Cell Culture and NMN treatment
Human peripheral blood from healthy donors were diluted with PBS at the ratio of 1:1, lymphoprep (MP Biomedicals) was used to centrifugate and isolated the blood by low-density centrifugation. Human peripheral blood mononuclear cells (PBMC) were activated with anti-CD3 and anti-CD28 beads and transduced with retrovirus on two consecutive days by centrifugation on retronectin-coated culture dish (Thermo). Six days after transduction, T cells were cultured in X–VIVO15 serum-free medium (Lonza) which containing 5% Human Serum AB (Gemini Bio) and IL-2 (SL PHARM). For weekly stimulations, cells were treated by100uM NMN (St. Louis, MO, USA N3501). This research was approved by the Beijing Shijitan Hospital Institutional Review Board and informed consent was obtained from all the participants. All methods were performed in accordance with the relevant guidelines and regulations
NALM-6 was obtained from ATCC and cultured in RPMI-1640 medium (Lonza) supplemented with 10% fetal bovine serum (Biosera), 10,000 IU/mL penicillin/10,000 μg/mL streptomycin (EallBio Life Sciences) at 37°C in a humidified 5% CO2 incubator. NALM-6 cells were transduced to express firefly luciferase-GFP.
The firefly luciferase-GFP expressed NALM-6 cells were served as the target cells. The effector (E) and tumor target (T) cells were co-cultured in triplicates. The E/T ratio was 0.5:1 using 96-well plates with 1x 104 target cells in a total volume of 200 μl T cells medium each well. Target cells alone were plated at the same cell density to determine the maximal luciferase expression.24 hours later, 20μl luciferase substrate (Bright-Glo, Promega) was added to each well directly. Emitted light was detected on IVIS Series III imaging system (Lunma), and quantified using Living Image software (Lunma).
Antigen provide and proliferation assays
NALM-6 expressing firefly luciferase-GFP cells were used as antigen-presenting cells. For adequate antigen to sustain the cell alive, 1x 105NALM-6 cells and 1x 106 CAR-T cells were co-culture in 24-well plates within XVIVO 15, 5% human serum AB and 200 U IL-2 /ml. Every 7 days, cells were counted with trypan blue.
Flow cytometry was performed and analyzed on a FACS Canto Plus instrument (BD Biosciences). The following antibodies were used to define antigen expression by flow-cytometry:CD3APC-CY7(BDBiosciences,560176);CD4-V450(BDBiosciences562424);CD8-FITC(BDBiosciences555634);CCR7-PECY7(BDBiosciences,557648);45RO-BV605(biolegend,304238);CD107a-FITC(Beckton-Dickinson Pharmingen, San Diego, CA, USA). For flow cytometry for detection of apoptosis, aliquots of 105 cells were washed and incubated in the dark for 15 min in PBS containing either 2 μl of annexin V-FITC (to detect early apoptosis) and 2ul 7-AAD/ml (to detect late apoptosis), Samples were analyzed 20 min later by FAC-Scan flow cytometry.
The CAR-T cells treated with/without NMN were co-cultured with NALM-6 cells at an E: T ratio of 0.5:1 for 24 hours. The medium from the co-culture model was collected and centrifuged at 1500rpm to collect the supernatant. IFN-γ and IL-6 cytokines in the harvested supernatant were measured using a commercial ELISA kit in accordance with the manufacturer’s instruction (R&D system).
NAD+ and NADH levels were measured using NAD+/NADH assay kits according to the manufacturer’s instructions (Abbkine CheKine™). The principle of the method is based on glucose dehydrogenase cycling reaction. The product absorbance was measured at 565 nm, is proportionate to the NAD+ concentration of the sample25 .
Telomerase activity measure
Telomerase activity of the samples were assayed using TRAPEZE Gel-Based Telomerase Detection Kit (Merck S7700). Briefly, 1μg protein was mixed with TRAP PCR mixture for PCR amplification and the products were electrophoresed on a 12 % non-denaturing polyacrylamide gel and chemiluminescence detection.
DNA Isolation and Measurement of Telomere Length
The genomic DNA was extracted from the CD19 CAR-T cells treated with/without NMN. FastPure Blood DNA Isolation Mini Kit V2 (Vazyme) was used following theinstruction book. The NanoDrop spectrophotometer (Thermo Fisher Scientific) was used to detected the DNA concentration. Telomere length was assessed by Real-time quantitative PCR. Briefly, the primers F:CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT , R:GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT was used to amplify the telomeres (T) of the CD19 CAR-T cells.
F: CAGCAAGTGGGAAGGTGTAATCC R:CCCATTCTATCATCAACGGGTACAA were used to amplify the single-copy gene as S. The relative telomere length was measured by comparing the ratio of T repeat copy number and S copy number, expressed as the telomere length (T/S) ratio.
