Mice
Male and female mice were used for the study. CD45.1+ OT-I or P14 TCR-transgenic mice were housed together. CD45.2+ male and female C57BL/6N or C57BL/6JNifdc mice aged 6–8 weeks were purchased from Vital River as recipients. Female NCG (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) mice aged 6–8 weeks were purchased from GemPharmatech. Rosa26-Cas9 mice were provided as a gift from the W. Sheng laboratory at the University of Zhejiang. We crossed Rosa26-Cas9 mice with OT-I transgenic mice to generate Cas9+ OT-I mice for CRISPR–Cas9 screening in tumour antigen-specific CD8+ T cells. Four-week-old Klhl6+/− mice were purchased from Cyagen. The Klhl6+/− mice were crossed with OT-I, P14 or C57BL/6N mice to generate Klhl6−/− OT-I/P14 mice or Klhl6−/− mice for subsequent experiments. All mice were kept in a specific-pathogen-free facility, and all animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of Suzhou Institute of Systems Medicine (ISM-IACUC-0151-R and ISM-IACUC-20240098). Mice were housed in standard conditions, with 12 h/12 h light/dark cycles, a controlled temperature of 22–24 °C and humidity of 60%, with unrestricted food and water availability, and were examined daily. All mice were used at 6–16 weeks old. All tumour burdens did not exceed the permission of the Institutional Animal Care and Use Committee of Suzhou Institute of Systems Medicine. Age-matched and sex-matched mice were assigned randomly to experimental and control groups.
Cell lines
Human embryonic kidney 293T (HEK293T) cells were purchased from the American Type Culture Collection (ATCC, CRL-3216) and maintained in DMEM (Gibco, C11995500BT) supplemented with 10% fetal bovine serum (FBS) (Gibco, 16000044) and 1% penicillin–streptomycin (P/S) (Gibco, 15140122). The mouse melanoma cell line B16 was transduced to express OVA 257-264 antigen (a gift from Bo Huang laboratory) and maintained in DMEM with 10% FBS and 1% P/S. HepG2 cells (ATCC, HB-8065) were transduced to express human NY-ESO antigen (HepG2-ESO) and cultured in DMEM with 10% FBS and 1% P/S. Jurkat (ATCC, TIB-152) and EL4 (ATCC, TIB-39) cell lines were cultured within the complete Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% FBS, 1% P/S, 1% GlutaMAX (Gibco, 35050061), 10 mM HEPES (Gibco, 15630130), 1% non-essential amino acids (Gibco, 11140076), 1 mM sodium pyruvate (Gibco, 11360070) and 50 μM β-mercaptoethanol (Sigma, M6250). HEK293T, Jurkat, HepG2 and EL4 cells were pre-authenticated by ATCC by short tandem repeat (STR) sequencing. B16-OVA cells were frequently monitored based on their morphological features but have not been authenticated by STR. All cell lines were routinely tested for mycoplasma contamination.
Plasmids
Mouse Klhl6, Tox and Ppargc1a genes were amplified from the complementary DNA (cDNA) library of mice OT-I T cells, and human KLHL6 and TOX genes were amplified from human peripheral blood mononuclear cells (PBMCs). Retroviral plasmid (MSGV-Thy1.1-Klhl6, MSGV-Thy1.1-Ppargc1a and MSGV-Thy1.1-Vector) and packaging vector (pCL-Eco) plasmid were used to produce retroviruses in HEK293T cells using 293 Transfection Reagent (Mirus, MIR 2700), which were then transduced into OT-I CD8+ T cells. The MESV-shCtrl-GFP (Addgene, 85587) was used for Tox or Pgam5 KD. Primer sequences used for Tox and Pgam5 KD can be found in Supplementary Table 6. The retroviral plasmids (MSGV-NGFR-KLHL6, MSGV-Thy1.1-1G4 TCR and MSGV-NGFR-Vector) and packaging vectors (pHIT60 and RD114) were used to produce retroviruses, which were used to transduce PBMCs and Jurkat cells. Lentivirus vectors (pCCLc-MND-Thy1.1-Klhl6 and pCCLc-MND-Thy1.1-Vector) and packaging vector (PA2X and VSV-G) plasmids were used for lentivirus production in HEK293T cells using Liposomal Transfection Reagent for transduction into EL4 cell line. For transient expression experiments in HEK293T cells, the vector plasmid pcDNA4/TO or pFLAG-CMV-4 was used according to the experimental need.
