Flow cytometric analysis of innate lymphoid cells: challenges and solutions.

INTRODUCTION

The three groups of helper innate lymphoid cells (ILCs), namely ILC1, ILC2 and ILC3, have been identified by flow cytometry by combinations of cell surface markers. Here, we review various ways ILCs are currently identified, focusing on potential problems and their solutions. The first step to identify all ILCs is to exclude other lymphocytes and myeloid cells by their lineage-specific markers (Lin). However, the Lin cocktail varies in various studies, and the definition of Lin- population containing ILCs is often ambiguous, resulting in contamination of Lin cells, particularly T cells.

METHOD

We have designed combinations of cell surface markers to identify ILC populations in various tissues of B6 mice by flow cytometry. To minimize T cell contamination, TCR/CD3ϵ antibodies were used separately from the Lin cocktail. ILCs identified by surface markers are confirmed by the expression of the transcription factors GATA3, RORγt, T-bet and Eomes.

RESULT

ILC1s in the B6 mouse liver are identified by Lin-NKp46NK1.1TCR/CD3ϵCD49aCD49b. However, defining ILC1s in other tissues remains a challenge. ILC2s in the lung are identified by LinTCR/CD3ϵ Thy1CD127ST2 whereas ILC2s in the small intestine and liver are identified by LinTCR/CD3ϵThy1GATA3RORγt. ILC3s in B6 mouse spleen, liver, lung and small intestine are identified by LinTCR/CD3ϵ Thy1CD127RORγt.

DISCUSSION

The ILC population is heterogeneous and the strategies to identify ILCs have to be designed for each ILC population and tissue. Excluding T cells in all cases is crucial, and a combination of transcription factors GATA3, RORγt, T-bet, and Eomes should be used to identify ILCs. Using CD3ϵ/TCRs in a different fluorochrome not in Lin cocktail minimizes contamination of T cells specifically identify individual ILC populations in various tissues.