FOXO1 affects CAR T cell stemness and metabolism

Isolation and Preparation of Healthy Donor T Cells for a Blood Transplant Experiment at Children’s Hospital of Philadelphia

The coats were obtained from anonymous donors, or from people who consented to their blood being used in the experiment, under the University Institutional Review Board-exempt protocol. CD3+ cells were isolated using the RosetteSep Human T Cell Enrichment Kit, Lymphoprep density gradient medium and SepMate-50 tubes according to the manufacturer’s protocol (STEMCELL Technologies). The University of Pennsylvania Human Immunology Core was used to obtain the CD3+ healthy donor T cells for the experiments at Children’s Hospital of Philadelphia. All T cells werepreserved in a medium.

HER2-transduced tumour cells from American Type Culture Collection ( ATCC) for study of Mycoplasma and colon adenocarcinoma

The colon adenocarcinoma cell line was given to the mouse MC 38. The mouse breast carcinoma cell line E0771 was obtained from R. Anderson. The MC38 and E0771 tumours cell lines were transduced with a mouse stem cell vaccine to express a truncated human HER2 antigen that lacks signalling components. Transduced tumour cell lines are referred to as MC38-HER2 and E0771-HER2. OVCAR-3 and MCF7 tumour cells were obtained from the American Type Culture Collection. PCR analysis was used to verify that tumour lines were negative for Mycoplasma.

The CAR constructs used in this study include CD19.28ζ, CD19.BBζ, anti-GD2 HA.28ζ and Her2.BBζ. Gene Blocks were created from the codon-optimized Tcd1, FOXO1, FOXO13A, and P2A ribosomal skip sequence. The only thing that has a P2A ribosomal skip sequence is the tNGFR-only construct. The FOXO1DBD construct was generated by two-step mutagenic NEBuilder HiFi DNA Assembly (New England BioLabs). All plasmids were amplified by transformation into Stellar Competent Escherichia coli (Takara Bio), and sequences were validated by sequencing (Elim Biopharmaceuticals).

The American type Culture Collection ( ATCC) has two retroviral packaging cell lines. The cells were kept in a RPMI medium with 10% heat-inActivated Fetal bovine Serum, 1 mM of stearic acid, 2 mM of glutamine, and 0.1 mM of non-essential acids. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. The cells were kept in a humidified incubator with a small amount of CO2. The PA317 cell line was maintained in DMEM (Gibco) supplemented with 2 mM glutamine and 100 U ml−1 penicillin and 100 μg ml−1 streptomycin and was maintained in a humidified incubator at 37 °C with 10% CO2.

A total of 5 × 104 CAR T cells were co-cultured with 5 ×104 tumour cells in 200 μl of complete T cell medium (AIM-V or RPMI) without IL-2 in a 96-well plate, all in triplicate. Twenty-four hours after co-culture, culture supernatants were collected, diluted 20- to 100-fold and analysed for IL-2 and IFNγ using ELISA MAX kits (BioLegend) and Nunc Maxisorp 96-well ELISA plates (Thermo Fisher Scientific). Absorbance readings were collected with a plate reader or the Synergy H1. The co-culture medium used for FOXO1i had concentrations of AS1842856 used during T cell expansion.

CD8+tNGFR+ CAR T cells were isolated using the EasySep Human CD8+ T Cell Isolation Kit. A total of 150,000 CD8+ T cells were slow-frozen in BamBanker (Bulldog Bio) cell preservation medium. 100,000 CART cells were washed in ice cold PBS and subjected to nuclei isolation through a 10 mM lysis buffer. NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin and 1% BSA. 50 l lysis buffer was added after each sample and cells were resuspended using pipetting. Nuclear pellets were resuspended in a transposase reaction containing two Tn5 transposses, as well as H2O, 2 and 12.5 l. The reaction was put to use for 30 min at 37 C. The reaction was stopped by the addition of 75 μl TE buffer and 500 μl PB buffer (QIAGEN), followed by column purification per the manufacturer’s recommendation (QIAGEN, Minelute Kit). The columns had 21 l DNA, 25 l Phusion master mix, and 2 l of each barcoded PCR primer. Each sample had 15 cycles run for it. Reactions were cleaned up with AMPure XP beads according to the recommendations of the manufacturer. Libraries were quantified with a Qubit fluorometer and fragment analysis was performed with Bioanalyzer. The libraries were read on the NovaSeq 6000.

