UC Davis

Efficient IgG responses are the basis for many vaccines strategies. B cells differentiate into high affinity antibody-producing plasma cells because of activation steps that involve antigen processing and presentation (via MHC class II molecules) to CD4+ "helper" T cells. Current understanding is that this process involves protein antigens becoming captured by B cells via their B cell receptor (BCR), which consists of membrane-bound IgM and associated signaling chains CD79a and CD79b (IgM-BCR). Following antigen-capture, the IgM-BCR antigen complex is internalized, protein antigens are degraded into peptides and peptides are then integrated into MHCII molecules and presented on the B cell surface to CD4 T cells. Following antigen presentation to cognate CD4 helper T cells, B and T cells will become further activated and enter differentiation programs, including the development of IgG-secreting plasma cells. Understanding the mechanisms that support maximal IgG response induction is significant, as it can be exploited for vaccine development and design as well as lead to a better understanding of immunity to infections.

The immunoglobulin isotype M (IgM) is present on cell surfaces of all developing B cells as IgM-BCR. Additionally, IgM is present in a secreted form (sIgM). IgM is the evolutionary most conserved antibody isotype and is present in all jawed vertebrates. Previous experiments have shown that mice lacking sIgM and infected with influenza virus had reduced virus-specific serum IgG, increasing their susceptibility to death and inability to clear infection. Together, this suggests that sIgM is critical for effective IgG responses.

Although sIgM has been reported to bind various cell surface receptors, there is only one receptor that it binds with high affinity: the FcμR, a receptor highly expressed on B cells. Previously, our lab showed that mice lacking the FcμR (FcμR-/-) have significantly reduced IgG responses after influenza infection, like sIgM-deficient mice. This dissertation was designed to test the hypothesis that the FcμR mediates the uptake and processing of sIgM-antigen complexes, augmenting presentation of antigen to helper T cells and thereby enhancing subsequent IgG responses. My dissertation work confirmed this hypothesis and in addition identified a novel function for the FcμR in supporting IgM-BCR antigen complex internalization, and antigen processing and presentation.

Chapter 1 reviews the literature on the structure and function of sIgM and the FcμR. The functions of secreted IgM have been extensively studied and are outlined in this review with focus on potential roles and connections to IgG secretion, as well as its recently discovered structure and interaction with AIM/CD5L. The FcμR, was described originally as a receptor that prevents Fas mediated apoptosis but was later rediscovered as a receptor that binds exclusively to the Fc region of IgM. The major questions raised in the review and its focus revolve around how sIgM-FcμR interactions together support maximal IgG responses.

Chapter 2 describes establishment of a system that can model the dynamics of T-dependent B cell responses in vitro and in vivo. For that, I generated a conjugate protein antigen, hen-egg lysozyme (HEL) fused to ovalbumin (OVA), for which previously described HEL-specific BCR- and OVA-specific T cell receptor (TCR)-transgenic mice were bred and utilized. The BCR transgenic “SWHEL” mice are gene “knock-in” mice that express and secrete HEL-specific IgM and can undergo class-switch recombination to secrete other Ig isotypes, such as IgG. The SWHEL mice were backcrossed onto mice, which have been described previously, that lack the FcuR. The “OT-II” mouse contains a transgene encoding OVA-specific TCR presented in the context of MHC-II I-Ab, as expressed in C57BL/6 mice. In this system, B cells either expressing or not the FcuR bind to HEL, internalize, and process the HEL-OVA conjugate and present OVA peptides in MHC-II I-Ab to OT-II CD4 T cells, which are then activated to proliferate. To track antigen via confocal and STED microscopy, as well as flow cytometry, the fluorescent molecule BODIPY was conjugated to whole HEL protein. Chapter 2 outlines the generation of these reagents and assessment for their use in subsequent experiments, which are outlined in Chapter 3.

Chapter 3 demonstrates reduced serum IgG responses to subcutaneous immunization with HEL-OVA conjugate in SWHEL transgenic mice lacking the FcuR compared to SWHEL controls that express the FcuR, as measured by ELISA. Given that up to 20% of B cells in the SWHEL mouse are HEL-binding, mixed bone marrow irradiation chimeric mice were generated that contained 2% SWHEL transgenic B cells with or without the FcuR, which were similarly assessed for their IgG responses but in the context of more physiological frequencies of antigen-specific B cells. The results were consistent with those obtained with total SWHEL transgenic mice. To investigate what role the FcμR plays in the B cell’s ability to internalize and present antigen to helper (CD4+) T cells, HEL-OVA construct was used containing a small peptide that when integrated into I-Ab was recognized by a commercially available monoclonal antibody. The results demonstrated significant reductions in MHC-peptide complex surface expression and reduced antigen-presentation of B cells to CD4 T cells in the absence of the FcuR. Utilization of STED and confocal microscopy assessed the protein kinetics and localization of the internalized IgM-BCR, antigen and FcμR as well as other intracellular antigen marking the pathway of endocytosis, such as RAB11, LAMP-1, and EEA1. Collectively, the results demonstrate the need for FcuR expression in IgM-BCR internalization and antigen processing. Finally, addition of HEL-specific sIgM was shown to further enhance antigen-presentation by FcuR sufficient but not deficient B cells. Thus, this chapter demonstrates that the FcμR acts as a chaperone for IgM-BCR complexed with antigen and a second, albeit less prominent role for the receptor in binding antigen-sIgM complexes further enhancing antigen presentation to CD4+ T cells.