The SA-β-gal staining of CD19 CAR-T cells treated with/without NMN was investigated using SA-β-gal staining kit (Cell Signaling Technology). CD19 CAR-T cells were fixed and washed in 1.5ml tubes and incubated with the staining solution at 37°C overnight. Four random fields each well were imaged using the phase-contrast microscope (Olympus). Mean percentage of SA-β-gal-positive cells was calculated.
Quantitative real time polymerase chain reaction (Q-PCR)
Total RNA was extracted with TRIZOL reagent (Invitrogen) and reversed-transcribed using the Prime Script RT Master Mix Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. The quantity and purity of RNA were measured using Nanodrop spectrophotometer (Thermo Fisher Scientific). The reverse transcription products were used as the templates and subjected to Q-PCR amplified using SYBR Green PCR Master Mix (Thermo Fisher Scientific) with gene-specific primers. The primer sequences for Q-PCR analysis are listed in the Supplementary Material. GAPDH was used as the internal control. Relative gene expression was calculated using the 2− ΔΔCt method. Primer sequences are shown in Table 1.
Total RNA was extracted from 1x106 cells by using TRIZOL reagent (Invitrogen) and the cDNA was synthesized using the cDNA Reverse Transcription kit (Thermo Fisher Scientific).PCRs were performed using the following primers:SIRT1:F:- GCAGGTTGCAGGAATCCAAA.R:GGCAAGATGCTGTTGCAAAG.β-actin: F: GGGTCAGAAGGATTCCTATG, R:GGTCTCAAACATGATCTGGG.ThePCR cycle conditions were 94℃for 2 min, 55℃for 1 min, and 72℃for 1 min for a total of 35 cycles. Samples were resolved on a 1% agarose gel and visualized with ethidium bromide.
We used 6 to12week-old NOD/SCID mice, under a protocol approved Beijing shijitan hospital in Capital Medical University Institutional Animal Care and Use Committee. All relevant animal use guidelines and ethical regulations were followed. Mice were inoculated with 2x 106NALM-6 expressing firefly luciferase-GFP cells by tail vein injection using 200μl 1× PBS, followed by 2x 107 CD19 CAR-T cells injected for 3 days. The CD19 CAR-T cells treated with NMN (100uM) 24 hours in advance. NALM-6 expressing firefly luciferase-GFP produce even tumor burdens, NMN was administered intraperitoneal injection at 300 mg/kg/day. Bioluminescence imaging was performed by injecting mice weekly via tail vein with luciferin and quantifying luminescence using IVIS Lunma Series III imaging. The study is reported in accordance with ARRIVE guidelines.
2 µg total RNA samples were used as the input material for generating the sequencing libraries, The NEBNext®Ultra™ RNA Library Prep Kit was used (#E7530L, NEB). In brief, poly-T oligo-attached magnetic beads was used to purify mRNA from total RNA. Under the condition of elevated temperature and NEB Next First Strand Synthesis Reaction Buffer (5X), divalent cations was used to generate fragmentation. Random hexamer primers and RNase H was used to synthesize first-strand cDNA. Second strand cDNA synthesis was subsequently performed using buffer, DNA polymerase I, dNTPs and RNase H. The library fragments was purified by QiaQuick PCR kits and the product was eluted with EB buffer, and then terminal repair, A-tailing, and adapters were implemented. Index-coded samples was performed on a cBot Cluster Generation system with TruSeq SR Cluster kit, v3-cBot-HS (Illumina Inc). Afterwards, the library was subjected to an Illumina Nova Seq 6000 System platform (Illumina Inc). Raw data were first processed using custom Perl scripts. Clean data were read by removing reads containing poly-N with 5′- adapter contaminants, without 3′- adapter or the insert tag, or containing poly-A, -T, -G or -C from raw data, as well as low-quality reads. The genes expressed differentially were subjected to KEGG pathway, gene ontology (GO) and subcellular localization analysis using DAVID Bioinformatics Resources 6.8.
The statistical significance of differences between two groups was assessed with the two-tailed paired or unpaired t test. Comparisons of more than two groups were performed use the one-way ANOVA with multiple comparison tests. Data are shown as the mean ± standard deviation (SD). Difference were marked as NS, P > 0.05; *P < 0.05; **P < 0.01, and ***P < 0.001.In vivo experiment, P value of radiance based on Wilcoxon rank-sum test. The excel Kaplan-Meier and non-parametric Wilcoxon p value was used for survival curve comparison. All the data obtained from the study was analyzed using SPSS 22.0 (IBM, USA).