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. 2023 May 22;11(5):e006649. doi: 10.1136/jitc-2022-006649

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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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CRISPRa-engineered IL-15 secreting NK cells eradicate MYC-overexpressing B-ALLs in vitro. (A)Fold change in IL-15 transcript levels by qPCR in dCas9-VP64-GFP⁺ NK-92 cells transduced with control sgRNA-RFP (Control NK) or IL-15 sgRNA-RFP (IL-15 NK) and levels of secreted IL-15 by ELISA in culture supernatant of control NK and IL-15 NK cells stimulated with PMA and Ionomycin for 24 hours. (B)Cell counts of control and IL-15 NK cells cultured in the presence and absence of rhIL-2 (100 U/mL) for 96 hours. (C, D)Specific cytotoxicity of control and IL-15 NK cells by flow cytometry using (C)K562, (D)SEM (KMT2A-rearranged), KOPN8 (KMT2A-rearranged), MHH-CALL4 (hypodiploid, CRLF2-rearranged), P493-6, and VAL as target cell lines. Effector: target=10:1. (E)Specific cytotoxicity of control and IL-15 NK cells by flow cytometry against MYC-overexpressing (MYC^ON) and MYC-inactivated (MYC^OFF) P493-6 cells. MYC was inactivated by treatment of P493-6 cells with 0.2 µg/mL of doxycycline for 24 hours. p=0.0108, control (MYC^ON) vs IL-15 NK (MYC^ON); p=0.0037, control (MYC^OFF) vs IL-15 NK (MYC^OFF). All experiments were conducted in three technical and three biological replicates. One representative of three biological replicates of each experiment is shown. Comparisons between any two groups were conducted using Student’s t-test (A–E).Exact p values are provided whenever significant (<0.05) or trending to significance (0.05<p<0.1). B-ALL, B-cell acute lymphoblastic leukemia; NK, natural killer; ns, not significant.