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. 2018:1803:353-370.
doi: 10.1007/978-1-4939-8549-4_21.

Isolation and Identification of Innate Lymphoid Cells (ILCs) for Immunotoxicity Testing

Elia D Tait Wojno  1 , Celine A Beamer  2
Affiliations

Isolation and Identification of Innate Lymphoid Cells (ILCs) for Immunotoxicity Testing

Elia D Tait Wojno et al. Methods Mol Biol. 2018.

Abstract

Innate lymphoid cells (ILCs) comprise a family of innate immune cells that orchestrate mucosal immune responses: initiating, sustaining, and even curbing immune responses. ILCs are relatively rare (≤1% of lymphocytes in mucosal tissues), lack classical cell-surface markers, and can be divided into three subsets (type 1-3 ILCs) based on differences in cytokine production, phenotype, and developmental pathway. Because ILCs can only be identified by combinations of cell-surface markers and cytokine production, multicolor flow cytometry is the most reliable method to purify, characterize, and assess the functionality of ILCs. Here, we describe the methods for cell preparation, flow cytometric analysis, and purification of murine ILCs from the lung.

Keywords: Flow cytometry; ILC2; ILC3; Innate lymphoid cells; Mouse; Respiratory tract.

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Figures

Figure 1
Figure 1
Typical workflow to isolate and identify total ILCs from the murine lung.
Figure 2
Figure 2
Typical aspect of the lymphocyte-rich interphase between medium and Fico-lite after centrifugation.
Figure 3
Figure 3
Typical enrichment of CD90.2+ cells from lung digests. (A) The frequency of live, CD90.2+ cells was analyzed from the single cell suspension pre-enrichment, the CD90.2−fraction, and the CD90.2+ fraction after the first and second magnetic enrichment columns. (B) A representative overlay shows the frequency of CD90.2+ cells before magnetic separation (light grey line), the CD90.2− cells (dark grey line), and the CD90.2+ cells (black line).
Figure 4
Figure 4
Staining strategy to identify bulk lung ILCs (CD90.2+CD45+Lineage−CD127+CD11b−). Lung ILC2s are identified as ST2+CD4−, whereas ILC3s are ST2−CD4+/-. Cells were first gated for live propidium iodide (PI) negative, singlet leukocytes. See Table 2 for antibody panel.
Figure 5
Figure 5. Transferred ILC2s drive type 2 inflammation in the lung of ILC2-deficient mice
CD45.1-expressing C57BL/6 mice were treated for 5 days with 300 ng recombinant murine IL-33. On day 6, CD45.1+ Lineage(CD3/CD4/CD5/CD11b/CD11c/CD19/NK1.1)−CD90.2+CD25+ CD127+IL-33R+ ILC2s were sort-purified, and 1×105 ILC2s were transferred intravenously to Rag2−/−cγ−/− CD45.2-expressing mice every 7 days for 4 transfers. The day after the first transfer, transferred Rag2−/−cγ−/− mice, untransferred Rag2−/−cγ−/− mice, and Rag2−/− mice were infected with 500 N. brasiliensis L3 larvae. On day 32 following infection, (A) transferred ILC2s could be identified by flow cytometry in the Rag2−/−cγ−/− lung, and (B) IL-5 levels were measured by ELISA in the lung homogenate. Data are representative of 2 experiments, n = 2–4/group in each experiment; mean±sem.
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