deletion of a sequence from the FOXO1 DNA-binding domain was used to examine the role of FOXO1 in CAR T cell function. CAR T cells were removed from activated beads by magnetic separation on day 4. Twenty microlitre reactions were prepared with one million CAR T Cells in a P3 buffer before they were killed by the P3 primary cell kit. Ribonucleoproteins were prepared by complexing 0.15 ng of sgRNA targeting FOXO1 or AAVS1 (Synthego) with 5 µg Alt-R S.p. It’s called Cas9 Adding the cell suspension to each reaction requires the use of a genotypic substance. A previously valid sgRNA sequence was used for AAVS1 edits. For FOXO1, two separate sgRNAs were used in tandem, at equal concentrations (5′-UUGCGCGGCUGCCCCGCGAG-3′ and 5′-GAGCUUGCUGGAGGAGAGCG-3′). sgRNA56 was used for bulk-Rab-seq experiments performed at CHOP. A separate sgRNA was designed and used for in-vivo experiments. The Lonza 4Dnucleotide was used for the pulsed reaction. Cells were recovered immediately in 260 µl of warm complete AIM-V medium supplemented with 500 U ml−1 IL-2 in round-bottom 96-well plates and expanded into 1 ml fresh medium within 24 h. Cells were maintained at 0.5 × 106 cells per ml to 1.0 × 106 cells per ml in well plates until day 14–16 for functional and phenotypic characterization. The knockout efficiency was determined by the staining of the cell signal and the flow cytometry.

CAR T cells were washed twice in the same FACS buffer and stained with anti-freeze and fluorophores for 30 minutes. Cells were washed twice with FACS buffer before analysis. The FoxP3 Transcription Factor Staining Buffer Set is the same set of reagents as the initial surface stain. Anti-human FOXO1 (clone C29H4) and anti-human TCF1 (C36D9) antibodies were purchased from Cell Signaling. The 1A7 anti-14G2a idiotype antibody used to detect the HA CAR was obtained from the NCI and conjugated using the Dylight 650 antibody labelling kit (Thermo Fisher Scientific). The anti-FMC63 idiotype antibody was manufactured by GenScript and fluorescently conjugated using the Dylight 650 antibody labelling kit. Cell-surface antibodies were used at a 1:100 dilution during staining, with the exception of anti-14g2a and anti-FMC63, which were used at a 1:1,000 dilution. Intracellular antibodies were used at a 1:50 dilution and live/dead staining was used at a 1:1,000 dilution. Cells were analysed with either a BD Fortessa running FACS Diva software, or a Cytek Aurora using SpectroFlo v.3.1.0. Downstream analyses were performed using the FlowJo software. All reagents are listed in Supplementary Table 2. A representative gating strategy for FOXO1KO and FOXO1OE experiments is shown in Supplementary Fig. 1. In experiments in which we stained for Annexin V, cells were gated on all singlets, excluding debris but not excluding dead or dying T cells. For MFI quantification, background subtraction was performed using either unstained or FMO samples. The MFI quantification in Extended Data Fig. 1e was not background subtracted owing to negative MFI values in some control samples.

CAR T cells were cultured with Tumour cell targets at a 2:1 ratio for 24 h. After overnight incubation, supernatants were collected and an equivalent number of tumour cells were reseeded into the incubations for another 24 h. This process was repeated one final time before cells were collected for analysis by flow cytometry and supernatants were analysed by cytometric bead array (CBA) using either mouse or human cytokine Flex sets (BD Biosciences) according to the manufacturer’s instructions.

Sea Horse Bioscience Analyzer XFe 96 was used for the analyses. 0.2 106 Cells were resuspended in the assays medium supplemented with 11 mMglucose and 2 mM glutamine and plated on a Cell-Tak microplate. The values of Mitochondrial activity and glycolytic parameters were measured by the oxygen consumption rate and ECAR, respectively, with the use of real-time injections. Respiratory parameters were calculated according to the manufacturer’s instructions (Seahorse Bioscience). Supplementary Table 2 contains reagent sources.

