CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma

Abstract

Background Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH, or CIS) as a critical negative regulator in NKCs. Methods The GBM tumor environment featured with immunological aspect was analyzed with Cancer immunogram and GlioVis. We generated human primary CIS-deleted NKCs (NK dCIS) using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting genome sites on CIS coding exons. The genome-edited NKCs underwent microarray with differential expression analysis and gene set enrichment analysis (GSEA). The anti-GBM activity of the genome-edited NKCs was evaluated by apoptosis induction effects against allogeneic GBM cells and spheroids. We further detected in vivo antitumor effects using xenograft brain tumor mice. Results We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. Conclusion The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Characterization of HIF-1α Knockout Primary Human Natural Killer Cells Including Populations in Allogeneic Glioblastoma

“…The expressions of the following NK cell immunity-related genes were assessed: NK-activating receptors, NK inhibitory receptors, chemokines, chemokine receptors, cytotoxicity markers, inflammatory cytokines, immunosuppressive effectors, anti-apoptotic markers, and proliferation markers. These genes were selected from our previously published study [ 28 ]. Among the HIF KO NKP in hypoxic conditions, NCR2 (NKp44) was downregulated and ITGAL was upregulated significantly ( Figure 5 A); LILRB1 and CD33 were significantly downregulated ( Figure 5 B); CCL2 and CCL5 were upregulated and CCL28 and CXCL16 were downregulated ( Figure 5 C); CCR5 and CX3CR1 were upregulated and CCR3 and CXCR4 were downregulated ( Figure 5 D); GZMK (granzyme K) was downregulated, while PRF1 was upregulated ( Figure 5 E); TNF was upregulated ( Figure 5 F).…”

“…The AIM-V medium containing 3000 IU/mL rhIL-2 was refilled as required. Genome ed iting was conducted as previously described with minor modifications [ 28 , 29 ]. Expanded NK cells (3 × 10 6 ) were electroporated to RNP complexes (targeted sgRNA/tracrRNA and recombinant Cas9, IDT) using a Human NK Cell Nucleofector Kit (VPA-1005; Lonza, Basel, Switzerland) and electroporation program X-001.…”

“…The genome-edited NK cells were harvested 4 days after electroporation, their DNA was extracted using a QIAamp DNA mini kit (Qiagen, Hilden, Germany), and T7E1 mismatch detection assays were conducted using an Alt-R Genome Editing Detection Kit (IDT) as described previously [ 28 , 29 , 42 ]. Briefly, the OT and OF sites and adjacent sequences from the genomic DNA were amplified using KOD FX enzyme solution (TOYOBO, Osaka, Japan).…”

“…It is now possible to expand highly purified NK cells from human peripheral blood (PB) with high efficiency [ 27 ]. This expansion technique is coupled to cell populations that have undergone CRISPR/Cas9 gene-specific knockout protocols, allowing NK phenotypes to be quantified, as demonstrated with the negative regulator gene CIS [ 28 ] and inhibitory receptor gene TIM3 [ 29 ]. In the present study, we aimed to induce and expand human NK cells with HIF-1α knockout by CRISPR/Cas9.…”

Understanding the immunosuppressive microenvironment of glioma: mechanistic insights and clinical perspectives

“…Annexin A1 (ANXA1) is implicated in DC maturation and is related to worse outcomes in patients with GBM [ 322 ]. Silencing cytokine-inducible SH2 (CIS) containing protein in NK cells increases production levels of IFN-γ and TNF-α, enhancing cancer cells apoptosis mediated by allogeneic NK cells and improving overall survival in mice with GBM [ 323 ]. GBM cells can secrete LDH5, which induces natural-killer group 2 member D (NKG2D) ligands upregulation, leading to NKG2D downregulation in NK cells [ 196 ].…”