Cell lines and primary cells
Diffuse large B-cell lymphoma cell line SU-DHL-4, Burkitt lymphoma cell lines NAMALWA, RAMOS, RAJI and CA46, mantle cell lymphoma cell line JEKO-1, B lymphoblastic lymphoma cell line HMy2.CIR, B-ALL cell line NALM6, acute myeloid leukemia cell line MOLM13 were purchased form ATCC or DSMZ and preserved in our lab and all verified before being used in following experiments. All cell lines were cultured with RPMI 1640 medium containing 10% fetal bovine serum (FBS, Gibco, USA) in humidified 5% CO2 at 37 °C. Primary human tumor cells were obtained by magnetic bead sorting from peripheral blood of 2 clinical patients who suffered relapse after CD19 CART treatment, 1 case of B-NHL and 1 case of B- ALL, with informed consent obtained from all subjects, in accordance with the Declaration of Helsinki. Helsinki.
Generation of human CD22 CAR T cells
Lenti-X™293T cells were co-transfected with the vector encoding CD22 CAR, and the viral supernatants were collected 72 h later. The lentiviral concentrate was collected by ultracentrifugation and then aliquoted and stored at -80 °C. Peripheral blood samples were taken from healthy blood donors, and the T cells were separated from PBMCs using CD3 microbeads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) following the manufacturer’s instructions. Then, cells were stimulated with Dynabeads™ Human T-Activator CD3/CD28 (Gibco, Grand Island, NY, USA) at a 1:1 ratio in CTS™ OpTmizer™ medium (Gibco, Grand Island, NY, USA) containing 2 mM l-glutamine (Gibco, Grand Island, NY, USA) and 200 IU/mL rhIL-2 (PeproTech, Rocky Hill, NJ, USA). Within 24 h, the T cells were transfected with concentrated lentivirus at a multiplicity of infection (MOI) ranging from 3 to 5. Then, the T cells were cultured at a density of 5×105 to 1×106/mL.
NAMALWA Xenograft in vivo studies
The functionality of CD22 CAR T cells were evaluated in vivo in NAMALWA xenograft with NCG mice (Gempharmatech, Nanjing, China) aged 6-8 weeks. The female mice received 1x106 GFP+ Luciferase+ NAMALWA cells intravenously on day 0. 7days later, the mice were divided into three groups and were intravenously injected with CD22-CAR-transduced T cells at the indicated quantity. To monitor leukemia burden, luciferin-D (YEASEN, Shanghai, China) was injected into mice intraperitoneally and imaged 8 minutes later using In Vivo Imaging System (IVIS) technology (Caliper Life Sciences, USA). Bioluminescent signal flux (luminescence) for mice was measured using Living Image Version 4.1 software. All animal experiments were approved by the Institutional Committee of Animal Care of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. All experiments were performed at the animal experimental center of Tongji Medical College. All methods were carried out in accordance with relevant guidelines and regulations and reported in accordance with ARRIVE guidelines.
Chidamide treatment
Chidamide was purchased from Med Chem Express (USA), dissolved in Dimethyl sulfoxide (DMSO) at the recommended concentration according to the instructions, and stored at -80 °C. For vitro experiments, Chidamide was administered to cells at the indicated concentration, and the cells were centrifuged and replaced with fresh medium containing the corresponding concentrations of Chidamide every 3 days, while the control group was treated with DMSO at an equal volume. For tumor/T cells pretreatment, Chidamide was administered to cells at a indicated concentration, DMSO vehicle control was administered to control-treated cells at an equal volume. Prior to co-culture, Chidamide- and DMSO- treated cells were washed three times in sterile PBS. For all in vivo experiments, Chidamide was diluted in DMSO, PEG300, Tween80 and saline in turn and orally administered at 3 mg/kg (30ug/20g mouse). For control-treated groups, an equal volume of DMSO was diluted and administered in the same manner.
Flow Cytometry
FACS analysis of cell surface CAR and protein expression was performed using a CytoFLEX S flow cytometer (Beckmam Coulter, CA, USA). CD22-CAR was detected by incubation with APC-F(ab)2 (Jackson Immunoresearch, USA). The following human monoclonal antibodies were used for detection of cell surface proteins: CD22-APC, CD22-PE, CD19-PE, CD45-PerCP/Cy5.5, CD3-APC/Cy7, CD8-Pacific Blue, CD4-APC, CD45RO-PE/Cy7, CD45RA-FITC, CCR7-APC (all from BioLegend). CD22 site density was quantified by Quantity-PE beads (BD Biosciences, USA) following instructions. Both GFP-expressing cells and CFSE dye-labeled cells were identified through the FITC channel.
Proliferation and apoptosis assays
Cell lines and primary cells were assayed for cell proliferation using the CCK8 kit (Vazyme Biotech, Nanjing, China), and cell proliferation inhibition rate (%) = (OD treated - OD blank)/(OD control - OD blank) × 100%. Apoptosis was detected by AnnexinV/7-AAD apoptosis assay kit (BD Biosciences, USA), and the apoptosis rate was detected by flow cytometry after cell staining. The proliferation of T cells was detected by CFSE. CD22-CAR-transduced T cells and mock-transduced T cells were labeled with CFSE separately and co-cultured with different target cells at a 1:1 ratio in 24-well plates with IL-2-free T cell complete medium (TCM). 4 days later, Cells were stained with CD3-APC/Cy7, and the cell division index (CDI) was calculated to compare the proliferation differences of T cells in each group.
