BCR-mediated Internalization Research Articles

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.

Defects in Germinal Center Selection in SLE.

Germinal centers (GCs) are the primary site at which clonal expansion and affinity maturation of B cells occur. B cells encounter antigen and receive T cell help in the GC light zone (LZ) and then migrate to the dark zone where they proliferate and undergo somatic mutation before cycling back to the LZ for further rounds of selection. Tolerance to autoantigens is frequently lost de novo as GC B cells undergo class switching and somatic mutation. This loss of tolerance is regulated by a variety of mechanisms including cell death, failure to compete for T cell help, and failure to differentiate into effector cells. Systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nucleic acid antigens. While defects in tolerance occur in the naïve repertoire of SLE patients, pathogenic autoantibodies also arise in the GC by somatic mutation from non-autoreactive precursors. Several B cell defects contribute to the loss of GC tolerance in SLE, including polymorphisms of genes encoded by the Sle1 locus, excess TLR7 signaling, defects in FcRIIB expression, or defects of B cell apoptosis. Extrinsic soluble factors, such as Type-1 IFN and B cell-activating factor, or an increased number of T follicular helper cells in the GC also alter B cell-negative selection. Finally, defects in clearance of apoptotic debris within the GC result in BCR-mediated internalization of nucleic acid containing material and stimulation of autoantibody production by endosomal TLR-driven mechanisms.

Multivalent Antigens for Promoting B and T Cell Activation.

Efficacious vaccines require antigens that elicit productive immune system activation. Antigens that afford robust antibody production activate both B and T cells. Elucidating the antigen properties that enhance B-T cell communication is difficult with traditional antigens. We therefore used ring-opening metathesis polymerization to access chemically defined, multivalent antigens containing both B and T cell epitopes to explore how antigen structure impacts B cell and T cell activation and communication. The bifunctional antigens were designed so that the backbone substitution level of each antigenic epitope could be quantified using (19)F NMR. The T cell peptide epitope was appended so that it could be liberated in B cells via the action of the endosomal protease cathepsin D, and this design feature was critical for T cell activation. Antigens with high BCR epitope valency induce greater BCR-mediated internalization and T cell activation than did low valency antigens, and these high-valency polymeric antigens were superior to protein antigens. We anticipate that these findings can guide the design of more effective vaccines.

HAX1 deletion impairs BCR internalization and leads to delayed BCR-mediated apoptosis.

Deletion of HAX1 in mice causes a severe reduction in the numbers of lymphocytes in the bone marrow and in the spleen. Additionally, B220+ B progenitor cells in the bone marrow are reduced, suggesting an important function of HAX1 in B cell development. HAX1 is thought to play a protective role in apoptotic processes; therefore, we investigated the role of HAX1 in bone marrow B progenitor cells and splenic B cells. We did not observe an effect on the survival of Hax1−/− bone marrow cells but detected enhanced survival of splenic Hax1−/− B cells upon in vitro starvation/growth-factor withdrawal. To explain this apparent inconsistency with previous reports of HAX1 function, we also studied the B cell receptor (BCR)-induced apoptosis of IgM-stimulated splenic naïve B cells and found that apoptosis decreased in these cells. We further found impaired internalization of the BCR from Hax1−/− splenic B cells after IgM crosslinking; this impaired internalization may result in decreased BCR signaling and, consequently, decreased BCR-mediated apoptosis. We measured HAX1 binding to the cytoplasmic domains of different Ig subtypes and identified KVKWI(V)F as the putative binding motif for HAX1 within the cytoplasmic domains. Because this motif can be found in almost all Ig subtypes, it is likely that HAX1 plays a general role in BCR-mediated internalization events and BCR-mediated apoptosis.

PDE4 Inhibitors Block TLR7 and TLR9-Driven Signaling, Proliferation and Cytokine Secretion in CLL Cells As Well As Proliferation Driven by Exposure to Apoptotic Cells

AbstractAbstract 1774A growing body of literature supports the concept that at least a subset of chronic lymphocytic leukemia (CLL) B cell-receptors (BCRs) are reactive with antigens exposed on apoptotic cells. Work done in the autoimmunity field suggests that through their BCRs, autoreactive B cells can internalize RNA and DNA-containing autoantigens, thereby activating endosomal Toll-like receptor (TLR) 7 and TLR9, respectively, with subsequent TLR-mediated signaling, proliferation and cytokine secretion. cAMP signaling modulates inflammatory signaling in immune system cells and our prior studies have shown that PKA, EPAC and CREB-mediated cAMP signaling can be potently induced in CLL cells with inhibitors of the cyclic nucleotide phosphodiesterase (PDE) PDE4 family in the absence of additional exogenous stimulators of adenylate cyclase. We therefore examined the ability of PDE4 inhibitor-mediated cAMP signaling to regulate TLR7 and 9-mediated signaling in CLL cells. Treatment of CLL cells with the TLR7 and 9 agonists R848 and CpG-B oligonucleotide, respectively, induced IRAK1 degradation and activation of three signaling pathways known to be downstream of TLR7 and 9: the transcription factors NFkB and IRF5 as well as the MAP kinase JNK. The prototypic PDE4 inhibitor rolipram inhibited R848-induced TLR7 and CpG-B-induced TLR9 signaling in CLL cells as judged by Western analysis and immunofluorescent studies of NFkB and IRF5 nuclear translocation as well as JNK phosphorylation. Rolipram also blocked R848 and CpG-B-induced CLL proliferation and TNF secretion. In contrast, rolipram augmented R848 and CpG-B-induced CLL IL-10 synthesis, in keeping with prior studies documenting cAMP response elements in the IL-10 promoter. To test whether RNA and/or DNA antigens exposed in apoptotic cells might induce proliferation in CLL cells through BCR-mediated internalization and subsequent activation of TLR7 and TLR9, we irradiated CLL cells and added them in a graded fashion to live autologous CLL cells. Such exposure to irradiated cells undergoing apoptosis drove CLL proliferation in a manner that was sensitive to TLR7 and TLR9 antagonists, IRAK4 kinase inhibitors and the PDE4 inhibitor rolipram. Our work supports the concept that BCR and TLR-driven responses to apoptotic RNA and DNA-containing complexes may drive proliferation in CLL cells and that PDE4 inhibitors and other agents that target TLR signaling may prove to be effective therapeutic agents in this disease. Disclosures:No relevant conflicts of interest to declare.

