Figure 3. Antigen-capturing CX3CR1-GFP^bright cells are Bronchus-Associated Macrophages (BAMs).

(A) Representative flow cytometric analysis of CX3CR1-GFP^bright OVA^bright cells (top) versus CD11c-YFP^bright cells (bottom). Cells were pre-gated to exclude B cells, neutrophils, eosinophils, and AMs (CD19^– Ly6G^– Siglec-F^–). (B) Quantification of the frequency of CD24⁺ CD64^– (“CD24⁺”) or CD64⁺ CD24^–/lo (“CD64⁺”) cells among CX3CR1-GFP^bright OVA^bright cells (top) or CD11c-YFP^bright cells (bottom) that were gated as in (A). (C–D) Representative two-photon microscopy of MHC-II (blue) and CD64 (red) antibody staining in precision-cut vibratome lung sections from CX3CR1-GFP (green) mice, showing a region containing an airway as a 3D fluorescence opacity rendering of a 54 μm z-stack (C) as well as single z-plane images of cells to confirm colocalization of molecules (D); note C and D are from different imaging fields. In (C and D), single color and merged images are shown. In (C), the bronchial airway was visualized by transmitted illumination and the approximate boundaries are denoted by dashed lines, with the airway lumen denoted by * in the merged image. In (C), most CX3CR1-GFP^bright CD64⁺ cells are MHC-II^hi (solid arrowheads), yet some cells are MHC-II^lo (open arrowheads). (E) Representative two-photon microscopy of CD64 antibody staining (red) in vibratome lung sections from CD11c-YFP (green) mice, showing a MIP of a 15 μm z-stack. Mice in all panels were unimmunized. Each data point represents one mouse (B). Data in all panels are representative of ≥2 experiments. Scale bars, 20 μm.

Figure 3—figure supplement 1. CD11c antibody labeling, CD11c-YFP and CX3CR1-GFP expression on CD64⁺ IMs.

(A) Representative flow cytometric analysis of CD11c-YFP reporter expression (upper histogram) versus CD11c antibody surface staining (bottom histogram) in the same sample, comparing OVA^bright (OVA⁺⁺) BAMs (blue) to DCs (red) gated as shown (left). (B) Representative flow cytometric analysis comparing the fluorescence intensity of the CX3CR1-GFP reporter in cDC2s (red), MHC-II⁺ CD64⁺ MerTK⁺ cells (blue), and monocytes (green). Data are representative of two experiments (A) and four experiments (B).

Figure 3—figure supplement 2. Phenotypic comparison of cDCs, BAMs and AMs.

Representative flow cytometric analysis of marker expression on all CD24⁺ DCs (red), CD64⁺ MHC-II⁺ BAMs (blue) and AMs (green). cDCs and BAMs were gated as CD19^– Ly6G^– Siglec-F^– CD11c⁺ MHC-II⁺ then CD24⁺ (cDCs) versus CD64⁺ (BAMs) as in (Figure 3A). AMs were gated as CD19^– CD11c⁺ Siglec-F⁺. (A) GFP expression on cells from the lung of a Zbtb46-GFP mouse. Background signal in a GFP-negative control mouse is shown in gray. (B–E) Antibody surface staining for CD11b (B), MerTK (C), CD169 (D), and CD206 (E) on each cell population in a wild-type mouse. Background signals in Fluorescence Minus One (FMO) controls are shown in gray. (F) Antibody surface staining for Siglec-F on each indicated cell population in a wild-type mouse. Data are representative of ≥2 experiments.