GoKawiilEthics and study approval
All experiments detailed in this manuscript were approved by the University of Pittsburgh Institutional Animal Care and Use Committee (20077737 and 23073380). The use of retroviral vectors was approved by the Institutional Biosafety Committee. Research involving human participants was approved by the institutional review board (21090049).
Animals
C57BL/6J inbred mice were purchased from Jackson Laboratories. Experimental mice were maintained under specific-pathogen-free conditions in accordance with the University of Pittsburgh Institutional Animal Care and Use Committee, certified by the Association for Assessment and Accreditation of Laboratory Animal Care. Procedures were performed under their guidelines. Male and female, 6- to 8-week-old mice were used in all the experiments. C57BL/6J mice were bred in-house, at a Charles Rivers facility (starting October 2020), or obtained from the Jackson Laboratory. NOD.Cg-Prkdc scid ll2rg tm1Wjl /SzJ/Arc (NSG), Rag2−/− and Ldlr−/− mice were purchased from the Jackson Laboratory.
Cell lines and cell culture
B16, B16-OVA, A549 and NALM6 cell lines were cultured in complete RPMI media (RPMI (Gibco, 11875-093), 10% fetal bovine serum (FBS), 2 mM l-glutamine, 100 U ml−1 penicillin–streptomycin, 1× non-essential amino acids, 1 mM sodium pyruvate, 5 mM HEPES, β-mercaptoethanol). Plat-E and 293 GP cell lines were cultured in complete Dulbecco’s modified Eagle medium (DMEM (Gibco, 11995-065), 10% FBS, 2 mM l-glutamine, 100 U ml−1 penicillin–streptomycin, 1× non-essential amino acids, 1 mM sodium pyruvate, 5 mM HEPES, β-mercaptoethanol). The NALM6 cell line was a gift from TCR2 therapeutics. Cell lines were free of mycoplasma contamination. Cell cultures were maintained at 37 °C with 5% CO 2 in a humidified incubator.
Chemical reagents
Rapamycin (mTORC1 inhibitor; Sigma, 553210) was used at 5 mg kg−1 dose in vivo wherever mentioned. Puromycin (Sigma, P8833) was used at 10 mg ml−1 concentration in vitro. OVA peptide (257–264) (SIINFEKL) (Genscript, RP10611) was used at 10 μg ml−1. Itraconazole was used at 2 μM concentration in vitro. For blocking the insulin receptor in vivo, S961 (1 mg kg−1; MedChemExpress, HY-P2093) was intraperitoneally (i.p.) injected during the fasted and fed periods.
Mouse T cell isolation and culture
Mice were fasted (by removal of food without interruption of water supply) or fed ad libitum. To isolate CD8+ T cells from mice, the spleen and three lymph nodes (axillary, brachial and inguinal) from both the right and left sides were harvested. The spleen was minced, treated with ACK lysis buffer (0.15 M NH 4 Cl + 1.0 mM KHCO 3 + 0.1 mM Na2-EDTA) for 2 min to lyse the erythrocytes and mixed with pooled and minced LN cells. Naive (CD62LhighCD44low) CD8+ T cells were then purified from total pooled lymphocytes according to the manufacturer’s instructions (Miltenyi Biotec, 130-096-543). For in vitro expansion, naive CD8+ T cells were stimulated with anti-CD3 and CD28 mAb-coated dynabeads (Thermo Fisher Scientific, Gibco, 11452D).
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