CD107a release assay
The cell fractions positive for the CD22 CAR were adjusted to 30% by adding untransfected T cells. CAR T cells were washed to remove IL-2 and resuspended in RPMI medium and then were co-cultured with different target cells at a 2:1 ratio at 37 ºC in an incubator. CD107a-PE/Cy7 antibody (BioLegend, CA, USA) was initially added at 20 µl/mL. One hour after incubation, monensin (Golgi-Stop, BD Biosciences, NJ, USA) was added to a final concentration of 6 mg/mL, and incubation was allowed to last an additional 3 h. The cells were then stained with CD3-APC (BioLegend, CA, USA) and CD8-Pacific Blue (BioLegend, CA, USA) antibodies, and CD107a expression on the CD3 and CD8 double-positive cells was detected.
In Vitro Cytotoxicity
The calcein release assay was performed to detect the cytotoxicity of CAR T cells in vitro. The target cells (NALM6 and NAMALWA cells pretreated with Chidamide, NALM6 and NAMALWA cells pretreated with DMSO) positive for CD22 and target cells (MOLM13 cells) negative for CD22 were labeled with calcein (Aladdin, Shanghai, China) and then co-cultured with effector cells (untransfected T cells and CAR22-transfected T cells) in 96-well plates at different ratios (9:1, 3:1, and 1:1). Wells with co-cultured target cells and PBS served as spontaneous release wells, and wells with co-cultured target cells and lysis solution were taken as maximum release wells. The cultures were centrifuged 3 h after incubation, and the supernatants were transferred to another 96-well plate. The fuorescence of each well (F) was measured on a microplate reader, and the tumor-killing efficiency was calculated according to the following formula: lysis (%) = (F experimental wells - F spontaneous release)/(F maximal release - F spontaneous release) × 100%.
Detection of Intracellular Cytokines
As in the CD107a assay, different effector cells (untransfected T cells and CAR22-transfected cells) were co-cultured with different target cells (NALM6 and NAMALWA cells pretreated with Chidamide, NALM6 and NAMALWA cells pretreated with DMSO, and MOLM13 cells) at a 2:1 ratio in 96-well plates and incubated at 37 ºC for 24 hours. Plates were spun at 1200 RPM for 6 minutes to pellet the cells, and cytokine concentrations in the culture supernatants were measured using ELISA kit (NEOBIOSCIENCE, China), including IL-2, TNF-α, IFN-γ and GranzymeB following the manufacturer’s instructions.
Quantitative Real-Time Polymerase Chain Reaction Analysis
The appropriately treated cells (SU-DHL-4, NAMALWA, RAMOS, RAJI, CA46, JEKO-1, HMy2.CIR, and NALM6 cells) were collected and resuspended in Trizol agent for RNA extraction. cDNA was converted from 1 μg RNA using a reverse transcriptase kit (Vazyme Biotech, Nanjing, China). Gene expression was assessed using qRT-PCR following the instructions and the obtained data were analyzed by the 2−ΔΔCt method.
Western Blotting Analysis
Cytoplasmic proteins, cytosolic proteins and total proteins were extracted using a protein extraction kit (abcam, UK) and proteins in all samples were quantified with Bicinchoninic acid protein assay. Proteins of equal amounts from all samples were separated with SDS/PAGE gel (Bio-Rad, USA) and transferred onto PVDF membrane (Bio-Rad, USA). Bands were sealed with 5% skim milk, incubated in primary antibodies (anti-CD22 and anti-GAPDH, CTS, SUA) at 4 °C overnight, then in secondary antibodies (HRP-goat anti-rabbit IgG, CTS, SUA) at room temperature for 1 h, and then examined and analyzed by using ChemiDoc™ XRS+ with Image Lab™ Software (Bio-Rad, USA).
Transcriptome sequencing and data analysis
Three biological replicates RNA samples were collected From NAMALWA and NALM6 cell lines which were grouped as control or treated with 0.5 μM Chidamide for 3 days. Total RNA was used for RNA-seq analysis. cDNA library construction and
sequencing were performed by Beijing Genomics Institute using BGISEQ-500 platform. High-quality reads were aligned to the human reference genome (GRCh38) using Bowtie2. The levels of expression for each of the gene were normalized to fragments per kilobase of exon model per million mapped reads (FPKM) using RNA-seq by Expectation Maximization (RSEM)46. Pathway analysis was performed using the GSEA software developed by the Broad Institute47. The significance of DEGs was confirmed with the BGI bioinformatics service using the combination of the absolute value of log2-ratio ≥1 and P ≤ 0.05 in this research.
Data analysis
SPSS 25.0 software (IBM, NY, USA) was used for data analysis. Comparison of quantitative data between groups was performed using Student’s t test, one-way ANOVA, or a nonparametric test. P < 0.05 was considered statistically significant (*P < 0.05; **P < 0.01; ***P < 0.001), and various statistical graphs were used. were plotted using Graphpad Prism 8.0 software.