Chromatin-bound and soluble proteins were separated as previously described23. In brief, cytoskeletal (CSK) buffer was prepared using 100 mM 300mM sucrose, 3mM MgCl2, 10 mM PIPES, 4% IGEPAL CA- 630. After washing, cells were lysed with buffer for 20 minutes on ice. The sample was pre-warmed at 1,500g for 5 min, and then it wascentrifugation to separate the sample into two parts. The proportion of conjugates was determined by the DCProteinASSy. The remaining pellet was washed twice with the buffer and then centrifuged for 5 minutes. The resuspended proteins were boiled in the 1Pierce Reducing Sample Buffer for 5 min before being resuspended in a new buffer. The soluble fraction was supplemented with Pierce Reducing Sample Buffer to achieve 1× and boiled for 5 min. For immunoblotting, equal amounts of soluble and chromatin-bound fraction for each sample were analysed by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad, 1704158). For 30 min, the Membranes were blocked in 5% milk from the TBST (1 Tris buffered saline containing 0.1% Tween-20). After washing with liquid, the cells were placed in a container and immersed in the anti-FOXO1 antibody at 4 C. Next, membranes were washed with TBST and incubated with anti-mouse (1:10,000, Cell Signaling, 7074) or anti-rabbit (1:10,000, Cell Signaling, 7076) IgG conjugated to horseradish peroxidase for 1 h at room temperature. The ChemiDoc Imaging System and Image Lab Touch Software v. 3.0 were used to allow for visualization of themembranes. After visualization, membranes were stripped using a mild stripping buffer (1.5% glycine, 0.1% SDS, 1% Tween-20, pH 2.2). The previous steps were repeated for detection of soluble (1:5,000 GAPDH; Cell Signaling, 97166, clone D4C6R) and chromatin-bound (1:1,000 Lamin A; Cell Signaling, 86846, clone 133A2) fraction loading controls. Densitometry analyses were done using the same software as before.

NOD/SCID/Il2rg/ mice under APLAC- or CHOP ACUP-approved protocols. Engraftment, expansion and clearance of 142B tumours by tail vein injection, and

NOD/SCID/Il2rg−/− (NSG) mice were bred, housed and treated under Stanford University APLAC- or CHOP ACUP-approved protocols. The six to eight-week old mice were healthy, immunocompromised, drug and testnaive and unused in other procedures. The mice were kept at a barrier facility that had a 12-h light–dark cycle and had a temperature of 20–23 C or 20–26. There were a lot of mice in each cage and they had ample food and water. Liquid feeds were used to facilitate eating for sick mice. Mice were monitored daily by trained VSC and DVR staff under the supervision of a veterinarian who reported excess morbidity immediately and/or euthanized mice for humane reasons. If the end-point criteria were met, mice were euthanized if they had 142B tumours larger than 1.2 cm, or if evidence of extensive disease occurred. Tumour injection sites were chosen so as not to interfere with the mouse’s normal body functions, such as ambulation, eating, drinking, defecation and/or urination. In Nalm6-bearing mice, 2 × 105 to 1 × 107 cells in 100–200 μl of sterile PBS were engrafted by tail vein injection (TVI). In 143B osteosarcoma models, 1 × 106 to 3 × 106 cells in 100 μl sterile PBS were engrafted by intramuscular injection into the flank. Mice were randomized prior to CAR T cell infusion to ensure equal tumour burden across groups. The main text noted that TVI had enfranchised the CAR T cells. Nalm6 engraftment, expansion and clearance were measured by intraperitoneal injection of luciferin and subsequent imaging by a Spectrum IVIS bioluminescence imager and quantified using Living Image software v.4.7.3 (Perkin Elmer), or by a Lago X imager and quantified using Aura software v.4.0.7 (Spectral Instruments Imaging), all under isoflurane anaesthesia. The size of the 142B tumour was measured. T cell and Tumor injections were done by technicians who were blinded to treatments and outcomes.

