Fig 2: Transcriptional and Epigenetic Basis for Enhancing CAR19-iNKT Cell Reactivity(A) CD1D mRNA quantification by qPCR in CLL cells from two patients upon ATRA treatment (10-6 M) for 0–96 hr.(B and C) Bar charts (B) and flow cytometry histograms (C) showing CD1d expression on malignant B cells upon ATRA treatment and mean fluorescent intensity (MFI) analysis of CD1d expression in comparison with isotype control.(D) Cytotoxicity of second- and third-generation CAR19-T and -NKT cells against aGalCer-pulsed CLL cells pre-treated with 0.1% DMSO control or 10-6 M ATRA. Error bars represent SEM of triplicate assays.(E) ChIP-qPCR assay for H3K4me3 and H3K27me3 enrichment in the promoter of CD1D using IgG as control in U266 cells. GAPDH is an active gene control, while HOXA2 is a repressed gene control. ChIP data are shown as a percentage of the input chromatin.(F) ChIP-re-ChIP qPCR assay showing fold enrichment of H3K27me3 or IgG control after immunoprecipitation (IP) against H3K4me3.(G) ChIP-qPCR assay against RNA Pol II for Ser5 over Ser2 phosphorylated form at the promoter of the indicated genes.(H) ChIP-qPCR assay against RARa, EZH2, and Ig control at the promoters of the genes shown.(I) ChIP-re-ChIP qPCR assay showing enrichment of EZH2 or IgG control after IP against RARa in U266 cells for –(I) (n = 3).(J) qPCR quantification of CD1D mRNA in U266 cells treated with 0.1% DMSO, 10-6 M GSK343, 10-6 M ATRA or 10-6 M GSK343 plus 10-6 M ATRA. Values are normalized to CD1D mRNA expression levels in normal peripheral PB B cells (n = 3). ND, not detectable.(K and L) Relative MFI analysis (K) and histogram depiction (L) of CD1d expression in comparison with isotype control in U266 cells from the same experiment shown in (J).Error bars represent SEM. See also Figure S4.

Fig 3: Tumor targeting of AlloHSC-iNKT cells through engineered chimeric antigen receptors (CARs)(A) Experimental design to generate BCMA CAR-engineered AlloHSC-iNKT (AlloBCAR-iNKT) cells in vitro. BCMA, B cell maturation antigen; BCAR, BCMA CAR; Retro/BCAR-tEGFR, retroviral vector encoding a BCMA CAR gene as well as a truncated epidermal growth factor receptor (tEGFR) reporter gene. tEGFR was used as a staining marker indicating BCAR expression.(B) FACS analysis of BCAR expression (identified as tEGFR+) on AlloBCAR-iNKT at 72 h after retrovector transduction. Healthy donor PBMC-T cells transduced with the same Retro/BCAR-tEGFR vector (denoted as BCAR-T cells) were included as a staining control.(C–F) In vitro killing of human multiple myeloma cells by AlloBCAR-iNKT cells. MM.1S-CD1d-FG, human MM.1S cell line engineered to overexpress human CD1d as well as FG dual reporters. PBMC-T, BCAR-T, and AlloHSC-iNKT cells were included as effector cell controls. (C) Experimental design. (D) Diagram showing the tumor-targeting triple mechanisms of AlloBCAR-iNKT cells, mediated by NK activating receptors, iNKT TCR, and BCAR. (E) Tumor cell killing by the indicated effector cells with/out the addition of aGC (n = 4). (F) Tumor cell killing by AlloBCAR-iNKT cells with/out the blockade of DNAM-1 (n = 4). Tumor cell killing was analyzed at 8-h after co-culture (effector/tumor ratio 5:1).(G–N) Studying the in vivo antitumor efficacy of AlloBCAR-iNKT cells in an MM.1S-CD1d-FG human multiple myeloma xenograft NSG mouse model. Tumor-bearing mice injected with BCAR-T cells or no cells (vehicle) were included as controls. (G–J) Low-tumor-load condition. (G) Experimental design. (H) BLI images showing tumor loads in experimental mice over time. (I) Quantification of (H) (n = 5). (J) Kaplan-Meier survival curves of experimental mice over a period of 4 months after tumor challenge (n = 5). (K–N) High-tumor-load condition. (K) Experimental design. (L) BLI images showing tumor loads in experimental mice at day 38. (M) Quantification of tumor load in experimental mice over time (n = 5). (N) Kaplan-Meier survival curves of experimental mice over a period of 4 months after tumor challenge (n = 5).Representative of two experiments (K–N) and three experiments (A–J). Data are presented as the mean ± SEM. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by one-way ANOVA (E, F, I, and M), or log rank (Mantel-Cox) test adjusted for multiple comparisons (J and N). See also Figure S2.

