CAR-T cells targeting CD38 and LMP1 exhibit robust antitumour activity against NK/T cell lymphoma

Cell lines

The cell lines used in this study, NKYS, YT and KAI3, were obtained from Dr. Wing C. Chan (City of Hope Medical Center, Los Angeles, USA). Dr. Norio Shimizu and Yu Zhang of Chiba University provided SNK6 cells. SNT16 cells were a gift from Guangzhou Bairui Biomedical Technology Company, Ltd. NKYS, YT, KAI3 and SNT16 cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco, USA) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). Additional IL-2 (100 IU/ml) was added to the medium for NKYS and KAI3 cells. SNK6 cells were cultured in RPMI-1640 medium with 10% immune cell serum replacement (Gibco, USA), IL-2 (200 IU/ml) and antibiotics. All cells were cultured in an incubator (Thermo Fisher, USA) at 37 °C and 5% CO₂. All cell lines tested negative for mycoplasma contamination, and cell-surface markers for these cell lines were validated by flow cytometry.

Western blot analysis

Cells were lysed in NP40 lysis buffer and then centrifuged to harvest the protein supernatant. Twenty micrograms of proteins was resolved by SDS‒PAGE and transferred to the polyvinylidene fluoride membranes (Amersham Biosciences, USA). The membranes were blocked in Tris-buffered saline Tween buffer containing 5% (w/v) non-fat milk at room temperature for 1 h and then incubated with primary antibodies including anti-LMP1 (Abcam, ab78113) and anti-GAPDH (ProteinTech, 60,004–1-lg) at 4 °C overnight and then with secondary antibodies for 1 h at room temperature. Band images were digitally captured with a ChemiDocTM XRC + system (Bio-Rad Laboratories, USA).

Immunofluorescence

NKYS, YT, KAI3, SNK6 and SNT16 cells were fixed in formalin and blocked in phosphate buffer solution (PBS) with 5% goat serum and 0.1% Triton X-100. Then, the cells were incubated overnight at 4 °C with an anti-LMP1 antibody (Abcam, ab78113). After washing 3 times with PBS, the cells were incubated with a Cy3-labelled goat-anti-mouse secondary antibody and DAPI. After washing with PBS, the cells were analysed under a fluorescence microscope (Nikon, Japan) at × 40 magnification.

Immunohistochemistry

Tumour tissues of NKTCL patients or tumour-bearing mice were fixed in formalin, decalcified and paraffin-embedded. Following antigen retrieval, the sections were blocked with 0.3% H₂O₂ in methanol. The sections were boiled for 10 min in citrate buffer and blocked with serum-free protein block. Then, slides were incubated with anti-CD38 (Servicebio, GB114831), anti-LMP1 (Abcam, ab78113), anti-CD3ε (Servicebio, GB13014) or anti-CD56 (Servicebio, GB12041) primary antibodies and horseradish peroxidase (HRP)-labelled secondary antibody. For EBV-encoded RNA (EBER) in situ hybridization, the sections were treated with gastric enzymes and dehydrated with alcohol after dewaxing. Then, the sections were incubated with EBER probe and HRP-labelled anti-digoxin antibody. After incubation with HRP-labelled antibodies, the sections were washed three times with PBS and stained with DAB colour-developing solution. Then, the sections were counterstained with haematoxylin, dehydrated through a graded alcohol series, cleared in xylene and covered with coverslips. The percentage of positive cells was scored as follows: 0, negative expression (the frequency of positive cells was less than 5%); 1, weakly positive expression (the frequency of positive cells was 5% ~  < 25%); 2, positive expression (the frequency of positive cells ranged from 25 to < 50%); and 3, strongly positive expression (the frequency of positive cells was ≥ 50%).

Lentiviral chimeric antigen receptor constructs and generation of lentiviral particles

The CAR sequences included the single-chain antibody variable region gene fragment (scFv) domain(s) of CD38- and/or LMP1-specific antibodies, a CD8a transmembrane domain, a 4-1BB costimulatory domain and a CD3ζ motif. The CAR sequences were synthetically produced (GENEWIZ, Suzhou, China) and cloned into the pEF-MCS-P2A-EGFP vector. Among these, the LMP1-CAR sequence was cloned and fused with or without EGFP. CD38-CAR and two Tan-CAR sequences were not fused with EGFP.

293 T cells were cultured in DMEM medium supplemented with 10% FBS and antibiotics. Then, pRSV-Rev, pLP-VSVG, pCMV-Gag-Pol vectors and CAR constructs (or vector) were transfected into 293 T cells using PEI transfection reagent (Sigma, USA). Seventy-two hours after transfection, cell-free supernatants were collected and concentrated using an ultrafiltration device (Merck Millipore, USA).