Peripheral blood was taken from mice with isoflurane anaesthetized. Fifty microlitres of blood was labelled with surface antibodies, lysed using FACS Lysing Solution (BD) and quantified using CountBright Absolute Counting Beads (Thermo Fisher Scientific), then analysed on a BD Fortessa cytometer. The mice were euthanized and the tissues were washed twice in PBS. sputlums were put in a 6 cm petri dish and then washed through a sterile 70 m cell strainer. Tumours were mechanically and chemically dissociated with Collagenase IV and DNAse in HBSS and incubated at 37 °C with shaking for 30 min. Cells were mashed through a sterile 70-µm cell strainer before washing with PBS. The cells were spun down to 450g and then treated with aCK lysis buffer. Cell suspensions were washed with PBS and CAR T cells were isolated using the EasySep Release Human CD45 positive selection kit. The cells were stained with markers of interest and analysed on the Cytek Aurora using the SpectroFlo software.

A total of 106 T cells were pelleted after being flash-frozen. Pellets were thawed on ice and processed using either an RNEasy Plus Mini Kit or an AllPrep DNA/RNA Micro Kit (for simultaneous DNA and RNA isolation) (QIAGEN) according to the manufacturer’s instructions. Total RNA was quantified using either a Qubit Fluorometer or a DeNovix DS-11 FX Spectrophotometer/Fluorometer and sequenced using a 150 bp paired-end read length and around 50 million read pairs per sample (Novogene).

Fastq files were generated from the sequence files for ATAC-seq experiments. Next, quality control of files were performed using FASTQC (v0.11.5). Adaptor trimming of paired-end reads was performed with NGmerge (v0.3) where required59. The reads to the reference human or mouse were aligned using Bowtie2. The SAM files were converted toBAM files using the Samtools view command, which sorted and indexed the SAM files, and which marked potential duplicates with the Samtools markdup. Peak calling was performed with either MACS2 (v2.1.1) or Genrich (v0.6.0) packages. The ATAC peaks were annotated using either the annotatePeaks.pl function from the ChIPseekerR package or the annotations from the Homer package. The BigWig files were converted using the bam function. BigWig files were then imported into Integrative Genomics Viewer (IGV, v2.7.0) for visualization of specific loci. To generate IGV style track plots from BigWig files, the package trackplot was used60. The HOMER makeTagDirectory command was used to generate tag directories, and the findPeaks command was used to identify peaks, with the control tag directory set to respective control groups. The findMotifsGenome tool and settings identify de novo motifs from peaks identified. The enriched motifs from the database were identified with the help of the ChromVAR R package.

The human CD45+ cells were isolated through the process of NGFR selection, and the Her2+ tumours were collected from five mice per condition. Tumour-infiltrating CAR T cells were further purified using human CD3+ TILs. A total of 20,000 CAR TILs were pooled across five groups of mice. Cells were barcoded and sequencing libraries were generated using the 10X Chromium Next GEM Single Cell 3’ v.3.1 kit (10X Genomics) according to the manufacturer’s instructions. Libraries were sequenced at the CHOP High Throughput Sequencing Core on an Illumina NovaSeq 6000 with an average read depth of 50,000 reads per cell.

The libraries were processed using the pepatac pipeline and had default options. Fastq files were trimmed to remove the adapters and then pre-aligned to the mitochondrial genome to exclude reads. To make sure the accuracy of downstream analysis was maintained, multiple mapping reads were removed from the dataset. Bowtie2 was used to align the reads. SAMtools was used to identify uniquely aligned reads, and Picard was used to remove duplicate reads. The file was deduplicated and aligned for downstream analysis. Peaks in individual samples were identified using MACS2 and compiled into a non-overlapping 500-bp consensus peak set. The peaks were ranked by significance based on their 500 bp width. The peaks that overlap with the region were picked by ranks, and the most significant peak was retained. The peak-sample count matrix was generated using ChrAccR with the default parameters of the run_atac function. Signal tracks for individual samples were generated within the pepatac pipeline. These tracks were then merged by group using WiggleTools to produce a comprehensive view of the data across all samples.

