Natural Killer Cells Generated From Human Induced Pluripotent Stem Cells Mature to CD56brightCD16+NKp80+/-In-Vitro and Express KIR2DL2/DL3 and KIR3DL1

Abstract

The differentiation of human induced pluripotent stem cells (hiPSCs) into T and natural killer (NK) lymphocytes opens novel possibilities for developmental studies of immune cells and in-vitro generation of cell therapy products. In particular, iPSC-derived NK cells gained interest in adoptive anti-cancer immunotherapies, since they enable generation of homogenous populations of NK cells with and without genetic engineering that can be grown at clinical scale. However, the phenotype of in-vitro generated NK cells is not well characterized. NK cells derive in the bone marrow and mature in secondary lymphoid tissues through distinct stages from CD56brightCD16- to CD56dimCD16+ NK cells that represents the most abandoned population in peripheral blood. In this study, we efficiently generated CD56+CD16+CD3- NK lymphocytes from hiPSC and characterized NK-cell development by surface expression of NK-lineage markers. Hematopoietic priming of hiPSC resulted in 31.9% to 57.4% CD34+CD45+ hematopoietic progenitor cells (HPC) that did not require enrichment for NK lymphocyte propagation. HPC were further differentiated into NK cells on OP9-DL1 feeder cells resulting in high purity of CD56brightCD16- and CD56brightCD16+ NK cells. The output of generated NK cells increased up to 40% when OP9-DL1 feeder cells were inactivated with mitomycine C. CD7 expression could be detected from the first week of differentiation indicating priming towards the lymphoid lineage. CD56brightCD16-/+ NK cells expressed high levels of DNAM-1, CD69, natural killer cell receptors NKG2A and NKG2D, and natural cytotoxicity receptors NKp46, NKp44, NKp30. Expression of NKp80 on 40% of NK cells, and a perforin+ and granzyme B+ phenotype confirmed differentiation up to stage 4b. Killer cell immunoglobulin-like receptor KIR2DL2/DL3 and KIR3DL1 were found on up to 3 and 10% of mature NK cells, respectively. NK cells were functional in terms of cytotoxicity, degranulation and antibody-dependent cell-mediated cytotoxicity.

Human-Induced CD49a+ NK Cells Promote Fetal Growth

“…Ex vivo-induced CD49a + iNK cells can mimic dNK cell function in vivo. Several attempts have been made to expand NK cells with phenotypic similarities to dNK cells (27,53). However, it is unclear whether these NK cells induced by different pathways have in vivo functions that promote embryonic development.…”

Natural killer cells in sepsis: Friends or foes?

“…In humans, about 5-15% of lymphocytes are defined as NK cells in peripheral blood, and tissue-specific subpopulations are found in the spleen, liver, and lung (57)(58)(59)(60)(61). Generally, human NK cells can be divided into two subpopulations by the expression of CD56 and CD16 on the cell membrane (62,63). About 90% of all NK cells in human peripheral blood are CD56 dim CD16 bright , whereas only 10% are CD56 bright CD16 -/dim (64).…”

Adoptive Immunotherapy: A Human Pluripotent Stem Cell Perspective

“…Recently, hPSCs have emerged as an alternative cell source for NK cells to address these issues. Due to their innate killing ability, many studies have shown the superior antitumor activities of hPSC-derived NK cells without additional engineering approaches that confer antigen-specific activity [Woll et al, 2005[Woll et al, , 2009Larbi et al, 2012;Hermanson et al, 2016;Zeng et al, 2017;Mat-subara et al, 2019;Zhu et al, 2020a, b;Cichocki et al, 2020;Goldenson et al, 2020;Euchner et al, 2021;Woan et al, 2021;Du et al, 2022;Jung et al, 2022]. A few strategies were developed to enhance in vivo persistence and antitumor activities of hPSC-derived NK cells.…”