Mice
Gsk3αfl/fl, Gsk3βfl/fl, Ctnnb1fl/fl (Jax stock 022775), Gzmb Cre (Jax stock 003734), P14 (Jax stock 037394), CD8KO (Jax stock 002665), B6 CD45.1+ (Jax stock 002014), Rosa26LSL − Cas9 (Jax stock 028551) mice were previously reported [36]. Unless otherwise noted, Gsk3αfl/flGsk3βfl/flGzmb Cre mice were analyzed at 8 to 12 weeks of age, while other mice were used as donors or recipients at 8 to 12 weeks of age. All mice were bred and housed under specific pathogen-free conditions. All animal experiments were approved by the Animal Care and Use Committee of Xiamen University.
Adoptive transfer and LCMV infection
For the adoptive transfer experiments, 1×105 naïve P14 cells were purified by fluorescence-activated cell sorting and adoptively transferred into host mice 1 day before LCMV-Arm infection. For acute viral infection, mice were injected intravenously with 2×105 plaque-forming units LCMV-Arm.
Cell culture and cellular assays
CD8+ T cells were cultured in RPMI medium supplemented with 10% FBS (EXCELL Biotech), 25 mM HEPES (Sigma), 100 units/mL penicillin-G, 100 µg/mL streptomycin (Gibco), 50 µM β-ME. For adoptive cell transfer, total splenocytes isolated from WT and DKO P14 cells were activated with gp33 peptides (2ng/mL) in the presence of IL-2 (20ng/mL). Five days later, total CD8+ T cells (1×106 cells per genotype) were inoculated intravenously into recipient mice carrying subcutaneous B16-gp33 tumors (1×106 cells injected 5 days before T cell transfer). For in vitro activation, CD8+ T lymphocytes were purified from spleen and peripheral lymph nodes by negative selection. Naive CD8+ T cells were activated with plate-bound anti-CD3 (2 µg/mL) and anti-CD28 (2 µg/mL).
Tumor model and treatment
B16-F10 melanoma, MC38 and LLC-1 cells were cultured in complete DMEM medium (Gibco) supplemented with 10% FBS containing 100 units/mL penicillin-G, 100 µg/mL streptomycin (Gibco) and Non-Essential Amino Acids (Gibco). Tumor cells (1×106) were injected subcutaneously into the flanks of mice. TILs were isolated by dissociating tumor tissue in the presence of 1 mg/mL collagenase D for 1 hr before centrifugation on a discontinuous Percoll gradient (Cytiva). Isolated cells were then used in various assays of T cell function. For the experimental metastasis assay, 1×106 B16-gp33 cells in a volume of 700 µL PBS were injected intravenously into the tail vein. After 18 days, the mice were killed via cervical dislocation, and their lungs were removed and rinsed in phosphate-buffered saline. The number of B16-gp33 colonies on all five lobes of the lung were counted macroscopically. All mice used are C57BL/6 background, both male and female, 8–12 weeks of age, 25 (± 5) g. Checkpoint blockade therapy consisting on a combination of 200 mg of 200 mg of anti-PD-1 (RMP1-14, BioXCell) antibodies per dose or isotype control antibodies was administered on day 6, 9 and 12 after tumor inoculation.
Antibodies and reagents
The following anti-mouse fluorochrome-conjugated antibodies were used for flow cytometric analysis: anti-TCR Vα2 (B20.1), anti-CD4 (GK1.5), anti-CD8a (53 − 6.7), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-CD45.1 (A20), anti-CD45.2 (104), anti-CD62L (MEL-14), anti-CD69 (H1-2F3), anti-CD71 (R17217), anti-CD127 (A7R34), anti-KLRG1 (2F1), TIM3 (RMT3-23), LAG3 (eBioC9B7W(C9B7W)), anti-IFNγ (XMG1.2), anti-TNFα (MP6-XT22), anti-granzyme B (NGZB), anti-Perforin (eBioOMAK-D), anti-TOX (TXRX10) and anti-Eomes (Dan11mag) were from eBioscience. Anti-PD-1 (29F.1A12), anti-TIGIT (1G9), anti-T-bet (4B10), anti-Bcl2 (3F11) and anti-Ki67 (16A8) were from BioLegend. Anti-Tox/Tox2 (E6G5O) was from Cell Signaling. The following anti-mouse biotinylated antibodies were used for CD8+ T cells purified: anti-B220 (RA3-6b2), anti-CD4 (GK1.5), CD11b (M1-70), CD11c (N418), anti-CD25 (PC61.5), anti-CD44 (IM7), anti-NK1.1 (PK136), anti-Ly-6G/Ly-6C (RB6-8C5), anti-F4/80 (BM8) and anti-TER119 (Ter119) were all from BioLegend. The following antibodies were used for Western blot: anti-GSK3α/β (D75D3), anti-β-actin (13E5), anti-Tox/Tox2 (E6G5O) and anti-NFAT1 (D43B1) were from Cell Signaling Technology; Anti-GAPDH (1E6D9), anti-LaminB1 (3C10G12) and anti-β-Tubulin (1D4A4) were from Proteintech; Anti-Bach2 (7A4) was from Abcam. PMA (phorbol 12-myristate 13-acetate) was from Sigma. Ionomycin was from Yeasen. Recombinant murine IL-2 and IL-7 were from Novoprotein. Fixation/Permeabilization Solution Kit and Annexin V/7-AAD Kit were from BD bioscience. Foxp3 Transcription Factor Buffer Set was from eBioscience. EdU Flow Kit was from Beyotime.
