Human B2M Knockout Cell Line-K562

The human K562 leukemia cell line, used as a scaffold for artificial antigen-presenting cells (aAPCs) for in vitro lymphocyte expansion, normally does not express major histocompatibility complex (MHC) molecules. However, when stimulated with human T lymphocyte culture supernatant, K562 cells upregulate beta-2 microglobulin (B2M) and MHC class I expression, thereby inducing allo-specific T cells. Here, researchers disrupted the B2M locus in K562 cells by CRISPR/Cas9 and obtained MHC class I-negative K562 cells. A K562-based MHC class I-negative aAPC line was further generated by zinc finger nuclease-mediated insertion of costimulatory molecules into the AAVS1 locus. This aAPC line can attenuate allogeneic immune responses but supports robust antigen-independent and CD19 antigen-specific chimeric antigen receptor-T cell expansion in vitro. These results suggest that B2M-knockout K562 cells provide a new scaffold for the construction of aAPCs and their broader application in adoptive immunotherapy.

K562 cells are commonly used as feeder cells for large-scale expansion of αβ-T cells. To test the stimulating function of B2M knockout K562-based aAPCs for antigen-independent T-cell expansion, PBMCs were treated with CD3/CD28 antibody-coated micro-Dynabeads for 1 week to activate T cells. T cells were then collected to mix with inactivated K562B or K562A cells at an 1:50 ratio and expanded in the presence of anti-CD3 antibody, OKT-3, for 2 weeks (Figure 1A). With this method, a total cell expansion of 1000- to 2000-fold was observed at the end of the 14-day culture in a representative study (Figure 1B). The researchers further investigated the composition of the cell population after 2 weeks of expansion (Figure 1C). The CD8+ αβ-T-cell population constituted the major portion (60–75%) followed by the CD4+ αβ-T-cell population (20–40%). CD3- CD56+ NK cells percentage was slightly increase from 3 to 13% during Dynabeads stimulation due to the loss of monocytes and other non-T/NK lymphocytes, but a decrease in the proportion of NK cells from 3 to 5% was observed during the 14-day T cell/K562 coculture. There was no obvious expansion of the γδ-T-cell population (1.5–3%). These studies suggest that B2M knockout in K562 does not affect its ability to function as aAPC for antigen-independent T cell expansion.

Figure 1. Antigen-independent T-cell expansion with β-2 microglobulin-knockout K562-based artificial antigen presenting cells. (Zha S, et al., 2019)