B Cell Receptors and Complement Receptors Target the Antigen to Distinct Intracellular Compartments 1 Laure A. Perrin-Cocon,* Christian L. Villiers, 2 * Jean Salamero, † Franc ¸oise Gabert,* and Patrice N. Marche* 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 con- taining 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 Immu- nology, 2004, 172: 3564 –3572. T he efficiency of Ag presentation depends not only on the efficiency of Ag internalization but also on the intracel- lular transport of Ags toward compartments equipped for processing and loading of antigenic peptides on MHC class II mol- ecules (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). Neo- synthesized 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- ecules are present throughout the endocytic pathway (9). The ad- dressing 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) catal- ysis, 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 mol- ecules (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 address- ing 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 se- verely impaired Ab responses to T cell-dependent Ags (23). Under *Laboratoire d’Immunochimie, De ´partement de Re ´ponse et Dynamique Cellulaires, Commissariat a ` l’Energie Atomique, Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 548, Universite ´ Joseph Fourier, Grenoble, France; and † Laboratoire de Compartimentation et Dynamique Cellulaire, Unite ´ Mixte de Recherche 144, Cen- tre 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 De ´le ´gation Ge ´ne ´rale de l’Armement (DSP 99-34-038) and the Ministe `re de l’E ´ ducation Nationale, de la Re- cherche, et de la Technologie (Programme de Recherche Fondamentale en Microbi- ologie et Maladies Infectieuses et Parasitaires) and by institutional grants from Institut National de la Sante ´ et de la Recherche Me ´dicale and Commissariat a ` l’Energie Atomique. L.A.P.-C. was initially supported by a fellowship from the Ministe `re de l’E ´ ducation 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, Labora- toire d’Immunochimie, De ´partement de Re ´ponse et Dynamique Cellulaires, Commis- sariat a ` l’Energie Atomique, 17 rue des Martyrs, F-38054 Grenoble, cedex 9, France. E-mail address: immuno@dsvsud.cea 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 re- ceptor; Lamp-2, lysosomal-associated membrane protein-2; PNS, postnuclear super- natant; MF, magnetic fraction; NP-40, Nonidet P-40; CIIV, class II-containing ves- icle; DAF, decay-accelerating factor; MCP, membrane cofactor protein. The Journal of Immunology Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00