Mitomycin d

For measuring the frequency of antigen-specific CD4⁺ T cells after Mtb infection, single cell suspensions from lungs were prepared and collected in complete IMDM. Lung cells were stimulated for 20 h with mitomycin-D (Sigma-Aldrich)-inactivated spleen cells from uninfected mice that had been pulsed with the MHC class II peptide ESAT-6_1–20 (Research Center Borstel). Detection of antigen-specific IFN-γ-producing CD4⁺ T cells was conducted using ELISPOT assay kits as described by the manufacturer (BD Bioscience and R&D Systems, respectively). Spots were automatically enumerated using an ELISPOT reader (ELISPOT 04 XL; AID) and the frequency of cytokine-producing cells was determined.

The frequency of antigen-specific CD4⁺ T cells in infected lungs was determined in an ELISPOT assay after enrichment of CD4 T cells as described [35 (link)]. Briefly, antigen-specific production of IFNγ and IL-17A was quantified using an ELISPOT assay kit according to the manufacturer’s instructions (BD Bioscience and R&D Systems, Wiesbaden-Nordenstadt, Germany, respectively). Serially diluted CD4 T cells were added to mitomycin-D (Sigma-Aldrich, Hamburg, Germany)-inactivated spleen cells from uninfected wild type mice as antigen-presenting cells and incubated with Mtb ESAT6_1–20 (10 µg/mL; Research Center Borstel, Germany) and recombinant mouse IL-2 (10 U/mL; Peprotech, Hamburg, Germany). After 20 hrs at 37 °C, the cells were washed, incubated with secondary mAbs. After development, spots were counted in an ELISPOT reader (EliSpot 04 XL; AID, Straßberg, Germany) and the frequency of the ESAT6_1–20-specific CD4⁺ T cells was calculated.

“Hanging drop” spheroids were generated with hNF, hAF and hLSF according to a modified protocol published by Ruedel et al. [78 (link)]. Cells were trypsinized, adjusted to 50,000 cells/mL in DMEM, and mixed with 20% methocel (6 g methyl cellulose; Sigma-Aldrich, Munich, Germany, 250 mL DMEM). A total of 25 mL of the cell suspension were dropped onto the cover of a 9 cm petri dish (Greiner Bio One; Frickenhausen, Germany) filled with DPBS. The cover dish was inverted and incubated under a humidified atmosphere of 8% CO₂ at 37 °C for 72 h. “Hanging drop” spheroids were carefully inverted and treated with CAP for 2 min in the petri dish or remained untreated. Subsequently, spheroids were harvested and collected in a 15 mL Falcon™ tube (Fisher Scientific GmbH; Schwerte, Germany) filled with DMEM. Harvested spheroids were embedded into a collagen matrix (1 part × 10 minimum essential medium, 1 part 7.5% sodium bicarbonate solution; Sigma-Aldrich, Munich, Germany), and rat tail Coll I (BD Biosciences, Heidelberg, Germany) at a final concentration of 2.5 mg/mL and covered with 2 mL of DMEM and 1.6 mg/mL of mitomycin D (Sigma-Aldrich, Munich, Germany) per well. Migration of fibroblasts out of the spheroid was photographed, and the migrated area was determined immediately (0 h) as well as 24 and 48 h after the start of the assay.