Fig 2: Evaluation of the tissue microenvironment in a mouse model of Mycobacterium avium infection.A Schematic diagram of the NTM-infected mouse model. Six-week-old male BALB/c mice were intratracheally infected with M. avium and euthanized 6 weeks later. B Histological image of lung tissue stained with hematoxylin and eosin (H&E) and Ziehl–Neelsen (Z–N). Scale bars: 250 µm and 50 µm. C Double staining image of PD-L1 immunostaining with Z–N. Scale bar: 50 µm. D Double staining image of PD-1 immunostaining with Z–N. Scale bar: 50 µm. E Representative images obtained by multiplexed immunohistochemical consecutive staining on single slide (MICSSS) with pseudo-fluorescence transformation, showing CD3, CD8, PD-1, Iba1, and PD-L1, along with merged images, in lung tissue from NTM-infected mice. Scale bar: 50 µm. F Positional plot of the neighborhoods color-coded by the region type. Scale bar: 2 mm. G Heatmap of the number of positive cells in each region normalized by the analyzed area. These results are representative of two independent experiments. H MICSSS pseudo-fluorescence image for Iba1 and PD-L1 (red: PD-L1; green: Iba1). Scale bar: 50 µm. I Comparison of the number of Iba1+ cells and PD-L1 positivity rate in macrophages between non-infected and infected areas using the HALO image analysis software program (n = 8 per group). J MICSSS pseudo-fluorescence image for CD3 and PD-1 (orange: CD3; yellow: PD-1). Scale bar: 50 µm. K Comparison of the number of CD3+ cells and the PD-1 positivity rate in lymphocytes between non-infected and infected areas using the HALO image analysis software program (n = 8 per group). L MICSSS pseudo-fluorescence images of CD3, PD-1, CD25, and Ki-67, along with merged images, in lung tissue from NTM-infected mice. Scale bar: 50 µm. M Comparison of the number of PD-1+, CD25–, Ki-67– cells between non-infected and infected areas using the HALO image analysis software program (n = 8 per group). Data are expressed as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001. Differences between groups were examined for statistical significance using the Mann-Whitney U test.