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. 2008 Nov 24;205(12):2839–2850. doi: 10.1084/jem.20081430

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© 2008 Jakubzick et al.

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Figure 4. — EGFP reporter expression in LysM-Cre × Rosa26-stop^floxEGFP mice by DCs that migrate into lymph nodes from upstream organs in response to inflammatory stimulation. (A) TRITC was applied to skin epicutaneously in a contact-sensitizing solution, and TRITC⁺ cells in the draining lymph nodes 18 h later were identified (left dot plot, gated area) and further analyzed. The flow cytometry plot on the right shows an overlay of TRITC⁺ cells (black) over all live lymph node cells (gray) with respect to the expression of CD11c and CD11b. The percentage of EGFP⁺ cells within TRITC⁺ and TRITC⁻ lymph node DCs was normalized to the percentage of EGFP⁺ Ly-6C^hi monocytes in the same mouse. Graph shows individual mice per data point. TRITC⁻ DCs refers to a gate on TRITC⁻ CD11c⁺ DCs that excludes the CD11b^hiCD11c^int gate that is considered to contain mainly monocytes (reference 37). The TRITC⁻ CD11c⁺ grouping includes all DCs and the putative monocyte gate, thereby containing all TRITC⁻ CD11c⁺ and CD11c^int cells in the lymph node. (B) PBS or LPS was delivered intranasally (I.N.). 24 h later, live cells from the lung-draining lymph nodes were gated to identify CD11c^hi CD11b^lo cells, and gated CD11c^hi CD11b^lo cells were examined for CD103 or CD8α expression, revealing three subpopulations: CD103⁺ CD8α⁻ (CD103⁺) lung-derived DCs, CD8α⁺ DCs, and double-negative DCs. Shown is the number (C) or percentage (D) of the CD103⁺ CD8α⁻ (CD103⁺) DCs, CD8α⁺ DCs, and double-negative DCs within the CD11c^hi CD11b^lo gated population (from the FACS dot plot, top left) from the lung-draining lymph nodes treated with LPS (black squares) or with PBS (gray circles) to maintain near steady-state conditions. (E) Graph shows the effect of LPS treatment, used to induce DC migration from the lung, on the expression of EGFP by various populations, normalized to Ly6C^hi monocytes within the same mouse. BAL and lung were gated on CD11c^hiCD11b^loCD103⁺ DCs (CD103⁺). Lung-draining lymph node was gated on CD11c^hiCD11b^loCD103⁺ DCs (CD103⁺), CD11c^hiCD11b^loCD103⁻ DCs (CD103⁻), and plasmacytoid DCs. Horizontal bars represent means. (F) Percentage of blood monocyte subsets and lymph node DC populations that expressed EGFP with or without intranasal LPS administration (from mice in E). In each graph, all mice studied are compiled, and each data point corresponds to one mouse. Two to three mice per group were handled per experiment. Data represent the mean ± SEM.

	
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