MHC class II antigen processing in B cells: accelerated intracellular targeting of antigens.

Processing and presentation by Ag-specific B cells is initiated by Ag binding to the B cell Ag receptor (BCR). Cross-linking of the BCR by Ag results in a rapid targeting of the BCR and bound Ag to the MHC class II peptide loading compartment (IIPLC). This accelerated delivery of Ag may be essential in vivo during periods of rapid Ag-driven B cell expansion and T cell-dependent selection. Here, we use both immunoelectron microscopy and a nondisruptive protein chemical polymerization method to define the intracellular pathway of the targeting of Ags by the BCR. We show that following cross-linking, the BCR is rapidly transported through transferrin receptor-containing early endosomes to a LAMP-1+, beta-hexosaminadase+, multivesicular compartment that is an active site of peptide-class II complex assembly, containing both class II-invariant chain complexes in the process of invariant chain proteolytic removal as well as mature peptide-class II complexes. The BCR enters the class II-containing compartment as an intact mIg/Igalpha/Igbeta complex bound to Ag. The pathway by which the BCR targets Ag to the IIPLC appears not to be identical to that by which Ags taken up by fluid phase pinocytosis traffick, suggesting that the accelerated BCR pathway may be specialized and potentially independently regulated.     

Phosphoinositide 3-kinase activation by Igbeta controls de novo formation of an antigen-processing compartment

Antigens that bind B cell antigen receptor (BCR) are preferentially and rapidly processed for antigen presentation. The BCR is a multimeric complex containing a signaling module composed of Igalpha and Igbeta. Signaling pathways implicated in antigen presentation through the BCR are ill defined. Here we demonstrate that phosphoinositide 3-kinase (PI3K) inhibitors preclude antigen presentation induced by BCR or Igbeta but not Igalpha. Unraveling the mechanisms responsible for this inhibition, we show that PI3K inhibitors block neither antigen internalization nor degradation. Rather PI3K inhibitors block de novo formation of a multivesicular antigen processing compartment, which is induced by triggering of the BCR or Igbeta. Strikingly, we found using fluorescent probes binding specifically to PI3K products that BCR and Igbeta but not Igalpha induce PI3K activation in endocytic compartments wherein antigen is transported. Altogether, these results strongly suggest that Igbeta couples the BCR to PI3K activation that is instrumental for de novo formation of the antigen processing compartment and efficient antigen presentation.     

Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igalpha

Ags that cross-link the B cell Ag receptor are preferentially and rapidly delivered to the MHC class II-enriched compartment for processing into peptides and subsequent loading onto MHC class II. Proper sorting of Ag/receptor complexes requires the recruitment of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif tyrosines of the B cell Ag receptor constituent Igalpha. We postulated that the Igalpha nonimmunoreceptor tyrosine-based activation motif tyrosines, Y(176) and Y(204), contributed to receptor trafficking. Igalpha(YDeltaF(176,204))/Igbeta receptors were targeted to late endosomes, but were excluded from the vesicle lumen and could not facilitate the presentation of Ag to T cells. Subsequent analysis demonstrated that phosphorylation of Y(176)/Y(204) recruited the B cell linker protein, Vav, and Grb2. Reconstitution of Igalpha(YDeltaF(176,204))/Igbeta with the B cell linker protein rescued both receptor-facilitated Ag presentation and entry into the MHC class II-enriched compartment. Thus, aggregation accelerates receptor trafficking by recruiting two separate signaling modules required for transit through sequential checkpoints.     

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.     

Analysis of the individual contributions of Igalpha (CD79a)- and Igbeta (CD79b)-mediated tonic signaling for bone marrow B cell development and peripheral B cell maturation

The individual contribution of Igalpha and Igbeta for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Igalpha and Igbeta participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Igalpha and Igbeta under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Igalpha or Igbeta and Igalpha/Igbeta heterodimers with targeted Tyr-->Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2(-/-) primary pro-B cell lines and in gene transfer experiments in the muMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Igalpha/Igbeta ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Igalpha and Igbeta chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.     

