Optimal Identification of Human Conventional and Non-conventional (CRTH2-IL7Rα-) ILC2s Using Additional Surface Markers.

Abstract Background Human type-2 innate lymphoid cells (ILC2s) are identified by coupled-detection of CRTH2 and IL7Rα on lineage-negative cells. Type-2 cytokine production by CRTH2-IL7Rα- ILCs is unknown. Objective To identify CRTH2-IL7Rα- type-2 cytokine producing ILCs and their disease relevance. Methods We studied human blood and lung ILCs from asthmatic and control subjects by flow cytometry, ELISA, RNA-seq, qPCR, adoptive transfer to mice and measurement of airway hyperreactivity by Flexivent. Results We found that IL5 and IL13 were expressed not only by CRTH2+ but also by CRTH2-IL7Rα+ and CRTH2-IL7Rα- (DN: double negative) human blood and lung cells. All three ILC populations expressed type-2 genes and induced airway hyperreactivity when adoptively transferred to mice. The frequency of type-2 cytokine+ IL7Rα and DN ILCs were similar to that of CRTH2 ILCs in the blood and lung. Their frequency was higher in asthmatic patients compared to disease controls. Transcriptomic analysis of CRTH2, IL7Rα and DN ILCs confirmed the expression of mRNA for type-2 transcription factors in all three populations. Unexpectedly, the mRNA for GATA3 and IL5 correlated better with mRNA for CD30, TNFR2, ICOS, CCR4 and CD200R1 than CRTH2. Using a combination of these surface markers, especially CD30/TNFR2, we identified a previously unrecognized ILC2 population. Conclusions The commonly used surface markers for human ILC2s leave a majority of type-2 cytokine-producing ILC2s unaccounted for. We identified top GATA3-correlated cell-surface expressed genes in human ILCs by RNA-seq. These new surface markers such as CD30 and TNFR2 identified a previously unrecognized human ILC2 population. This ILC2 population is likely to contribute to asthma.