流式细胞术 Flow cytometry 

CytoFLEX (Beckman Coulter Inc) was used to perform fluorescent expression analysis. Cells were harvested on the following days after transfection and stained with mouse anti-human antibody labeled by fluorescence for 10 minutes at room temperature in the dark as follows: TCR α/β-PE/Cy7 (IP26, Biolegend), TCRα/β-PE (IP26, Biolegend), β2-microglobulin (B2M)-PE (2M2, Biolegend), β2-microglobulin (B2M)-FITC (2M2, Biolegend), CD279(PD-1)-APC (EH12.2H7, Biolegend), CD279(PD-1)-PE (EH12.2H7, Biolegend), HLA-A2-FITC (BB7.2, BD Pharmingen).  

Surveyor nuclease assay and sequencing 

The levels of genomic disruption of TRAC, B2M, PD-1 in T cells or CAR-T cells were determined by surveyor nuclease assay using surveyor mutation detection kit (Integrated DNA Technologies, Inc). The percentage target disruption was quantified by densitometry and calculated as described [2]. The PCR products were also sequenced for TIDE (Tracking of Indels by Decomposition) analyses using specially designed software provided as a simple web tool (available at http://tide.nki.nl). The PCR primers used for the amplification of target loci and sequencing are listed in Supplementary Table 1. The purified PCR products were ligated with pEASY blunt cloning vector usingpEASY Blunt Cloning Kit (Transgen Biotech) to detect mutant alleles. Ligation products were used for transformation and about 20-30 colonies per sample are sequenced using universal primer M13F.  

富集双敲除好三敲除的CAR-T细胞 Enrichment of DKO and TKO CAR-T cells 

DKO and TKO CAR-T Cells were enriched using EasySep PE selection kit (Stemcell Technologies) according to the manufacturer's instructions. Briefly, the gene modified CAR-T cells were labeled with PE-conjugated antibody (TCRα/β-PE, β2-microglobulin-PE, PD-1-PE) and anti-PE MicroBeads, and then the labeled cells were put into a magnetic field. Using this procedure, the magnetically labeled PE-positive cells were retained in the tube while the unlabeled DKO and TKO CAR-T cells could be recovered in the supernatant.  

ELISA Cytokine enzyme-linked immunosorbent assay (ELISA)  

Cytokine production by effector (CAR-T, DKO CAR-T, TKO CAR-T, T) cells was evaluated by co-incubation with target tumor cells (Daudi, Raji, K562-CD19, K562) at a 1:1 ratio (104 cells each) for 24 hours. Supernatants were harvested and IL-2 and IFN-γ levels were analyzed by ELISA (Biolegend).  

流式细胞毒检测 Flow-based cytotoxicity assay 

The cytolytic activity and specificity of CAR-T cells were assessed according to the flow cytometry-based cytotoxicity assay described in [3]. Lytic activities of effector cells were tested by Violet/AnnexinV and 7-AAD labeling cytotoxicity assay. Target tumor cells were labeled with 1 µM Celltrace Violet (ThermoFisher Scientific) for 25 min at 37°C in PBS. Labeling was stopped by adding 10 mL complete culture medium and incubated at 37 °C for 5 minutes and extensively washed in complete culture medium before seeding into the 48-well plates (Corning). Violet-labeled target cells were then incubated with effector cells by different effector to target ratio for 4 hours. FITC-AnnexinV and 7-AAD (Biolegend) were added to determine the ratio of dead target cells. Samples were analyzed by flow cytometry. Target cells were selected by gating on the Violet-positive cell population and further analyzed for different subpopulations. The percentages of cytotoxic activity was calculated using the following equation: %specific cell death={[%(Violet+AnnexinV++Violet+ AnnexinV-7-AAD+)-%spontaneous (Violet+AnnexinV++Violet+ AnnexinV-7-AAD+)]/[100%-% spontaneous (Violet+Annexin V++Violet+ AnnexinV-7-AAD+)]}×100% . 

在体实验 In vivo studies  

6-12-week-old NOD-Prkdcscid Il2rgnull (NPG) mice (VITALSTAR, Beijing, China) were injected with 2×105 Raji-ffluc cells via intraperitoneal injection in a volume of 50µL DPBS and 50µL matrigel matrix (Corning). Two days after injection, tumor engraftment was evaluated by serial biophotonicimaging using the Xenogen IVIS Imaging System (Perkin Elmer Life Sciences). Mice were injected intraperitoneally with 3 mg d-luciferin (Perkin Elmer Life Sciences), and then imaged 4 minutes later with an exposure time of 30 seconds. Luminescence images were analyzed usingLiving Image software (Perkin Elmer Life Sciences). The bioluminescence signal was measured as total photon flux normalized for exposure time and surface area and expressed in units of photons/s/cm2/steradian (p/s/cm2/sr). Mice with progressively growing tumors were segregated into treatment groups bearing comparable tumor loads and received 200 uL DPBS /mouse, 5x106 T cells /mouse, 5x106CAR-T cells /mouse, 5x106 DKO CAR-T cells /mouse intraperitoneally one day later. The tumor loads were evaluated 7 days after treatment.  

脱靶分析 Off target analysis 

The potential off target of each sgRNA is predicted using Benchling software using algorithm described in [4]. The top five targets for each sgRNA were amplified by PCR and subjected to Sanger sequencing. Sequencing results were analyzed using the TIDE method [5]. 

In exome sequencing experiments, we used NimbleGen SeqCap_EZ_Exome_v3+UTR exonic target sequences to capture and enrich human exonic region following the manufacture’s instruction. Briefly, we first built the DNA library and randomly fragmented DNA. The DNA fragments were then hybridized with the exome lipid chip forming complex. After hybridization, the library was purified, evaluated for its quality, and applied to sequencing. We used genomic alignment software (BWA [6]) to map the clean reads to the reference genome UCSChg19 and samtools [7] to sort the BAM file for mutation detection with high accuracy. Potential sequence variations were called using mutational analysis software GATK [8] against the hg19 genome and were then filtered by quality value, depth and reproducibility using default parameters. Indels called in the different samples were overlapped and presented as Venn diagrams. 100 bp window of genomic sequences surrounding called indels were extracted. All 20 mer sequences followed by NGG PAM from both strands were enumerated and aligned to the sgRNA spacers. The best alignment (with minimal number of mismatches) of each sgRNA spacer against each 100 bp window was reported with the number of mismatches.  

统计分析 Statistical analysis 

Graphpad Prism 5.0 (Graphpad software, San Diego, CA) was used for all statistical analysis. The mean ± S.E.M. was determined for each treatment group in the individual experiments. The onetailed Student t-test was used to determine the significances between treatment and control groups. P-values < 0.05 were significant.