Permanent silencing of NKG2A expression for cell-based therapeutics.
Natural killer (NK) and T-cell cytotoxicity is significantly reduced by signaling via CD94/NKG2A receptors. High levels of NKG2A on NK cells have been shown to compromise the graft-versus-leukemia effect in hematopoietic stem cell transplantation. We therefore evaluated the functional relevance of NKG2A silencing for the cytotoxic potential of genetically engineered NK and T cells. Lentiviral vectors containing short hairpin RNA (shRNA) sequences targeting NKG2A transcripts were used to transduce NKG2A(+) primary NK and T cells. NKG2A expression levels were measured by flow cytometry and real-time PCR. The effect of NKG2A silencing on the cytolytic potential of NK and T cells was evaluated in cytotoxicity assays using K562 and B lymphoblastoid cells as targets. Granzyme B mRNA transcript levels were detected by real-time PCR. The transduction of inducible RNAi cassettes containing the sequences for shRNAs targeting NKG2A reduced protein expression in NK and T cells by up to 95%. The cytotoxicity assays demonstrated that NKG2A silencing effectively enhanced NK and CD8+ T-cell lysis by up to 40% and 15%, respectively. However, lysis of K562 cells which lack human leukocyte antigen-E, the ligand of NKG2A, was associated with an upregulation of the natural cytotoxicity receptor NKp30 in NKG2A-silenced NK cells. Our data suggest that RNAi-mediated silencing of NKG2A in effector cells could improve the efficacy of cell-based immunotherapies but also show that indirect effects of NKG2A knockdown exist that have to be considered when designing therapeutic protocols with genetically engineered NK or T cells.
Figueiredo
Constança
C
Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
Seltsam
Axel
A
Blasczyk
Rainer
R
D019264:Adoptive Transfer; D001483:Base Sequence; D015550:Cell Degranulation; D002460:Cell Line; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D005434:Flow Cytometry; D053804:Granzymes / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D055654:NK Cell Lectin-Like Receptor Subfamily C / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism* / Q000502:physiology; D014162:Transfection
D055654:NK Cell Lectin-Like Receptor Subfamily C; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D053804:Granzymes