Neutrophils from the second wave reach the lung at the same time as effector T cells with which they establish intimate contacts.
(a) Lung tissue cells from wild-type C57BL/6 mice, infected with 5x106 CFU of BCG or 103 CFU of Mtb, were labeled on day 1, 3, 7 and 23 and analyzed by flow cytometry. The numbers of CD3+ CD4+ conventional T cells or CD3+ γδ + T cells per mouse were determined (n = 4–5), *P<0.05. (b) On day 1 and 23 after infection with BCG or Mtb, 105 total lung cells were cultured for three days in the presence of HK-BCG (equivalent to 106 CFU). IL-17A and IFN-γ concentrations present in the supernatant were determined by ELISA (n = 8–10). Mann Whitney *P<0.05, ***P<0.001. (c-f) Representative confocal images of day 1 (c, d) or 23 (e, f) lung 8 μm cryosections from mice infected with green fluorescent BCG Myc 409 (green) and labeled with anti-Ly-6G for neutrophils (red), and anti-CD3 for T cells (cyan) and counterstained with DAPI for nuclei (blue). Merge triple or quadruple fluorescence of lung sections observed under low (c, e) or high magnification (d, f) showed neutrophil and BCG co localisation and absence of T cells on day 1 whereas neutrophils and T cell clusters were observed on day 23 (e). Higher-magnification (f) showed that on day 23, neutrophils barely co localized with mycobacteria and established close interactions with T cells (white arrowheads). Images were acquired with a confocal Leica TCS SP8 microscope equipped with x63 objective and were analyzed with the Leica LAS AF software.