Membrane-targeted peptides derived from Igalpha attenuate B-cell antigen receptor function

Within the B-cell antigen receptor (BCR), heterodimers of Igalpha/Igbeta couple the receptor to intracellular signaling pathways. In the resting state, Igalpha associates with Src-family tyrosine kinases (SFTKs) which contain some basal activity. Upon engagement of the receptor, the SFTKs phosphorylate tyrosine residues in the BCR that recruit and activate the tyrosine kinase Syk, initiating signaling pathways. To test the hypothesis that disrupting the association between the resting receptor and the SFTKs would attenuate both basal and induced receptor activities, we expressed non-phosphorylatable membrane-targeted analogs of Igalpha (Igalpha/M) or Igbeta (Igbeta/M) in B lymphocytes. Both Igalpha/M and Igbeta/M inhibited BCR-induced calcium mobilization, but only Igalpha/M was able to diminish tyrosine phosphorylation. In an immature B-cell line, Igalpha/M attenuated both receptor-induced and basal apoptosis. Taken together, these data demonstrate the importance of the resting receptor complex and suggest therapeutic strategies for regulating receptor-mediated functions.     

Expression and plasma membrane localization of the mammalian B‐cell receptor complex in transgenic Nicotiana tabacum

The B‐cell antigen receptor (BCR), displayed on the plasma membrane of mature B cells of the mammalian immune system, is a multimeric complex consisting of a membrane‐bound immunoglobulin (mIg) noncovalently associated with the Igα/Igβ heterodimer. In this study, we engineered transgenic tobacco plants expressing all four chains of the BCR. ELISA, Western blotting and confocal microscopy demonstrated that the BCR was correctly assembled in plants, predominantly in the plasma membrane, and that the noncovalent link was detergent sensitive. This is the first example of a noncovalently assembled plasma membrane‐retained heterologous receptor in plants. In B cells of the mammalian immune system, following antigen binding to mIg, BCR is internalized and tyrosine residues on Igα and Igβ are phosphorylated activating a signaling cascade through interaction with protein kinases that ultimately leads to the initiation of gene expression. Expression of the BCR may therefore be an important tool for the study of plant endocytosis and the identification of previously unknown plant tyrosine kinases. The specificity and diversity of the antibody repertoire, coupled to the signal transduction capability of the Igα/Igβ heterodimer, also indicates that plants expressing BCR may in future be developed as environmental biosensors.     

Dual requirement for the Ig alpha immunoreceptor tyrosine-based activation motif (ITAM) and a conserved non-Ig alpha ITAM tyrosine in supporting Ig alpha beta-mediated B cell development

Surface Ig (sIg) expression is a critical checkpoint during avian B cell development. Only cells that express sIg colonize bursal follicles, clonally expand, and undergo Ig diversification by gene conversion. Expression of a heterodimer, in which the extracellular and transmembrane domains of murine CD8alpha or CD8beta are fused to the cytoplasmic domains of chicken Igalpha (chIgalpha) or Igbeta, respectively (murine CD8alpha (mCD8alpha):chIgalpha + mCD8beta:chIgbeta), or an mCD8alpha:chIgalpha homodimer supported bursal B cell development as efficiently as endogenous sIg. In this study we demonstrate that B cell development, in the absence of chIgbeta, requires both the Igalpha ITAM and a conserved non-ITAM Igalpha tyrosine (Y3) that has been associated with binding to B cell linker protein (BLNK). When associated with the cytoplasmic domain of Igbeta, the Igalpha ITAM is not required for the induction of strong calcium mobilization or BLNK phosphorylation, but is still necessary to support B cell development. In contrast, mutation of the Igalpha Y3 severely compromised calcium mobilization when expressed as either a homodimer or a heterodimer with the cytoplasmic domain of Igbeta. However, coexpression of the cytoplasmic domain of Igbeta partially complemented the Igalpha Y3 mutation, rescuing higher levels of BLNK phosphorylation and, more strikingly, supporting B cell development.     

Cutting edge: a hypomorphic mutation in Igbeta (CD79b) in a patient with immunodeficiency and a leaky defect in B cell development

Although null mutations in Igalpha have been identified in patients with defects in B cell development, no mutations in Igbeta have been reported. We recently identified a patient with a homozygous amino acid substitution in Igbeta, a glycine to serine at codon 137, adjacent to the cysteine required for the disulfide bond between Igalpha and Igbeta. This patient has a small percentage of surface IgM(dim) B cells in the peripheral circulation (0.08% compared with 5-20% in healthy controls). Using expression vectors in 293T cells or Jurkat T cells, we show that the mutant Igbeta can form disulfide-linked complexes and bring the mu H chain to the cell surface as part of the BCR but is inefficient at both tasks. The results show that minor changes in the ability of the Igalpha/Igbeta complex to bring the BCR to the cell surface have profound effects on B cell development.     

