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B Cell Receptor and Toll-like Receptor Signaling Integrate to Control Distinct B-1a Responses to both Self-Antigens and the Microbiota

  • Author(s): Kreuk, Lieselotte Sarah Maria
  • Advisor(s): Barton, Gregory
  • et al.
Abstract

Abstract

B Cell Receptor and Toll-like Receptor Signaling Integrate to Control Distinct B-1a Responses to both Self-Antigens and the Microbiota

By

Lieselotte Sarah Maria Kreuk

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor Gregory Barton, Chair

B-1a cells are innate-like lymphocytes that play an important role in mediating tissue homeostasis and protection against infections. These cells are the main producers of “natural” IgM, spontaneously secreted serum antibodies predominately reactive to self-antigens like phosphatidylcholine (PtC) on dead or dying cells or bacterial antigens present in the intestinal microbiota. In addition, B-1a cells are a major source of microbiota-reactive IgG3 and IgA. Whether production of these antibodies relies on clonal activation through the B cell receptor (BCR) versus polyclonal activation from innate immune stimuli remains poorly understood.

In this dissertation, I aim to reconcile conflicting studies on the contribution of BCR and Toll-like receptor (TLR) signaling in regulating B-1a specificity, activation, and function. Through the use of an in vivo reporter mouse that serendipitously marked a subset of antibody-secreting activated B-1a cells, I demonstrate a skewing of both anti-PtC and anti-microbiota reactivities within this activated subset, suggesting ongoing BCR-mediated activation of B-1a cells at steady state. I also show that a microbiota is required for B-1a-derived microbiota-reactive IgM but not for reactivity to the self-antigen PtC, supporting a model where B-1a cell responses to foreign versus endogenous antigens are distinctly regulated.

Intriguingly, unlike in germ-free mice, TLR-deficient mice have a significant reduction in both PtC- and microbiota-reactive B-1a responses, although distinct subsets of TLRs regulate responses against self-antigens versus microbiota-derived antigens. Whereas Unc93b1-dependent nucleic acid sensing TLRs appear to regulate anti-PtC B-1a reactivity in the peritoneal cavity, TLR2 and TLR4 regulate anti-commensal B-1a-derived serum IgM. Because commensal bacteria, which can express LPS or surface peptidoglycans, provide an antigenic context for TLR4 and TLR2 stimulation, and dead or dying cells exposing phosphatidylcholine and nuclear material like DNA or RNA may be capable of stimulating Unc93b1-dependent nucleic acid sensing TLRs, I propose a model whereby dual BCR and TLR signaling integrate to control distinct B-1a responses.

In order to determine a more global picture of the role of distinct TLRs in regulating B-1a specificity, I performed heavy chain CDR3 sequencing from peritoneal cavity and spleen B-1a cells from mice lacking different subsets of TLRs. These studies identified several new heavy chain variable region genes regulated by nucleic acid sensing TLR signaling. Additionally, I identified a novel role for TLR2 and TLR4 in regulating general diversity of splenic B-1a cells.

Finally, I found that TLR activation of B-1a cells leads to the reduction of surface CD5, a negative regulator of BCR signaling, suggesting a potential mechanism rendering B-1a cells responsive to BCR¬-mediated stimulation. Altogether, the work presented in this dissertation reveals substantial heterogeneity with the B-1a cell subset and an updated understanding of the role of B cell receptor versus innate immune receptor stimulation in regulating B-1a specificity and function.

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