Fig 2: Development of HLA-ablated universal HSC-iNKT (UHSC-iNKT) cells and derivatives(A) Experimental design to generate UHSC-iNKT and BCMA CAR-engineered UHSC-iNKT (UBCAR-iNKT) cells. CRISPR, clusters of regularly interspaced short palindromic repeats; Cas 9, CRISPR-associated protein 9; gRNA, guide RNA; B2M, beta-2-microglobulin; CIITA, class II major histocompatibility complex transactivator.(B–D) FACS monitoring of UHSC-iNKT and UBCAR-iNKT cell generation. (B) Intracellular expression of iNKT TCR (identified as Vß11+) and surface ablation of HLA-I/II (identified as HLA-I/B2M-HLA-II-) in CD34+ HSCs cells at day 5 (72 h after lentivector transduction and 48 h after CRISPR-Cas9 gene editing). (C) Generation of iNKT cells (identified as iNKT TCR+TCRaß+ cells) during stage 1 ATO differentiation culture, stage 2 aGC expansion, and stage 3 CAR transduction. Healthy donor PBMC-T cells transduced with the same Retro/BCAR-tEGFR vector was included as a staining control (denoted as BCAR-T cells). (D) Purification of HLA-I/II-negative UHSC-iNKT cells using MACS.(E and F) Studying allogenic T cell response against UBCAR-iNKT cells using an in vitro MLR assay. Irradiated UBCAR-iNKT cells (as stimulators) were co-cultured with donor-mismatched PBMCs (as responders). Irradiated AlloBCAR-iNKT and conventional BCAR-T cells were included as stimulator cell controls. (E) Experimental design. PBMCs from three different healthy donors were used as responders. (F) ELISA analyses of IFN-? production at day 4 (n = 3).(G and H) Studying allogenic NK cell response against UHSC-iNKT cells using an in vitro MLR assay. UHSC-iNKT cells were co-cultured with donor-mismatched PBMC-NK cells. AlloHSC-iNKT cells were included as a control. (G) Experimental design. (H) FACS quantification of the indicated cells (n = 3).(I–L) Studying the in vivo antitumor efficacy of UBCAR-iNKT cells in an MM.1S-CD1d-FG human multiple myeloma xenograft NSG mouse model. (I) Experimental design. (J) BLI images showing tumor loads in experimental mice over time. (K) Quantification of (J) (n = 5). (L) Kaplan-Meier survival curves of experimental mice over a period of 4 months after tumor challenge (n = 8). Mice were combined from two independent experiments.Representative of two (I–L) and three (B–H) experiments. Data are presented as mean ± SEM. ns, not significant; ****p < 0.0001 by Student’s t test (H), one-way ANOVA (F and K), or log rank (Mantel-Cox) test adjusted for multiple comparisons (L). See also Figure S7.

Fig 3: DDX60 regulates the expression of MHC-I molecules in CRC cells. (A,B). Western blotting images showed the expression of DDX60, B2M, and Actin in vector control or DDX60-overexpressed DLD-1 (A) and HCT-15 (B). (C–F) FACS analysis of MHC-I expression in vector control or DDX60-overexpressed DLD-1 (A) and HCT-15 (B) with/without IFN? treatment. (G) Western blotting images showed the expression of DDX60, B2M, and Actin in NCI-H747 cells treated with siRNA of nontarget control (siCTL) or DDX60 for 48 h. (H,I) FACS analysis of MHC-I expression in control (siCTL) and DDX60-Knockdown NCI-H747 cells with/without IFN? stimulation. p values were calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. ns: p = 0.05; **: p < 0.01; ***: p < 0.001.

Fig 4: Construction of genetically modified HEK293 cells.a Schematic of donor cell construction. b Sanger sequencing of constructed double KO HEK293 cells. The deleted sequences were underlined. c GPC3 and B2M proteins were extracted and identified from wild-type HEK293 cells and double KO HEK293 cells. Experiments were repeated thrice. d The expression level of fusogen was measured by flow cytometry in double KO HEK293 cells and eFT-HEK293 cells. e pH-dependent fusogen-mediated cell-cell fusion. Cells were co-cultured for 6 h (Green: GFP, Red: PKH26 staining, and Blue: DAPI staining; scale bar is 40 µm). The red arrow indicates multinucleated polykaryons, and the green arrow indicates a HepG2 microcluster. Experiments were repeated five times.

Fig 5: Schematic of anti-GPC3 scoff and engineered fusogen co-expressing eFT-CNVs for cytosolic delivery of therapeutics.Intrinsic GPC3 and B2M are knocked out using CRISPR/Cas 9 followed by co-expressing anti-GPC3 scFv and engineered fusogen on HEK293 cell membranes. The eFT-CNVs are generated by mechanical extrusion of the donor cells and loaded with various drugs, e.g., nucleic acids, protein toxins, or chemotherapeutic agents, for on-target cytosolic delivery.