Primary mouse T cell isolation, viral transduction and culture
Naive OT-I T lymphocytes were isolated from the spleens and peripheral lymph nodes of male and female OT-I mice (6–8 weeks). Spleens and peripheral lymph nodes were collected, and mashed through a 70-μm filter, and red blood cells were lysed using red blood cell lysis buffer (BioLegend, 420301) followed by washing with 1× phosphate-buffered saline (PBS). CD8+ OT-I T cells were purified using a CD8+ Naive T cell isolation kit (BioLegend, 480043) according to the manufacturer’s instructions. Primary mouse T cells were counted and then resuspended in RPMI-1640 supplemented with 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids, 10 mM HEPES, 1% GlutaMAX, 1% P/S, 50 μM β-mercaptoethanol and mouse IL-2 (20 U ml−1, Peprotech, 212-12). Then, the resuspended CD8+ OT-I T cells were seeded at a concentration of 1 million cells per ml on 24-well plates with overnight-bound anti-mouse CD3 (2 μg ml−1, BioLegend, 100359) and anti-mouse CD28 (1 μg ml−1, BioLegend, 102121) antibodies. Cells were activated in 24-well plates for 48 h and then transferred out of the activation plates and passaged to new plates every 2 days with a concentration of 1 million cells per ml. For drug treatment experiments, DMSO (Sigma, D2650), 2 μM LFHP-1c (MCE, HY-139598) or 10 μM Mdivi-1 (Selleck, S7162) and 20 μM M1 (Selleck, S3375) were added to cultures daily starting on day 3 after T cell activation. In viral transduction, 7.5 × 105 OT-I cells were transduced with unconcentrated retroviral supernatant after 24 h of activation in 24-well plates coated with RetroNectin reagent (15 μg ml−1, Takara, T100B). Following centrifugation at 2,500 rpm for 90 min at 30 °C, T cells were cultured in the incubator for 24 h. The transduction was repeated 24 h later and then returned to fresh medium for culture. Drug-treated or retrovirus-transduced OT-I cells were sorted by flow cytometry and then adoptively transferred into recipient mice that were inoculated with B16-OVA tumour cells before transfer.
Human T cell isolation, viral transduction and culture
Human PBMCs from healthy donors were purchased from Sailybio and isolated using Lymphoprep (Cytiva, 17144003) according to the manufacturer’s protocol. Isolated PBMCs were cultured in RPMI-1640 medium supplemented with 5% Human Serum AB (Gemini, 100-512), 1% GlutaMAX, 1% non-essential amino acids, 1% P/S, 1 mM sodium pyruvate, 10 mM HEPES and 50 μM β-mercaptoethanol in the presence of human IL-2 (100 U ml−1, Peprotech, 200-02). PBMCs were activated by anti-human CD3 (1 μg ml−1, BioLegend, 317347) and anti-human CD28 (1 μg ml−1, BioLegend, 302943) monoclonal antibodies for 2 days and then underwent viral transduction. In brief, 1 × 106 PBMCs were transferred to a new 24-well plate and dually transduced by 1G4 TCR-specific and KLHL6-specific retroviral supernatant in the presence of 10 μg ml−1 polybrene (Sigma, TR-1003-G). Following centrifugation at 2,500 rpm for 90 min at 30 °C, PBMCs were cultured in the incubator for 24 h with fresh medium and then underwent repeated transduction. The transduced PBMCs were adoptively transferred into female NCG mice that were inoculated with HepG2-ESO tumour cells before transfer.
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