Insights into the Diversity of Calcium Signaling in B Lymphocytes

Open Access
- Author:
- Emrich, Scott
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 13, 2021
- Committee Members:
- Neil Christensen, Major Field Member
Salvatore Stella, Major Field Member
Laura Carrel, Outside Unit & Field Member
Mohamed Trebak, Chair & Dissertation Advisor
Donald Gill, Major Field Member
Ralph Keil, Program Head/Chair - Keywords:
- Calcium signaling
Immunity
B lymphocyte
Store-operated Ca2+ entry - Abstract:
- Regulation of intracellular calcium (Ca2+) signals is essential for immune cell function. The predominant mechanism of Ca2+ influx into lymphocytes is through the ubiquitous store-operated Ca2+ entry (SOCE) pathway. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels, composed of hexameric Orai proteins (Orai1-3) in the plasma membrane and endoplasmic reticulum (ER)-resident stromal interaction molecules (STIM1-2). The fundamental role of SOCE in immunity is underscored by patients with loss-of-function mutations in Orai1 and STIM1, who manifest with severe combined immunodeficiency and autoimmunity resulting from defective innate and adaptive immune cell populations. Extensive research has defined critical roles for SOCE and CRAC channels in T cell subsets and their contribution to disease in these patients. In contrast, downstream signaling pathways activated by SOCE in B cells, factors that regulate CRAC channel activity, and the interplay of Orai isoforms in B cell biology is largely enigmatic. We investigated the role of individual Orai isoforms in B cell biology utilizing multiple Orai-deficient cell lines and B cell-specific Orai knockout mice. We reveal that expression of each Orai isoform is dynamic upon B cell activation and that Orai3 is an essential component of the native CRAC channel in B cell lines and primary B lymphocytes. Orai1 plays a critical role in the activation of multiple NFAT isoforms and its contribution becomes more prominent in robustly activated B cells. SOCE through Orai1/Orai3 heteromeric channels critically regulates the proliferation, survival, and metabolic reprogramming of primary B cells upon antigenic stimulation. SOCE in immune cells is also regulated by the mitochondria through a variety of mechanisms. We recently identified the mitochondrial Na+/Ca2+/Li+ exchanger (NCLX), which extrudes Ca2+ out of the mitochondria, as a novel regulator of SOCE. The contribution of NCLX-mediated Ca2+ extrusion to the immune response in health and disease is unknown. Here, we demonstrate that lymphocytes display unique mitochondrial Ca2+ extrusion dynamics compared to adherent cells. Using NCLX-deficient cell lines and B cell-specific NCLX knockout mice, we uncover that loss of NCLX inhibits SOCE, while BCR-induced Ca2+ oscillations and ER Ca2+ handing is unperturbed. We reveal that NCLX is a critical regulator of B cell metabolism, mitochondrial Ca2+ handing, and mitochondrial ultrastructure. Loss of NCLX in B cells diminished foreign antigen-driven and pathogen-induced germinal center responses in vivo. Collectively, these findings provide novel insights into the complex role of SOCE and mitochondrial Ca2+ signaling in B cell effector function and further establish these Ca2+ signaling mechanisms as key regulators of the adaptive immune response.