B Cell Receptor-Mediated Internalization of Salmonella: A Novel Pathway for Autonomous B Cell Activation and Antibody Production

The present paradigm is that primary B cells are nonphagocytosing cells. In this study, we demonstrate that human primary B cells are able to internalize bacteria when the bacteria are recognized by the BCR. BCR-mediated internalization of Salmonella typhimurium results in B cell differentiation and secretion of anti-Salmonella Ab by the Salmonella-specific B cells. In addition, BCR-mediated internalization leads to efficient Ag delivery to the MHC class II Ag-loading compartments, even though Salmonella remains vital intracellularly in primary B cells. Consequently, BCR-mediated bacterial uptake induces efficient CD4(+) T cell help, which boosts Salmonella-specific Ab production. BCR-mediated internalization of Salmonella by B cells is superior over extracellular Ag extraction to induce rapid and specific humoral immune responses and efficiently combat infection.

B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo.

Highly regulated activation of B cells is required for the production of specific antibodies necessary to provide protection from pathogen infection. This process is initiated by specific recognition of antigen through the B cell receptor (BCR), leading to early intracellular signaling followed by the late recruitment of T cell help. In this study we demonstrate that specific BCR uptake of CD1d-restricted antigens represents an effective means of enhancing invariant natural killer T (iNKT)-dependent B cell responses in vivo. This mechanism is effective over a wide range of antigen affinities but depends on exceeding a tightly regulated avidity threshold necessary for BCR-mediated internalization and CD1d-dependent presentation of particulate antigenic lipid. Subsequently, iNKT cells provide the help required for stimulating B cell proliferation and differentiation. iNKT-stimulated B cells develop within extrafollicular foci and mediate the production of high titers of specific IgM and early class-switched antibodies. Thus, we have demonstrated that in response to particulate antigenic lipids iNKT cells are recruited for the assistance of B cell activation, resulting in the enhancement of specific antibody responses. We propose that such a mechanism may operate to potentiate adaptive immune responses against pathogens in vivo.

Functional and structural requirements for the internalization of distinct BCR‐ligand complexes

Antigen (Ag) binding to the BCR rapidly initiates two important events: a phosphorylation cascade that results in the production of secondary signaling intermediaries and the internalization of Ag-BCR complexes. Previous studies using anti-BCR antibodies (Ab) have suggested that BCR signaling is an essential requirement for BCR endocytosis and have further implicated lipid rafts as essential platforms for both BCR functions. However, published data from our laboratory indicate that lipid rafts and consequently raft-mediated signaling are dispensable for BCR-mediated internalization of Ag-specific BCR. Therefore, we investigated the relationship between BCR signaling and endocytosis by defining the role of early kinase signaling in the BCR-mediated internalization of a model Ag (haptenated protein). The results demonstrate that Src kinases and Syk-mediated BCR signaling are not essential for BCR-mediated Ag internalization. Moreover, by comparing Ag and Ab, it was determined that while both localize to clathrin-coated pits, the internalization of Ab-BCR complexes is more susceptible to inhibition of signaling and highly sensitive to disruption of lipid rafts and the actin cytoskeleton compared to Ag-BCR complexes. Thus, these results demonstrate that the nature of the ligand ultimately determines the functional requirements and relative contribution of lipid rafts and other membrane structures to the internalization of BCR-ligand complexes.

Inhibition of B-cell receptor-antigen complex internalization by Fc γRIIB1 signals

Membrane-expressed immunoglobulins are B-cell receptors (BCR) for specific antigens (Ag). Upon Ag engagement of the BCR, B-cells are activated to internalize Ag-BCR complexes, process Ag and subsequently present Ag-peptides loaded in class II MHC. Due to the specific nature of the BCR, the cognate interaction between T-cells expressing Ag-specific T-cell receptor and these Ag-presenting B-cells occur in a highly regulated and precise manner. Accordingly, efficient control of T-cell activation may be achieved through regulation of Ag presenting B-cells. A potent form of regulation of lymphocyte responses is mediated by Ig end-product and anti-idiotypic antibodies via Fc-dependent mechanisms. In this communication, the authors present data that an anti-idiotype (anti-Id) Ab inhibits BCR-mediated internalization of specific Ag. Coupling of BCR to the cytoskeleton was also abortive in anti-Id Ab-treated B-cells. Inhibition by anti-Id Ab was dependent upon the presence of Fc γRIIB1 on B-cells. As a result of anti-Id Ab suppression, B-cells were unable to initiate Ca 2+responses in Ag-specific T-cells. The results suggest that co-crosslinking of Fc γRIIB1 and BCR inhibits cytoskeletal coupling and internalization of the Ag-BCR complex thereby preventing specific Ag presentation by B-cells. Anti-Id Ab may mediate a negative regulatory mechanism that suppresses B-cell-mediated Ag-specific T-cell activation.