Ig-independent Ig beta expression on the surface of B lymphocytes after B cell receptor aggregation

In order for humoral immune responses to develop, B cells must be able to recognize, bind, and internalize Ags. These functions are performed by the BCR, which is also responsible for initiating and transducing activation signals necessary for B cell proliferation and differentiation. We have examined surface expression patterns of individual components of the BCR following anti-Ig- and Ag-induced aggregation. Specifically, the localization and expression levels of the Ag-binding component, surface Ig (sIg), and the Igbeta component of the Igalpha/Igbeta signaling unit were investigated to determine their individual participation in the internalization and signal transduction. Using primary murine B cells, we found that while >95% of the sIg is internalized following anti-Ig-induced aggregation, 20-30% of Igbeta remains on the surface. These results suggest that sIg and Igbeta may function independently following the initial stages of signal transduction.     

The Syk-binding ubiquitin ligase c-Cbl mediates signaling-dependent B cell receptor ubiquitination and B cell receptor-mediated antigen processing and presentation

B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.     

The cytoplasmic domain of Ig alpha is necessary and sufficient to support efficient early B cell development

The B cell receptor complex (BcR) is essential for normal B lymphocyte function, and surface BcR expression is a crucial checkpoint in B cell development. However, functional requirements for chains of the BcR during development remain controversial. We have used retroviral gene transfer to introduce components of the BcR into chicken B cell precursors during embryonic development. A chimeric heterodimer, in which the cytoplasmic domains of chicken Igalpha and Igbeta are expressed by fusion with the extracellular and transmembrane domains of murine CD8alpha and CD8beta, respectively, targeted the cytoplasmic domains of the BcR to the cell surface in the absence of extracellular BcR domains. Expression of this chimeric heterodimer supported all early stages of embryo B cell development: bursal colonization, clonal expansion, and induction of repertoire diversification by gene conversion. Expression of the cytoplasmic domain of Igalpha, in the absence of the cytoplasmic domain of Igbeta, was not only necessary, but sufficient to support B cell development as efficiently as the endogenous BcR. In contrast, expression of the cytoplasmic domain of Igbeta in the absence of the cytoplasmic domain of Igalpha failed to support B cell development. The ability of the cytoplasmic domain of Igalpha to support early B cell development required a functional Igalpha immunoreceptor tyrosine-based activation motif. These results support a model in which expression of surface IgM following productive V(D)J recombination in developing B cell precursors serves to chaperone the cytoplasmic domain of Igalpha to the B cell surface, thereby initiating subsequent stages of development.     

T helper cell-dependent B cell activation.

Small, resting B lymphocytes are driven into the cell cycle as a consequence of receiving multiple signals from elements found within their local environment. The first of these signals results from the binding of specific antigen to membrane immunoglobulin (mIg) receptors on the B cells. Pursuant to antigen binding, signals are transduced and the B cell commences to endocytose and degrade the antigen. Fragments of the antigen are expressed on the B cell surface in noncovalent association with class II major histocompatibility complex (MHC) molecules. The antigen-class II MHC complex serves as a recognition complex for CD4+ helper T cells (Th). As a consequence of recognition, Th form stable physical conjugates with the B cells. Over an extended period of time the Th and B cells bilaterally signal one another. This interchange of signals results in the growth and differentiation of both cells. This review will discuss the sequence of events that culminate in the growth and differentiation of B lymphocytes to antibody-producing cells.     

Ig alpha/Ig beta complexes generate signals for B cell development independent of selective plasma membrane compartmentalization

Ligand-induced BCR association with detergent-resistant plasma membrane compartments (lipid rafts) has been argued to be essential for initiating and/or sustaining Igalpha/Igbeta-dependent BCR signaling. Because a fraction of the BCR and an even larger fraction of the preBCR associates with lipid rafts in the apparent absence of ligand stimulation, it has been proposed that raft-associated receptor complexes mediate the ligand-independent basal signaling events observed in resting B lineage cells. However, there is no direct evidence that localization of Igalpha/Igbeta-containing complexes to detergent-resistant membrane compartments is absolutely required for the signaling events that drive B cell development. To address these issues we have designed surrogate preBCR/Igalpha/Igbeta complexes that are incapable of ligand-induced aggregation and that are preferentially targeted to either raft or nonraft compartments. An analysis of their ability to promote the preBCR-dependent proB-->preB cell transition of murine B cell progenitors revealed that expression of these surrogate receptor complexes at levels that approximate that of the conventional preBCR can drive B cell development in a manner independent of both aggregation and lipid raft localization.     

