353.
61. Paccaud, J. P., W. Reith, B. Johansson, K. E. Magnusson, B. Mach, and
J. L. Carpentier. 1993. Clathrin-coated pit-mediated receptor internalization: role
of internalization signals and receptor mobility. J. Biol. Chem. 268:23191.
62. Cherukuri, A., P. C. Cheng, and S. K. Pierce. 2001. The role of the CD19/CD21
complex in B cell processing and presentation of complement-tagged antigens.
J. Immunol. 167:163.
63. Kozono, Y., R. Abe, H. Kozono, R. G. Kelly, T. Azuma, and V. M. Holers. 1998.
Cross-linking CD21/CD35 or CD19 increases both B7-1 and B7-2 expression on
murine splenic B cells. J. Immunol. 160:1565.
64. Grattone, M. L., C. L. Villiers, M. B. Villiers, C. Drouet, and P. N. Marche. 1999.
Co-operation between human CR1 (CD35) and CR2 (CD21) in internalization of
their C3b and iC3b ligands by murine-transfected fibroblasts. Immunology
98:152.
65. Hess, M. W., M. G. Schwendinger, E. L. Eskelinen, K. Pfaller, M. Pavelka,
M. P. Dierich, and W. M. Prodinger. 2000. Tracing uptake of C3dg-conjugated
antigen into B cells via complement receptor type 2 (CR2, CD21). Blood
95:2617.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
38.
uptake/internalization of antigen by antigen-specific and non-specific B cells.
Immunology 89:348.
Santoro, L., A. Reboul, I. Kerblat, C. Drouet, and M. G. Colomb. 1996. Monoclonal IgG as antigens: reduction is an early intracellular event of their processing
by antigen-presenting cells. Int. Immunol. 8:211.
Serra, V. A., F. Cretin, E. Pepin, F. M. Gabert, and P. N. Marche. 1997. Complement C3b fragment covalently linked to tetanus toxin increases lysosomal
sodium dodecyl sulfate-stable HLA-DR dimer production. Eur. J. Immunol.
27:2673.
Ahearn, J. M., M. B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou,
R. G. Howard, T. L. Rothstein, and M. C. Carroll. 1996. Disruption of the Cr2
locus results in a reduction in B-1a cells and in an impaired B cell response to
T-dependent antigen. Immunity 4:251.
Croix, D. A., J. M. Ahearn, A. M. Rosengard, S. Han, G. Kelsoe, M. Ma, and
M. C. Carroll. 1996. Antibody response to a T-dependent antigen requires B cell
expression of complement receptors. J. Exp. Med. 183:1857.
Wiersma, E. J., T. Kinoshita, and B. Heyman. 1991. Inhibition of immunological
memory and T-independent humoral responses by monoclonal antibodies specific
for murine complement receptors. Eur. J. Immunol. 21:2501.
Heyman, B., E. J. Wiersma, and T. Kinoshita. 1990. In vivo inhibition of the
antibody response by a complement receptor-specific monoclonal antibody.
J. Exp. Med. 172:665.
Hebell, T., J. M. Ahearn, and D. T. Fearon. 1991. Suppression of the immune
response by a soluble complement receptor of B lymphocytes. Science 254:102.
Gustavsson, S., T. Kinoshita, and B. Heyman. 1995. Antibodies to murine complement receptor 1 and 2 can inhibit the antibody response in vivo without inhibiting T helper cell induction. J. Immunol. 154:6524.
Carter, R. H., D. A. Tuveson, D. J. Park, S. G. Rhee, and D. T. Fearon. 1991. The
CD19 complex of B lymphocytes: activation of phospholipase C by a protein
tyrosine kinase-dependent pathway that can be enhanced by the membrane IgM
complex. J. Immunol. 147:3663.
Carter, R. H., and D. T. Fearon. 1992. CD19: lowering the threshold for antigen
receptor stimulation of B lymphocytes. Science 256:105.
Chavrier, P., R. G. Parton, H. P. Hauri, K. Simons, and M. Zerial. 1990. Localization of low molecular weight GTP binding proteins to exocytic and endocytic
compartments. Cell 62:317.
Guy, K., V. van Heyningen, B. B. Cohen, D. L. Deane, and C. M. Steel. 1982.
Differential expression and serologically distinct subpopulations of human Ia antigens detected by monoclonal antibodies to Ia ␣ and ␤ chains. Eur. J. Immunol.
12:942.
Roche, P. A., M. S. Marks, and P. Cresswell. 1991. Formation of a nine-subunit
complex by HLA class II glycoproteins and the invariant chain. Nature 354:392.
Sanderson, F., C. Thomas, J. Neefjes, and J. Trowsdale. 1996. Association between HLA-DM and HLA-DR in vivo. Immunity 4:87.
Salihi, A. A., F. Joisel, and M. Fontaine. 1982. Sensitive one-step haemolytic
assays of the fourth (C4) and fifth (C5) components of the human complement
system. Ann. Immunol. (Paris) 133C:299.
Perrin-Cocon, L. A., P. N. Marche, and C. L. Villiers. 1999. Purification of
intracellular compartments involved in antigen processing: a new method based
on magnetic sorting. Biochem. J. 338:123.
Fraker, P. J., and J. C. Speck. 1978. Protein and cell membrane iodination with
a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril.
Biochem. Biophys. Res. Commun. 80:849.
Saudrais, C., D. Spehner, H. de la Salle, A. Bohbot, J. P. Cazenave, B. Goud,
D. Hanau, and J. Salamero. 1998. Intracellular pathway for the generation of
functional MHC class II peptide complexes in immature human dendritic cells.
J. Immunol. 160:2597.
Perrin-Cocon, L. A., S. Chesne, I. Pignot-Paintrand, P. N. Marche, and
C. L. Villiers. 2001. Use of magnetic nanobeads for studies of intracellular antigen processing. J. Magn. Mater. Magnetism 225:161.
BCR AND CR INTRACELLULAR TARGETING OF Ags