CD47-dependent regulation of immune checkpoint gene ex-pression and MYCN mRNA splicing in murine CD8 and human Jurkat T cells

Elevated expression of CD47 in some cancers is associated with poor survival, related to its function as an innate immune checkpoint when expressed on tumors cells. In contrast, elevated CD47 ex-pression in cutaneous melanomas is associated with improved survival. Previous studies impli-cated protective functions of CD47 expressed by immune cells in the melanoma tumor microen-vironment. RNA sequencing analysis of responses induced by CD3 and CD28 engagement on wild type and CD47-deficient Jurkat T lymphoblast cells identified additional regulators of T cell func-tion that were also CD47-dependent in mouse CD8 T cells. MYCN mRNA expression was up-regulated in CD47-deficient cells but down-regulated in CD47-deficient cells following activa-tion. CD47 also regulated alternative splicing that produces two N-MYC isoforms. The CD47 ligand thrombospondin-1 inhibited expression of these MYCN mRNA isoforms as well as induction of the oncogenic decoy MYCN opposite strand (MYCNOS) noncoding RNA during T cell activation. Analysis of mRNA expression data for melanomas in The Cancer Genome Atlas identified signif-icant coexpression of MYCN with CD47 and known regulators of CD8 T cell function. Throm-bospondin-1 inhibited the induction of TIGIT, CD40LG and MCL1 mRNAs following T cell acti-vation in vitro. Increased mRNA expression of these T cell transcripts and of MYCN in melanomas was associated with improved overall survival. WT and CD47- Jurkat T cells were cultured routinely as described earlier(PMID: 21343308). CD3 Monoclonal Antibody (OKT3), eBioscience™ and anti-CD28 (CD28 Monoclonal Antibody (CD28.2), Functional Grade, eBioscience™ were purchased from ThermoFisher Life Technologies Corporation. For T cell activation assay, 10 cm plates (BD, Biosciences) were coated with 2mg/ml anti-CD3 and 4 μg/ml of anti-CD28 in PBS at 4°C overnight. Approximately 5x106 cells in 5 ml were plated for 1 h, 3 h and 6 h for each treatment using HITES medium, and 1 μg/ml of TSP1 was added either alone or in combination with anti-CD3 and anti-CD28. Cell pellets from each treatment were washed with PBS, and total RNA was extracted using TriPURE. The concentration of RNA and RNA quality was measured via Nanodrop.