Determining Molecular Mechanisms of SPI1-Mutated CVID

Previous work in our group described a novel cause of agammaglobulinemia (lack of circulating B cells) in certain patients due to heterozygous mutations in the SPI1 gene, which encodes transcription factor SPI1, also known as PU.1. Recently, we identified common variable immunodeficiency (CVID) patients with similar heterozygous mutations in the SPI1 gene. These patients possessed circulating B cells but lacked antibody titers and class-switched memory B cells. We were interested in understanding what the B cell subsets present in these patients look like relative to their healthy counterparts and the trends in their expression profiles at both the protein and transcript levels to better understand what is causing the spectrum of disease severity. We compared the transcripts of circulating B cells in the blood of an SPI1-mutated CVID patient and an age/sex matched healthy donor. We showed the appearance of a novel early transitional B cell population, an expansion of CD21loTBEThi B cells, and a stark decrease in mature naïve B cells. This confirmed our suspicion that there are key differences observable as soon as patient B cells enter circulation due to decreased SPI1 dosage. Upon thorough analysis of immunophenotyping data from our other SPI1 CVID patients in the cohort, we saw that all patients at the protein level showed a similar increase in transitional and CD21loTBEThi B cells, as well as a decrease in mature naïve B cells. Finally, we revisited bulk RNAseq data from three previously created isogenic pro B cell lines, edited to have normal SPI1 expression, monoallelic SPI1 expression, or no SPI1 expression. We saw a significant increase in TBET gene expression with decreasing SPI1 gene expression in each line, which was confirmed by flow cytometry that showed an increase in TBET correlating with the loss of SPI1 at the protein level. This suggested an inverse relationship between SPI1 and TBET, potentially explaining the developmental roadblock in the transitional B cells of SPI1 CVID patients.