Mifn γ

APCs were prepared from TLR2^−/− disrupted spleens that were T lymphocyte depleted with anti-CD 90.2 beads and LS Magnetic Bead Columns (Miltenyi Biotec) and then gamma irradiated with 2000 rads. For polyclonal activation 0.3 μg/ml CD3ε Abs (clone 2C11, Biolegend) was used or 0.3 μM of OVA peptide (ISQAVHAAHAEINEAGR, Sigma) for OT2 cells. Plate bound stimulation was conducted in 96 well flat bottom plates (Corning) with 0.3 μg/ml CD3ε Abs and soluble 1 μg/ml CD28 Abs (clone CD28.2, Biolegend). T_h1 polarization for mouse CD4+ T cells was conducted with 10 μg/ml mIL4 Abs (clone 11B11, Biolegend), 10 ng/ml mIFN-γ (Peprotech) and 10 ng/ml mIL-12 (Peprotech). T_h1 polarization for human CD4⁺ T cells was accomplished with CellXVivo Human Th1 Cell Differentiation Kit (R&D Systems) in accordance with manufacturers recommendations. For iT_reg development 0.5 ng/ml of mTGF-β1 (R&D) was used alone or in combination with T_h1 polarizing medium.

Target B16F10, TC-1, or B16-Trp2-KO cells were treated with 500 U/mL mIFN-γ (Peprotech) for 12 hr, then irradiated (120 Gy). Effector cells were splenocytes isolated from AIPV-treated mice that had rejected tumors 6 days after rechallenge with 10⁶ B16F10 cells. A Mouse IFN-γ ELISPOT Kit (BD) was used. Targets cells were seeded at 25,000 cells per well. Effector cells were seeded at 10⁶ cells per well. Plates were wrapped in foil and cultured for 24 hours then developed according to manufacturer’s protocol. Plates were scanned using a CTL-ImmunoSpot Plate Reader and data was analyzed using CTL ImmunoSpot Software.

BMDMs were differentiated in DMEM (Invitrogen) with 10% v/v FCS (Thermo Fisher Scientific), 10% MCSF (L929 cell supernatant), 10 mM HEPES (Invitrogen), and nonessential amino acids (Invitrogen). 1 day before infection, macrophages were seeded into 6-, 24-, or 96-well plates at a density of 1.25×10⁶, 2.5×10⁵, or 5×10⁴ per well. If required macrophages were pre-stimulated with PAM3CSK4, LPS O111:B4 (InvivoGen), mIFN-β or mIFN-γ (eBioscience). For infections with F. novicida, bacteria were grown overnight in BHI or TSB at 37 °C with aeration. The bacteria were added to the macrophages at multiplicity of infection (MOI) of 100, or as otherwise indicated. The plates were centrifuged for 15 min at 500 g to ensure comparable adhesion of the bacteria to the cells and placed at 37 °C for 120 min. Next, cells were washed and fresh medium with 10 μg/ml gentamycin (Invitrogen) was added to kill extracellular bacteria and plates were incubated for the desired length of time. Transfection with poly(dA:dT) or poly(dG:dC) was done as described previously^{29 (link)} or as indicated.