Panoply™ Human HLA-G Over-expressing Stable Cell Line

To address the unmet need for immunogenically matched materials in regenerative medicine, universal donor cells have been developed. Because forced expression of hypoimmunogenic genes suppresses immune responses, researchers have generated universal pluripotent stem cells (PSCs) by replacing endogenous β2-microglobulin (β2m) with β2m directly conjugated to human leukocyte antigen (HLA)-G, thereby simultaneously suppressing HLA-I expression and natural killer (NK) cell-mediated immune responses. These modified human PSCs retain their pluripotency and differentiation capacity. However, due to downregulation of antigen processing and presentation machinery (APM) genes, subsequently differentiated cells, particularly those of the neural lineage, no longer display HLA-G on the surface. Inducing APM gene expression by overexpressing NLR family CARD domain-containing 5 (NLRC5) or the activating subunit of nuclear factor κB (NF-κB) heterodimer (RelA) restores HLA-G surface expression and neural cell hypoimmunogenicity. These findings enhance the utility of low-immunogenic cells as universal donors and will aid in the development of off-the-shelf stem cell therapies.

To assess the potential for HLA-G to reduce immunogenicity, researchers performed calcein-AM release and NK cell degranulation assays using K562 and NK92 cells as effector cells. NK cell-mediated target cell-specific lysis increased with increasing E:T ratios (Figure 1E). The average percentages of specific lysis in control cells were 8.53%, 13.4%, 29.87%, 49.54%, and 57.87%, respectively, at E:T ratios of 0.33:1 to 10:1. In contrast, HLA-G-overexpressing K562 cells exhibited significantly lower cytotoxicity than control cells at all E:T ratios (0.36%, 2.81%, 11.48%, 26.12%, and 31.59%, respectively). NK cell degranulation assays assessed NK cell activation by detecting the plasma membrane marker CD107a (LAMP1). After co-culture with target cells, surface CD107a and the NK cell marker CD56 reacted with fluorescently labeled antibodies. The NK cell degranulation rate was significantly lower in HLA-G-overexpressing K562 cells than in wild-type K562 cells (Figure 1F,a). The average activation rate of NK cells co-cultured with HLA-G-overexpressing K562 cells was 15.6%, compared with 29.7% in co-cultured with wild-type K562 cells (Figure 1F,b). Therefore, ectopic expression of HLA-G in HLA-I-deficient cells inhibits NK cell-mediated immune responses.

Figure 1. Hypoimmunogenic Potential of HLA-G. (An J H, et al., 2022)