Fine-tuning B cell antigen receptor signaling by reactive oxygen species and diacylglycerol metabolism
- Author(s): Wheeler, Matthew Lewis
- Advisor(s): DeFranco, Anthony L
- et al.
Recognition of cognate antigen by the B cell antigen receptor (BCR) is one of the primary events that ultimately facilitate the production of pathogen-specific antibodies that are critical for host immunity. The process of B cell activation by antigen must be tightly regulated to ensure robust responses against pathogens while limiting responses directed at our own tissues. Much of this regulation is controlled at the level of signal transduction downstream of the BCR which requires a precise balance between signal amplification and feedback inhibition of pathways activated downstream of this receptor. The work presented in this thesis explores two distinct mechanisms by which signaling downstream of the BCR is regulated to govern the response of cells to cognate antigen stimulation.
Chapter 2 describes studies addressing the intracellular sources and physiological function of reactive oxygen species (ROS) produced in primary B cells in response to BCR stimulation. It was found that BCR stimulation of primary resting murine B cells induced the rapid production of ROS that occurred within minutes, and was maintained for at least 24 h following receptor stimulation. While the early production of ROS (0-2 h) was dependent on the Nox2 isoform of NADPH oxidase, at later stages of B cell activation (6-24 h) ROS were generated by a second pathway, which appeared to be dependent on mitochondrial respiration. B cells from mice deficient in the Nox2 NADPH oxidase complex lacked detectible early production of extracellular and intracellular ROS following BCR stimulation, but had normal proximal BCR signaling and BCR-induced activation and proliferation in vitro, and mounted normal or somewhat elevated antibody responses in vivo. In contrast, neutralizing both pathways of BCR-derived ROS with the scavenger N-acetylcysteine resulted in impaired in vitro BCR-induced activation and proliferation, and attenuated BCR signaling through the phosphatidylinositol 3-kinase pathway at later times. These results indicate that the production of ROS downstream of the BCR is derived from at least two distinct cellular sources and plays a critical role at the later stages of B cell activation by promoting sustained BCR signaling via the phosphatidylinositol 3-kinase signaling pathway, which is needed for effective B cell responses to antigen.
Chapter 3 describes studies addressing the role of metabolism of the second messenger diacylglycerol (DAG) by diacylglycerol kinase enzymes (DGKs), in modulating the magnitude of signaling by this second messenger downstream of the BCR. In the absence of DGKζ, the threshold for BCR signaling through the Ras-ERK MAP kinase pathway was markedly reduced in mature follicular B cells, resulting in exaggerated responses to antigen in vitro and in vivo. Inhibition of DAG signaling by DGKζ was especially important for limiting the number of antibody-secreting cells generated early in response to both T-independent type 2 antigens and T cell-dependent antigens. Furthermore, deficiency in DGKζ closely resembled the effects of increasing antigen affinity for the BCR during the T cell-dependent antibody response, strongly indicating that the magnitude of DAG signaling, likely through the degree of ERK activation, is important for translating the affinity of the BCR for antigen into the amount of antibody produced during early stages of an immune response.
Together, these studies provide novel insight into the mechanisms involved in fine-tuning BCR signaling to promote high quality humoral immune responses while avoiding immunopathological consequences such as autoimmunity or lymphoma.