Mice
CD45.2 (C57BL/6J) and B6.GFP (C57BL/6-Tg(UBC-GFP)30Scha/J) mice were purchased from the Jackson Laboratory (strains 000664 and 004354, respectively); CD45.1 (B6.SJL Ptprca), OT-I.CD45.1 (B6(Ly5.1)-[Tg]TCR OT-I-[KO]RAG1) and OT-I.CD45.2 (C57BL/6NAi-[Tg]TCR OT-I-[KO]RAG1) were obtained from the NIAID-Taconic exchange programme (strains 8478, 300 and 175, respectively). XCR1-DTR (B6.Cg-Xcr1tm2(HBEGF/Venus)Ksho) and XCR1-venus (B6.Cg-Xcr1tm1Ksho) transgenic mice15 were gifts from T. Kaisho. OT-I.GFP mice were cross-bred from OT-I.CD45.2 and B6.GFP mice and maintained as a homozygous strain in our laboratory. The majority of mice used were female and aged 6–16 weeks at the beginning of experiments, with a small number of experiments performed in male mice. No significant difference was observed between sex. Age-matched littermate mice were used to control for litter, cage and age effects. For imaging experiments, two to three mice were used per group in each experiment, and for flow cytometry experiments, three to six mice were used per group. Mice were randomly assigned for experimental groups, and animal experiments were not blinded as the procedures were performed by the same investigator. All mice were bred and maintained under specific-pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care (AAALAC)-accredited animal facility within NIAID and were used (including compliance with tumour size limit) under study protocol LISB-4E approved by NIAID Animal Care and Use Committee (NIH).
Tumour cell line generation
The KP-OVA tumour line was generated by lentiviral transduction of KP6-1B11 cells (a single colony subcloned from the KP1233 cell line derived from a KrasG12D/+Trp53−/− mouse14) with a lentivirus expressing full-length OVA (LRG-EFS-ZsGreen-P2A-OVA). pcDNA3-OVA (Addgene, 64599) was cloned into the lentiviral vector LRG-EFS-ZsGreen-P2A, a plasmid modified from LRG vector61, a gift from J. Shi, and transfected into HEK293T cells, a gift from T. Jacks, for lentiviral production. ZsGreen+ KP6-1B11 cells were sorted into 96-well plates containing single cells per well. Single-cell-derived clones with homogenous morphology and ZsGreen expression were selected and expanded for western blot and flow cytometry validation using anti-OVA (Invitrogen, PA1-196) and anti-mouse SIINFEKL–H-2Kb (25-D1.16) antibodies. The KP-OVA line was cultured and passaged in RPMI medium containing 10% FCS, l-glutamine (2 mM), penicillin (100 U ml−1), streptomycin (0.1 mg ml−1), sodium pyruvate (1 mM), HEPES (10 mM) and 2-mercaptoethanol (1 mM) at 37 °C under 6.5% CO 2 .
The MC38-OVA tumour line was generated by lentiviral transfection of MC38 cells with lentivirus expressing full-length OVA (pLV-EF1a-OVA-puro). The MC38 cell line was generously provided by M. Meier-Schellersheim (NIAID, NIH). pcDNA3-OVA (Addgene, 64599) was cloned into the pLV-EF1a-puro lentiviral vector. Transfection was performed on HEK293T cells (Takara, 632180) for lentivirus production. Infected cells were selected with puromycin (8 µg ml−1) and OVA expression was assessed by flow cytometry using anti-mouse SIINFEKL–H-2Kb (BioLegend, 25-D1.16). The MC38-OVA line was cultured and passaged in DMEM medium containing 10% FCS, l-glutamine (2 mM), penicillin (100 U ml−1), streptomycin (0.1 mg ml−1), sodium pyruvate (1 mM) and HEPES (10 mM) at 37 °C under 6.5% CO 2 . All HEK293T lines were tested negative for mycoplasma, but the tumour cell lines were not tested for mycoplasma.
Tumour induction and protein immunization
For tumour induction, mice were anaesthetized with isoflurane inhalation (2% induction, 1–1.5% maintenance). Mice were shaved on the left flank with a Wahl clipper (Kent Scientific), depilated using Nair hair removal cream and the shaved flank was washed thoroughly with water-soaked gauze. Tumour cells (KP-OVA and MC38-OVA) were collected by washing with PBS, incubated with 0.25% trypsin/EDTA (Thermo Fisher Scientific) at 37 °C for 5 min, then washed with prewarmed RPMI supplemented with 10% FCS. Then, 4 × 105 KP-OVA or 5 × 106 MC38-OVA cells were suspended in 20 µl Hanks’ balanced saline solution (HBSS) and intradermally injected into the left hind flank skin using a 30 G needle. Tumour volumes were measured using digital callipers and the volume was estimated using the formula: volume = ((width2 × length)/2). For OVA protein immunization, 25 µg ovalbumin (OVA EndoFit, Invivogen) and 12.5 µg poly(I:C) HMW (Invivogen) were reconstituted in 20 µl PBS and were injected intradermally as above, or subcutaneously in the left footpad.
Isolation of CD8+ T cells and adoptive cell transfer
Naive OT-I or wild-type T cells (B6.GFP, CD45.1 or CD45.2) were isolated from spleens and lymph nodes of donor mice with magnetic cell separation (MACS) using mouse CD8a+ T cell isolation kits (Miltenyi Biotec), according to the manufacturer’s instructions. Between 500 and 2 × 103 cells were injected intravenously in 200 µl HBSS into recipient mice at least 1 day before tumour induction or immunization. For CellTrace Violet labelling, CellTrace Violet dye (Thermo Fisher Scientific) was added at a final concentration of 5 µM to 1 × 107 cells per ml suspended in 0.1% BSA-containing PBS and incubated at 37 °C for 10 min, before addition of RPMI with 10% FCS to 10× staining volume and incubated further at 37 °C for 5 min. Cells were washed in RPMI before resuspension in HBSS for adoptive transfer.
In vivo antibody treatment
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