Extended Data Fig. 2: Representative gating strategy to identify immune cell subsets in BAL samples.

a, We developed a gating strategy that enabled us to quantify immune cell populations including monocytes, alveolar macrophage subsets and T cell subsets. We defined alveolar macrophages by their expression of CD206, subdividing them into early monocyte-derived alveolar macrophages (CD206^lo) and more mature (CD206^hi) alveolar macrophages. T cells were identified as CD3-positive and further subdivided into CD4⁺ and CD8⁺ T cells. T_reg cells were identified as CD3⁺CD4⁺CD25⁺CD127⁻. Neutrophils were identified as CD15⁺ cells. Monocytes were identified as HLA-DR⁺CD4⁺CD206⁻ cells. Of note, only CD206^hi alveolar macrophages were flow cytometry-sorted for bulk RNA-seq analysis (Figs. 2, 3); hence, early MoAMs (MoAM1 and MoAM2 in our single-cell RNA-seq data (Fig. 4a–h)) were not captured in bulk RNA-seq. A representative sample from a patient without neutrophilia is shown. Solid red arrows indicate direct sequential gating, dashed blue arrows indicate Boolean ‘not’ gates. Numbers on plots indicate the percentage of the parent population. Axis labels indicate laser line (UV, 355 nm; V, 405 nm; B, 488 nm; YG, 552 nm; and R, 640 nm), bandpass filter, fluorochrome and antigen/dye. b, Representative sample from a patient with neutrophilia illustrates loss of CD206^hi alveolar macrophages and influx of monocyte-derived CD206^lo alveolar macrophages. c, d, Contour plot and histogram overlays illustrating forward (FSC) and side scatter (SSC) properties of the CD3⁺ T cells (CD3), CD15⁺ neutrophils (CD15), monocytes, CD206^lo alveolar macrophages (CD206^lo) and CD206^hi alveolar macrophages (CD206^hi) in the representative sample from a patient without neutrophilia (c) and with neutrophilia (d). Note that neutrophils have higher side scatter than monocytes. e, Representative contour plots illustrating a sample with two distinct populations of CD206^hi alveolar macrophages. Single-cell RNA-seq analysis (Fig. 4a–h) suggests that CD206^hi alveolar macrophages (double-headed arrow) are bona fide tissue-resident alveolar macrophages.