Conference 2021 Video


Project title

Spi-C expression is dynamically regulated by external signals in B cells


Authors and Affiliations

Hannah L. Raczkowski1,2, Rodney P. DeKoter1,2

1. Department of Microbiology & Immunology and the Center for Human Immunology, Schulich School of Medicine and Dentistry, Western University, London, Canada
2. Division of Genetics and Development, Children’s Health Research Institute, Lawson Research Institute, London, Canada




Spi-C is a lineage-instructive E26 transformation-specific (ETS) family transcription factor closely related to PU.1 and Spi-B. Expression of Spi-C is important in antibody-generating responses, early B cell development, and red pulp macrophage generation. Spi-C expression is inducible by heme- and NF-κB-dependent pathways in B cells and macrophages. Recent reports implicate Spi-C in modulating inflammatory responses in macrophages, including in the context of chronic inflammatory diseases such as ulcerative colitis. The present research aims to examine the regulation of Spi-C in B cells and determine the biological relevance of its dynamic pattern of expression.


Primary B cells were enriched from mouse spleens and cultured for 24, 48, and 72 hours with signals of interest, including heme, LPS, anti-IgM antibodies, CD40L, and cytokines BAFF+IL-4+IL-5. RT-qPCR was performed to evaluate Spic expression and cells were counted at the indicated time points. To examine regulatory elements of Spic, dual-luciferase reporter assays were conducted to assess the Spic promoter and two putative enhancer regions.


Spic expression was significantly increased in B cells cultured with heme. Proliferative signaling through toll-like receptor 4, B cell receptors, or CD40 co-receptors caused robust downregulation of Spic. We then examined how cytokines BAFF, IL-4, and IL-5 affect Spic expression and found that each cytokine alone did not alter expression or B cell counts. However, culture with BAFF+IL-4+IL-5 significantly increased B cell counts, while decreasing Spic expression. Addition of actin polymerization inhibitor Cytochalasin D to B cells cultured with BAFF+IL-4+IL-5 partially rescued downregulation of Spic and blocked cell division. Luciferase assays indicated strong unidirectional promoter activity in WEHI-279 lymphoma cells, which was significantly decreased upon mutation of an NF-κB binding site. We next aim to characterize two prospective regulatory regions of Spic.


Taken together, these data indicate that Spi-C is dynamically regulated in response to external signals in B cells, with proliferative signals reducing its expression. The global identification of Spi-C target genes in lymphoid and myeloid cells will enhance knowledge of the molecular mechanisms of innate and adaptive immune responses, including in the context of human vaccination and diseases.