Fig 4: Enhanced Short- and Long-Term Reactivity of CAR19-iNKT Cells against B Lineage Malignancies(A) Second- and third-generation CAR19-T and CAR19-iNKT cell expansion (fold change) and absolute cell numbers (cell count) over a period of 3 weeks (n = 4). p value is for CAR19-iNKT versus CAR19-T cells using Friedman test. Error bars represent SEM.(B) Proliferation analysis of second- and third-generation CAR19-T and CAR19-iNKT cells in the presence (stimulated) or not (resting) of irradiated CD1d+CD19+ (C1R-CD1d) cells over 7 days. p value is for CAR19-iNKT versus CAR19-T cells using Friedman test.(C) Cytotoxicity of third-generation CAR19-T and -NKT cells against C1R-CD1d (representative of n = 3) and Farage lymphoma cell lines (representative of n = 2) pre-loaded or not with aGalCer. Error bars represent SEM of triplicate assays.(D) IncuCyte images of representative wells showing the final effector (gray) and live target cells (red) after 7 days. Effectors were second-generation CAR19-T and CAR19-NKT cells. Targets were CD19+ ARH-77-CD1d cells expressing mCherry red fluorescent protein. Scale bar represents 400 µm.(E) Seven-day trajectory of effector and target cell proliferation and elimination respectively as per (D). p value is for CAR19-iNKT versus CAR19-T cells using Friedman test.(F) Cytotoxicity of second-generation CAR19-iNKT, CAR19-T, and of untransduced iNKT cells against lymphoma cells from one patient with mantle cell lymphoma (MCL; top) and two patients with marginal zone B lymphoma (MZL; bottom) using three different T/iNKT cell healthy donors (A, B, and C). Error bars represent SEM of triplicate assays.**p < 0.01; ****p < 0.0001. Cc, cell confluency; FP, fluorescent protein; RCU, red calibrated units. See also Figures S2 and S3.

Fig 5: 3rdHSC-iNKT cells ameliorate GvHD through eliminating donor CD14+ myeloid cells through CD1d recognitionIn vitro mixed lymphocyte reaction (MLR) assay was performed using healthy donor PBMCs (responders) co-cultured with irradiated donor-mismatched allogeneic PBMCs (stimulators) with or without the addition of 3rdHSC-iNKT cells. Where applicable, purified anti-human CD1d antibody or its IgG isotype control was also added. To identify responders and stimulators by flow cytometry, HLA-A2+ responders and HLA-A2- stimulators were used in the study.(A) Experimental design.(B) ELISA analyses of IFN-? production in the indicated MLR co-cultures. Supernatant were collected and analyzed on day 4. N = 3.(C) FACS analyses of CD1d expression on the indicated cells. N = 4.(D) FACS detection of T, B, and CD14+ cells of responders in multiple MLR assays one day after MLR co-culture.(E) Quantification of (D). N = 4.Representative of 3 experiments. All data are presented as the mean ± SEM. ns, not significant, **p< 0.01, ***p< 0.001, and ****p< 0.0001 by one-way ANOVA.