Modulation of the intracellular inhibitory checkpoint SHP-1 enhances the antitumor activity of engineered NK cells

Inhibition of SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

“…The outcome of these molecular processes, however, demonstrated contradictory results, and the molecular regulation of SHP-1 in NK cells has not been fully addressed. We recently employed a mutant YTS-2DL1 knock-in line expressing the SHP-1 phosphor-mimetic serine to aspartic acid residue substitution (SHP-1 S591D), which exhibits increased antitumor NK function relative to WT SHP-1-expressing YTS cells ( Ben-Shmuel et al, 2021 ). To dissect the possible effect of SHP-1 phosphorylation on NK cell function, physiological activating and inhibiting interactions were induced with 721.221 target cells, and S591 phosphorylation patterns were examined in YTS-2DL1 cells and isolated primary NK cells expressing KIR2DL1 + (referred to as pNK-2DL1) from healthy human donors.…”

“…In order to demonstrate that PKC-θ regulation of SHP-1 via S591 influences SHP-1 substrate phosphorylation and activation, and not PKC-θ silencing per se, a mutant YTS-2DL1 line expressing an SHP-1 mutant that mimics the constitutively phosphorylated state, SHP-1 S591D (referred to as YTS-2DL1 SHP-1 S591D), was created utilizing CRISPR/Cas9, as previously reported ( Ben-Shmuel et al, 2021 ). In cells expressing this mutant, PKC-θ silencing would not be expected to impact SHP-1 activity ( Egelhoff et al, 1993 ; Huang and Erikson, 1994 ; Léger et al, 1997 ).…”

Inhibition of the SHP-1 activity by PKC-θ regulates NK cell activation threshold and cytotoxicity

“…The outcome of these molecular processes, however, demonstrated contradictory results, and molecular regulation of SHP-1 in NK cells has not been fully addressed. We recently employed a mutant YTS-2DL1 knock-in line expressing the SHP-1 phosphor-mimetic serine to aspartic acid residue substitution (SHP-1 S591D), which exhibits increased anti-tumor NK function relative to WT SHP-1expressing YTS cells (Ben-Shmuel et al, 2020). To dissect the possible effect of SHP-1 phosphorylation on NK cell function, physiological activating and inhibiting interactions were induced with 721.221 target cells, and S591 phosphorylation patterns were examined in YTS-2DL1 cells and isolated primary NK KIR2DL1 + cells (pNK-2DL1) from healthy human donors.…”

“…In order to demonstrate that PKC-θ regulation of SHP-1 via S591 influences SHP-1 substrate phosphorylation and activation, and not PKC-θ silencing per-se, a mutant YTS-2DL1 line expressing a SHP-1 mutant that mimics the constitutively phosphorylated state, SHP-1 S591D, was created utilizing CRISPR/CAS9, as previously reported (Ben-Shmuel, 2020). In cells expressing this mutant, PKC-θ silencing would not be expected to impact SHP-1 activity (Egelhoff et al, 1993;Huang et al, 1994;Léger et al, 1997).…”

“…CRISPR/CAS9 knockdown of SHP-1 in YTS cells was conducted according to published protocol and was performed as described in detail (Ran et al, 2013;Matalon, 2018;Ben-Shmuel, 2020).…”