Figure 2. IFN-γ boosts activation marker expression on MDM to a greater extent than AM.

Human AM (A, C, E) isolated from bronchoalveolar lavage fluid. PBMC were isolated from buffy coats and MDM (B, D, F) were differentiated and adherence purified for 7 days in 10% human serum. Cells were left unprimed (black) or primed with IFN-γ (red) or IL-4 (blue) (both 10 ng/ml) for 24 hr. AM or MDM were left unstimulated (circle) or stimulated with iH37Rv (MOI 1–10; square) or LPS (100 ng/ml; triangle). After 24 hr, cells were detached from the plates by cooling and gentle scraping and stained for HLAR-DR (A, B), CD40 (C, D), CD86 (E, F) and analysed by flow cytometry. Fold change of HLA-DR (G), CD40 (H) and CD86 (I) was calculated for AM (white bar) and MDM (black bar) based on the average of their respective no cytokine controls. Each linked data point represents the average of technical duplicates for one individual biological donor (n=8–9). Statistically significant differences were determined using two-way ANOVA with a Tukey (A–F) or Bonferroni post-test (G–I); *p≤0.05, **p≤0.01, p***≤0.001, ****p≤0.001.

Figure 2—figure supplement 1. Supplementary flow cytometry gating strategy.

PBMC were isolated from buffy coats and MDM were differentiated and adherence purified for 7 days in 10% human serum. Cells were placed in ice cold PBS and detached with gentle scraping. Cells were Fc blocked, treated with viability dye and stained with fluorochrome-conjugated antibodies specific for CD14, CD68, HLA-DR, CD40, and CD86. Cells were fixed with 2% PFA and analysed by flow cytometry. MDM were gated on the basis of forward (FSc) and side scatter (SSc), doublets were excluded using forwards scatter area versus width, live cells were gated on the basis of ZombieNearIR exclusion. MDM were CD14 and CD68 double positive. The expression of HLA-DR or CD40 were quantified by median fluorescence intensity (MFI). Representative dot plots show staining of unstimulated MDM and histograms show overlaid fluorescence of unstimulated cells compared with Mtb (iH37Rv) stimulated cells in the presence or absence of IFN-γ.