Data Sheet 1_SNP rs10748643 determines CD39 expression in T and NK cells through altering NFIC binding affinity rather than interfering with RNA splicing.docx

NTPDase-1 CD39, a dominant component of purinergic signaling, has proved to be a critical regulator of tissue microenvironment and cellular function particularly for T cells. We repeatedly use single-nucleotide polymorphism (SNP) rs10748643 genotype as a tag of CD39 expression in T cells in many studies. However, no study that profiles the impact of the SNP mutation on CD39 expression in all immune cells has been done yet. In this study, we used flow cytometry to measure CD39 expression in six types of immune cells across three genotypes. We revealed that SNP rs10748643 mainly determines CD39 expression in T and natural killer (NK) cells. In contrast, it has the least influence on monocytes, B cells, and granulocytes. These immune cells demonstrate constitutive CD39 expression, which is different from T and NK cells. We applied bioinformatic tools and bench experiments to disclose the underlying mechanism. We found that the amount of each variant is barely affected by SNP mutation in B cells, whereas almost all variants are significantly impacted by SNP mutation in T cells. Such discrepancy in T and B cells does not support the hypothesis that SNP rs10748643 interferes with the splicing efficiency of CD39 mRNA. MicroRNA regulation was also excluded. Transcriptional factor regulation was emphasized because conserved motif sequence of NFIC exactly covers the SNP site. Using Cleavage Under Targets and Tagmentation (CUT&Tag) technology, we found that the binding affinity of NFIC decreases when SNP mutates from A to G, causing the reduced inhibition of NFIC on CD39 expression in T cells. The NFIC inducer may be another potential approach to restrain CD39 expression for improving the immunosuppressive microenvironment.