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  • UC D br FC CD Biolegend A br


    FC: CD3 Biolegend 17A2
    Biolegend RM4-5
    eBioscience N418
    Biolegend HK1.4
    Biolegend UC3-10A6
    FC: IFN-g BD Bioscience XMG1.2
    FC: TNFa eBioscience MP6-XT22
    (Continued on next page)
    Biolegend IM7
    eBioscience LG.3A10
    FC: CD69 Biolegend
    FC: PD-1 Biolegend
    FC: RORgt
    FC: PLZF
    eBioscience Mags.21F7
    BD Bioscience B56
    Dr. Robert Tigelaar and Julia Lewis (Yale University, CT) 17D1
    IHC: Purified Rat Anti-Mouse Ly-6G
    BD Pharmingen 1A8
    IHC: Mouse mAb against human TCRd
    Santa Cruz H-41
    Bacterial and Virus Strains
    Adenovirus (Sftpc-Cre) Viral Vector Core, University of Iowa Ad5mSPC-Cre
    Chemicals, Peptides, and Recombinant Proteins
    Recombinant mouse IL-1b
    Recombinant mouse IL-23
    Recombinant Pimozide mouse IL-22
    Recombinant human amphiregulin
    Lipopolysaccharide from E. coli O111:B4 (LPS)
    Invivogen tlrl-eblps
    Peptidoglycan from S. aureus (PGN)
    Invivogen tlrl-pgns2
    Critical Commercial Assays
    Mouse IL-17A (homodimer) ELISA
    CellTiter-Glo Luminescent Cell Viability Assay
    Deposited Data
    16S sequencing
    Experimental Models: Organisms/Strains
    Jackson Laboratory 000664
    Jackson Laboratory 009088
    Jackson Laboratory, crossed to KP mice 026175
    Jackson Laboratory, crossed to KP mice 002014
    Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Tyler Jacks ([email protected]).
    Animals and In Vivo Procedures
    mice were used in all experimental groups in all mouse experiments. In most experiments, KP mice were infected with 2.5x108 pla-que-forming units (pfu) of Sftpc-Cre expressing adenovirus (Viral Vector Core, University of Iowa) or 30,000 transforming units of len-tiviral Pimozide co-expressing Cre and specific sgRNAs through intratracheal instillation between 8-12 weeks of age to initiate tumors, as previously described (DuPage et al., 2009). In the bacterial inoculation experiment, KP mice were infected with 2.0x108 pfu of Sftpc-Cre expressing adenovirus. Mice were randomized before treatments. For antibiotic treatment, mice were given a cocktail of ampicillin (1g/L, American Bioanalytical), neomycin trisulfate (1g/L, Sigma), metronidazole (1g/L, Sigma) and vancomycin (500 mg/L, Goldbio) (4Abx) or metronidazole alone in drinking water at indicated time points. For antibody-mediated depletion ex-periments, animals were treated every 2-3 days with i.p. injection of monoclonal antibodies (200 mg/mouse) directed against gd-TCR (UC7-13D5, BioXCell; or GL3, purified from the hybridoma generously provided by Dr. O’Brien, National Jewish Health, Denver) or IL-17A (17F3, BioXCell), and their isotype controls (BioXCell). For in vivo stimulation of gd T cells in the lung, recombinant mouse IL-1b (R&D Systems, 100 ng/mouse) plus IL-23 (R&D Systems, 100 ng/mouse), or LPS (Lipopolysaccharide from E. coli O111:B4, Invivogen, 2 mg/mouse) plus PGN (Peptidoglycan from S. aureus, Invivogen, 5 mg/mouse) were administered intratracheally to wild-type C57BL/6J mice. For bacterial inoculation experiment, 14 bacterial strains were isolated and cultured from late-stage lung tumors from SPF mice. They were mixed and intratracheally administered at a total dose of 107 CFU to a separate cohort of KP mice 3.5 weeks post tumor initiation. For bone marrow chimera experiments, KP mice on the CD45.1 background were irradiated with a Gammacell-40 Irradiator (5.5 Gy x 2, 3.5 hours apart), and then transplanted with bone marrow collected from wild-type or Myd88 knockout (Jackson Laboratory) donors on the CD45.2 background. Seven weeks post bone marrow reconstitution, mice were infected with 2.5x108 pfu of Sftpc-Cre expressing adenovirus for tumor initiation. All studies were performed under a Massa-chusetts Institute of Technology Committee on Animal Care-approved animal protocol, or an ICUC approved animal study protocol at NIAID, NIH (LSB-1E and LSB-4E). Mice were assessed for morbidity according to MIT Division of Comparative Medicine guidelines and were humanely euthanized when ill.