The actin cytoskeleton coordinates the signal transduction and antigen processing functions of the B cell antigen receptor

The B cell antigen receptor （BCR） is the sensor on the B cell surface that surveys foreign molecules （antigen） in our bodies and activates B cells to generate antibody responses upon encountering cognate antigen. The binding of antigen to the BCR induces signaling cascades in the cytoplasm, which provides the first signal for B cell activation. Subsequently, BCRs internalize and target bound antigen to endosomes, where antigen is processed into T cell recognizable forms. T helper cells generate the second activation signal upon binding to antigen presented by B cells. The optimal activation of B cells requires both signals, thereby depending on the coordination of BCR signaling and antigen transport functions. Antigen binding to the BCR also induces rapid remodeling of the cortical actin network of B cells. While being initiated and controlled by BCR signaling, recent studies reveal that this actin remodeling is critical for both the signaling and antigen processing functions of the BCR, indicating a role for actin in coordinating these two pathways. Here we will review previous and recent studies on actin reorganization during BCR activation and BCR- mediated antigen processing, and discuss how actin remodeling translates BCR signaling into rapid antigen uptake and processing while providing positive and negative feedback to BCR signaling.     

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR-Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II-invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR-Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II-peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation.     

Functional early endosomes are required for maturation of major histocompatibility complex class II molecules in human B lymphoblastoid cells.

Major histocompatibility complex (MHC) class II molecules are targeted together with their invariant chain (Ii) chaperone from the secretory pathway to the endocytic pathway. Within the endosome/lysosome system, Ii must be degraded to enable peptide capture by MHC class II molecules. It remains controversial exactly which route or routes MHC class II/Ii complexes take to reach the sites of Ii processing and peptide loading. We have asked whether early endosomes are required for successful maturation of MHC class II molecules by using an in situ peroxidase/diaminobenzidine compartment ablation technique. Cells whose early endosomes were selectively ablated using transferrin-horseradish peroxidase conjugates fail to mature their newly synthesized MHC class II molecules. We show that whereas transport of secretory Ig through the secretory pathway is virtually normal in the ablated cells, newly synthesized MHC class II/Ii complexes never reach compartments capable of processing Ii. These results strongly suggest that the transport of the bulk of newly synthesized MHC class II molecules through early endosomes is obligatory and that direct input into later endosomes/lysosomes does not take place.     

What to do with HLA-DO?

Antigenic peptide binding to MHC class II molecules in the endocytic pathway occurs via a multifactorial process that requires the support of a specialized lysosomal chaperone called HLA-DM. DM shows both in primary amino acid sequence and quaternary structure a high homology to both MHC class I and class II molecules. Like the peptide presenting class II molecules, DM is expressed in all professional antigen presenting cells. DM catalyzes the dissociation of peptides that do not bind stably to the class II peptide-binding groove, thereby leading to the preferential presentation of stably binding antigenic peptides. The recently discovered HLA-DO molecule is mainly expressed in B cells and associates with DM, thereby markedly affecting DM function. Like DM, the genes encoding the HLA-DO heterodimer lie within the MHC class II region and exhibit strong homology to classical class II molecules. This review evaluates the unique effects of DO on DM-mediated antigen presentation by MHC class II molecules and discusses the possible physiological relevance for the B cell-specific expression of DO and its function.     

Antigen-B Cell Receptor Complexes Associate with Intracellular major histocompatibility complex (MHC) Class II Molecules

Antigen processing and MHC class II-restricted antigen presentation by antigen-presenting cells such as dendritic cells and B cells allows the activation of naïve CD4+ T cells and cognate interactions between B cells and effector CD4+ T cells, respectively. B cells are unique among class II-restricted antigen-presenting cells in that they have a clonally restricted antigen-specific receptor, the B cell receptor (BCR), which allows the cell to recognize and respond to trace amounts of foreign antigen present in a sea of self-antigens. Moreover, engagement of peptide-class II complexes formed via BCR-mediated processing of cognate antigen has been shown to result in a unique pattern of B cell activation. Using a combined biochemical and imaging/FRET approach, we establish that internalized antigen-BCR complexes associate with intracellular class II molecules. We demonstrate that the M1-paired MHC class II conformer, shown previously to be critical for CD4 T cell activation, is incorporated selectively into these complexes and loaded selectively with peptide derived from BCR-internalized cognate antigen. These results demonstrate that, in B cells, internalized antigen-BCR complexes associate with intracellular MHC class II molecules, potentially defining a site of class II peptide acquisition, and reveal a selective role for the M1-paired class II conformer in the presentation of cognate antigen. These findings provide key insights into the molecular mechanisms used by B cells to control the source of peptides charged onto class II molecules, allowing the immune system to mount an antibody response focused on BCR-reactive cognate antigen.