The Production of Pro-angiogenic VEGF-A Isoforms by Hypoxic Human NK Cells Is Independent of Their TGF-β-Mediated Conversion to an ILC1-Like Phenotype

Circulating natural killer (NK) cells have been shown to adopt a type 1 innate lymphoid cell (ILC1)-like phenotype in response to TGF-beta and secrete VEGF-A when exposed to hypoxia. Although these changes are often considered to be linked attributes of tissue residency, it has yet to be determined if TGF-beta and hypoxia work in concert to coordinate NK cellular phenotype and angiogenic potential. Examination of human circulating NK cells treated with TGF-beta demonstrated heterogeneity in their potential to adopt an ILC1-like phenotype, as indicated by the upregulation of CD9 and CD103 on only a subset of cells in culture. Culturing NK cells in chronic hypoxia did not induce a similar ILC1-like conversion and did not enhance the degree of conversion for cells exposed to TGF-beta. Similarly, hypoxic culture of circulating NK cells induced VEGF-A secretion, but this production was not enhanced by TGF-beta. Fluorescentin-situhybridization flow cytometry demonstrated that hypoxia-induced VEGF-A production was uniform across all NK cells in culture and was not a selective feature of the cellular subset that adopted an ILC1-like phenotype in response to TGF-beta. Examination of VEGF-A isoforms demonstrated that hypoxia induces the production of pro-angiogenic VEGF-A isoforms, including VEGF-A(165)and VEGF-A(121), and does not stimulate any meaningful production of anti-angiogenic isoforms, such as VEGF-A(b)transcriptional variants or VEGF-Ax. In summary, TGF-beta-mediated ILC1-like conversion and hypoxia-induced VEGF-A production are discrete processes in NK cells and are not part of a linked cellular program associated with tissue residency.