Generation of CAR-T cells

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy donors by Ficoll-Paque density centrifugation. Then, T cells were isolated from PBMCs using magnetic cell separation (MACS) and CD3 Dynabeads (Miltenyi Biotec, Germany) and cultured in X VIVO-15 medium (LONZA, Switzerland) supplemented with 200 IU/ml IL-2 and CD3/28 Dynabeads (Gibco, USA).

T cells were transduced by incubation with a CAR lentivirus (CAR-T) or vector lentivirus (Control T). After immediate centrifugation for 90 min at 37 °C, the transduced cells were cultured at 37 °C and 5% CO₂ for 48 h. LMP1-CAR-EGFP fusion vector was used for the detection of transfection efficiency and in vitro cytotoxicity assay. LMP1-CAR vector without EGFP was used for the detection of T cell activation markers, cytokine production and in vivo experiments. The transfection efficiency of CD38-CAR or two Tan CAR-T cells was detected by fluorescence-activated cell sorter (FACS) using fluorescein isothiocyanate (FITC)-labelled recombinant human CD38 protein (ACROBiosystems, China).

Cell-mediated cytotoxicity assay

Serial dilutions of CAR-T or control T cells (effector) were co-incubated with NKTCL cell lines (target) at E:T (effector-to-target) ratios of 4:1, 2:1, 1:1 and 1:2 for 6 h. The cytotoxicity of CAR-T cells was determined by measuring lactate dehydrogenase (LDH) release by impaired tumour cells. LDH in the supernatant was detected with a CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega, USA).

To evaluate the cytotoxicity of T cells at a lower E:T ratio, NKTCL cell lines were labelled with carboxyfluorescein diacetate and succinimidyl ester (CFSE) and then co-incubated with CAR-T or control T cells at an E:T ratio of 1:2 for 18 h. Then, the cells were stained with Annexin V and PI (Keygen Biotech, Jiangsu, China) and analysed on a FACSCalibur flow cytometer.

Detection of T cell activation

CAR-T cells or control T cells were incubated with NKTCL cell lines (treatment group) at a ratio of 1:10 or with medium (blank group) for 24 h prior to detecting activation biomarkers of T cells. Then, different subsets of T cells were identified using fluorescein-conjugated antibodies specific for human CD4, CD8, CD25, CD69, CD38 and HLA-DR (BD Bioscience, USA).

Cytokine measurements

A cytometric bead array (CBA) was used to detect the inflammatory cytokines and granzyme B in the cell supernatant. In brief, 2 × 10⁴ CAR-T or control T cells were co-cultured with 2 × 10⁵ target NKTCL cells (treatment group) or medium (blank group) in a 200-μl volume for 24 h. Then, 50 μl of cell supernatant or cytokine standard dilutions with different concentration was incubated with specific capture beads recognizing IL-2, IL-5, IL-6, IL-13, granulocyte–macrophage colony-stimulating factor (GM-CSF), granzyme B, interferon (IFN)-γ and tumour necrosis factor (TNF)-α and phycoerythrin (PE)-labelled detection antibodies (BD Bioscience, USA) for 2 h. The beads were washed, and the fluorescence intensity of capture beads was analysed by a standardized flow cytometry assay. The standard curve was calculated according to the average fluorescence intensity of the standard dilutions. Then, the concentration of cytokines in the cell supernatant was calculated according to the standard curve.

In vivo experiment

The NSG mice (3–4 weeks old, female, 15–20 g) used in this study were obtained from the Shanghai Model Organisms Center (Shanghai, China). A total of 5 × 10⁶ NKYS-Luciferase cells were mixed with isopycnic Matrigel (Corning Incorporated, USA) and implanted subcutaneously in mice. The mice in treatment groups (n = 6 per group) received an intravenous tail vein injection of 4 CAR-T cells or control T cells on days 15 and 18 after tumour implantation, with a total of 1 × 10⁷ T cells infused per mouse (approximately 4 × 10⁶ transduced cells). Mice in the blank group were not treated. The tumour size was measured with a vernier calliper every 3 days for the first 50 days after implantation and daily after 50 days. The mice were given D-luciferin (150 mg/kg, i.p.) and anaesthetized with isoflurane. After 5 min, luminescence was detected using an in vivo imaging system (IVIS), and the intensity was quantitated and normalized with the Living Image software (PerkinElmer, MA, USA). A tumour size of 1000 mm³ or death of a mouse was considered an end event. After 75 days, the mice were euthanized by cervical dislocation.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The results are reported as the mean ± standard deviation or median and interquartile range for repeated measurements. Analysis of variance (ANOVA) followed by the Bonferroni post-test was applied to assess the differences among normally distributed samples, and the Mann‒Whitney test was used for non-normally distributed samples. A value of P < 0.05 was considered statistically significant.