Flow cytometry
Single-cell suspensions were prepared from mouse spleen, lymph nodes (LNs) and tumors, cells were passed through a 75 µm strainer, followed by lysis of erythrocytes with ACK (ammonium chloride-potassium). Tumors were excised, weighed, minced, and collagenase digested for 1 hr. Tumor-infiltrating leukocytes were isolated from tumors with Percoll (GE Healthcare). Fc block was performed by staining with anti-CD16/32 to avoid nonspecific binding. For cell surface marker staining, antibodies were combined in 30 µL FACS (fluorescence-activated cell sorting) buffer (PBS with 0.5% BSA and 0.05% NaN3), added to cells, and incubated at 4℃ for 30 min. For intracellular staining of transcription factor, after cell surface marker staining, cells were fixed and permeabilized for 30 min in the Fix/Perm buffer (eBioscience). After washing with the perm buffer, cells were stained with antibodies for 1 hr at room temperature. For intracellular staining of cytokines, cells were stimulated with PMA (phorbol 12-myristate 13-acetate; 50 ng/mL) and ionomycin (1 µg/mL) for 5 hr before cytokine staining with the BD Fixation/Permeabilization Solution Kit. All flow cytometry data was acquired on Fortessa or LSRFortessa X-20 (BD Biosciences), or a NovoCyte flow cytometer (ACEA Biosciences, Agilent) and analyzed with FlowJo software 10 (Treestar).
RNA sequencing and data analysis
Gsk3α fl/fl Gsk3β fl/fl P14 (WT CD45.2) and Gsk3αfl/flGsk3βfl/fl P14 Gzmb Cre (DKO, CD45.2) cells were injected intravenously (1×105 of each cell type) into B6 (CD45.1) hosts, followed by infection of the hosts 24 h later with LCMV-Arm. CD8+ T cells were sorted from hosts mice at day 5.5, total RNA was isolated by Qiagen RNesy Micro or Mini Kits following the manufacturer’s instructions. RNA was quantified with Nano Drops Nucleic Acid Analyzer. One microgram of RNA was used for RNA sequencing with a BGISEQ-500 instrument (BGI, Wuhan). Reads were aligned to the mm10 build of the Mus musculus genome with the Hisat2. The alignments were passed to StringTie, which assembled and quantified the transcripts in each sample and generated gene count matrices. Differentially expressed genes were analyzed using DESeq2 package. Genes were considered differentially expressed if they had an adjusted P value of 0.01 or less. Differentially expressed genes were analyzed with gene set enrichment analysis (GSEA) and Mouse Genome Database (MGD) (www.informatics.jax.org) to find enriched biological processes and signaling pathways.
Retroviral transduction
Plat-E packaging cells were transfected with 2 µg of retroviral vector along with PEI. At 48 and 72 hours after transfection, retrovirus-containing supernatants were collected and stored at -80℃. For transduction of retrovirus, naïve WT and DKO P14 cells were isolated from spleen cell samples, followed by enrichment by CD8 negative selection. After 24hr of activation with αCD3 (2 µg/mL) and αCD28 (2 µg/mL) in the presence of recombinant murine IL-2 (20 ng/mL), P14 cells were transduced with retroviral supernatants containing polybrene (1 µg/mL) by spin infection (1000g for 1h at 37℃).
Immunoblot analysis
For immunoblot, whole cell extracts were lysed in lysis buffer [20 mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1 mM EGTA] supplemented with Halt Protease and Phosphatase Inhibitor Cocktail. Lysates from equal numbers of cells were separated by 8% SDS PAGE and transferred to PVDF membranes (Merck Millipore), which were incubated with primary antibodies, followed by overnight incubation at 4℃. After washing three times in TBST buffer, HRP-conjugated goat anti-rabbit or goat anti-mouse antibody was incubated with the membrane. After washing three times in TBST buffer, protein bands were visualized with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore). Images were acquired with Amersham Imager 600 (GE Healthcare).
For the preparation of nuclear extracts, CD8+ T cells were washed twice in PBS, 1×107 cells were lysed in 0.15% NP-40 lysis buffer (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 0.15% NP-40) for 15 min on ice. The homogenates were centrifuged at a full speed (12,000g) for 3 min, and the supernatant was collected as cytoplasmic extracts. The nuclear pellet was washed three times with 0.15% NP-40 lysis buffer and resuspended in whole cell lysis buffer, followed by incubation on ice for 30 min.
Statistical analysis
All statistical analysis was performed with GraphPad Prism 9.0. Unpaired or paired Student tests and one-way ANOVA analysis were used in data analysis. Statistical significance is displayed as NS, not significant, *, P < 0.05; ** P < 0.01; ***, P < 0.001 and ****, P < 0.0001.