On the basis of our analysis of the peak-sample count matrix, the DESeq2 v.3.16 package was used to identify differential peaks across different conditions, with a threshold of an absolute log2-transformed fold change greater than 0.5 and P value less than 0.05. Adjusted P values were not used owing to donor variability. The vst function can be used to extract a variance-stabilized count matrix. Next, we corrected for batch effects by donor using the removeBatchEffect function in the limma library. Finally, we generated PCA plots using the corrected matrix with the plotPCA function using the top 2,000 most variable peaks. We aggregated differential peaks across conditions, standardized the peak signals using z-scores across samples and performed k-means clustering to generate a chromatin accessibility heat map. Motif enrichments of differential peaks and grouped peaks were searched with HOMER and findMotifsGenome.pl with default parameters. The gchromVAR package was used to enrich the regulatory elements. In brief, this method weights chromatin features by log2-transformed fold changes of cell-type-specific regulatory elements from a previous report9 and computes the enrichment for each cell type versus an empirical background matched for GC content and feature intensity.

Downregulated differential genes were intersecting in FOXO1KO cells and upregulated differential genes when creating the FOXO1 regulon gene set. Regulon enrichment scores were calculated by using ssGSEA in the GSVA R package.

The data objects of CAR T products profiled by single-cell ATAC-seq can be processed for regulon analyses. Mean fragments in peaks per cell were compared to consistency between donors. After examining quality control statistics, per-library enrichment and low data quality were the factors that led to the omission of donors. Using the epigenetic signature for FOXO1 and TCF1 overexpression (Fig. 2), we computed the per-cell epigenetic signature per factor using the chromVAR workflow as previously described for related T cell signatures derived from bulk experiments. To check for differences in the responder/non-responder associations, we performed an Ordinary least squares Regression with the per-cell Z-score against the donor’s BCA status at 6 months. Statistical significance was based on the Wald test statistic of the coefficient for the responder term in the two regressions for each factor.

In cases of significant differences between groups, the statistical analyses were done with either a one- or two-way analysis of variance or with a single- or multi-comparison test. In experiments in which same-donor samples were compared across two conditions, we performed a paired Student’s t-test. The survival curves were compared with each other. Statistical methods were not used to predetermine sample sizes.

This research and all study protocols have been approved and comply with the Peter MacCallum Animal Experimental Ethics Committee (AEC) ethical regulations regarding the use of animals. The Human Research Ethics committee at the Peter MacCallum Cancer Center approved studies using human peripheral blood mononuclear cells from healthy donors. The Australian Red Cross obtained informed consent.

The isotype control (1A3 clone, IgG2a, BE0254) and the Mouse Ikin antibodies were purchased from BioXcell. The cytokine IL-2 was obtained from the National Institutes of Health and purchased from Peprotech. I-7 and IL-15 were purchased from Peprotech. Where indicated, CAR T cells were stimulated with an anti-idiotype antibody that was custom made.

Mouse TC7, FOXO1 and ID3 were cloned into a mouse stem cell virus that also contained an mCherry marker gene. The viral packaging GP+E-86 cell line that produces the anti-HER2 CAR retrovirus was generated as previously described48. The anti-HER2 CAR construct was comprised of an extracellular scFv specific for human HER2, an extracellular CD8 hinge region, a CD28 transmembrane domain and an intracellular CD3ζ domain. GP+E-86 cell lines encoding both the anti-HER2 CAR and a transcriptional regulator were generated and the resulting anti-HER2 CAR packaging cells were sorted based on NGFR or mCherry expression by flow cytometry. Supernatants from these cells were used to transduce primary mouse T cells as previously described12 and following transduction, CAR T cells were maintained in supplemented RPMI medium with IL-7 (200 pg ml−1), IL-15 (10 ng ml−1) and β-mercaptoethanol (50 μM).

In C57BL/6 humans-HER2 mice, breast carcinoma cells were injected into the mammary fat pad 5 days before the treatment, or 5 days before the colon adenocarcinoma cells. The tumours were preconditioned with either 4 or 0.5 gallons of total body irradiation. The first dose of CAR T cells is given to the mice on two consecutive days followed by two more on the next two days. Tumour area was measured every 2–3 days following treatment. For IFNγ-blockade experiments, mice were dosed with 250 μg of anti-IFNγ or isotype control antibody 2A3 on days 0, 1 and 7 following CAR T cell treatment.

GraphPad Prism Analysis of Multi-dataset Tests: A Study of Student’s t-test and One-way ANOVA

Statistical analyses were performed using GraphPad Prism. Analyses performed include paired or unpaired Student’s t-test to compare two datasets, one-way ANOVA to analyse multiple datasets across a single timepoint and two-way ANOVA when analysing multiple sets of data across time.