Human CD117 (cKit)+ innate lymphoid cells have a discrete transcriptional profile at homeostasis and are expanded during filarial infection

Alexis Boyd, José M C Ribeiro, Thomas B Nutman, Alexis Boyd, José M C Ribeiro, Thomas B Nutman

Abstract

Since innate lymphoid cells (ILCs) have been found to play a role in the immune response to helminth parasites in rodents, we sought to determine their role in human helminth infection. By developing multicolor flow cytometry-based methods to identify and enumerate circulating ILCs and their subsets, we were able to identify a subset of cKit+ ILCs defined as Lineage (Lin)-/CD45+/cKit+/CD127+ that were significantly expanded in the filarial-infected individuals (p=0.0473) as were those cKit+ ILCs that produced IL-13. Additionally, the frequency of these cKit+ ILCs correlated with the frequency of IL-17 producing CD4+ T cells (Th17 cells; p=0.025). To investigate the function of cKit+ ILCs, sorted, highly purified human ILCs were subjected to transcriptional profiling by RNAseq and compared to appropriate control cells. These cKit+ ILCs expressed TLRs, a broad range of cytokines/cytokine receptors and MHC Class II molecules suggesting that these ILCs sense pathogens independent of other cell types. Functional analysis revealed expanded cKit+ ILC-specific transcription and ILC-specific microRNA precursors.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. cKit+ ILCs are increased in…
Figure 1. cKit+ ILCs are increased in filarial-infected individuals.
(A) Representative flow diagram from a filarial-infected individual demonstrating the cKit+ ILC gating strategy. Lin−/CD45+/cKit+/CD127+ cells were identified as cKit+ ILCs of which 28% were positive for IL-13 production. (B) Frequency of cKit+ ILCs (Lin−/CD45+/cKit/CD127+ cells) in filarial-uninfected (filled circles, n = 11) and filarial infected (open circles, n = 21). Horizontal bars are the geometric mean (GM) of the frequencies. (C) Frequency of IL-13+ cKit+ ILCs (Lin−/CD45+/cKit+CD127+/IL-13+ cells) in filarial-uninfected (filled circles, n = 11) and filarial-infected (open circles, n = 21) individuals. Horizontal bars are the geometric mean (GM) of the frequencies. (D) Correlation between the absolute eosinophil count and the frequency of IL-13+ cKit+ ILCs in the patients and uninfected individuals. The p value and r value from the Spearman rank correlation are shown.
Figure 2. The frequency of cKit+ ILCs…
Figure 2. The frequency of cKit+ ILCs in peripheral blood correlates with the frequency of Th17 cells.
Correlation of the frequencies of cKit+ ILCs and the frequencies of Th17 (A), Th2 (B), and Th1 (C) CD4+ T cells in 21 filarial-infected (circles) and 11 filarial-uninfected individuals (triangles). The p and r-values from the Spearman rank correlation analysis are shown. (D) Correlation of the frequencies of IL-13 producing cKit+ ILCs and Th2 CD4+ T cells from 8 filarial-infected and 6 uninfected individuals. The p-value and r-value from the Spearman rank correlation analysis are shown.
Figure 3. Cell sorting yields a purified…
Figure 3. Cell sorting yields a purified ILCs that produces Th2-, Th17- and Th1-related cytokines.
(A) Representative flow diagram of the strategy used for cell sorting of cKit+ ILCs (B) Sorted cKit+ ILCs (n = 5) cultured with media alone (closed circles-Unst) or with a cytokine cocktail consisting of rhIL-2, IL-7, IL-25 and IL-33 (open circles-Stim) for 3 days.
Figure 4. At homeostasis cKit+ ILCs express…
Figure 4. At homeostasis cKit+ ILCs express transcripts associated with the ILC2 and ILC3 subsets.
Transcripts differentially expressed between ILCs and lineage depleted PBMCs (control cells). The upregulated and downregulated transcripts from the RNASeq analysis were put into Ingenuity Pathway Analysis for graphical illustration. Pathway depicts genes that are upregulated (red) and downregulated (green) in cKit+ILCs in comparison to lineage-depleted PBMCs and their presumed location in the cell. The top of the figure depicts the cell membrane, with the nucleus towards the bottom of the figure.
Figure 5. The majority of expressed transcripts…
Figure 5. The majority of expressed transcripts in cKit+ ILCs are for signal transduction and transcription machinery.
(A) Volcano plot depicting all transcripts with those upregulated shown in red (n = 472) and those downregulated shown in blue (n = 2371). (B) Pie chart showing the functional classification of all transcripts identified. Sub-categories of the immunologically relevant genes are listed (B) with a full listing of all the genes classified as immunologically relevant in Table S1. (C) A heat map of the immunologically relevant transcripts of cKit+ ILCs (n = 6) compared to lineage depleted PBMCs (n = 2). (D) Sorted cKit+ ILCs (n = 6) cultured with media alone (closed circles – Unst) or with a cocktail of TLR ligands for TLR5, TLR6, TLR7 and TLR9 (open circles – Stim) for 3 days.
Figure 6. cKit+ ILCs express more genes…
Figure 6. cKit+ ILCs express more genes for nuclear regulation and transcription machinery and a unique microRNA signature.
(A) Pie chart depicting a functional analysis of the transcripts enriched in ILCs. Pulled out slices are functional categories significantly increased in ILCs compared to lineage depleted PBMCs. (B) Pie chart showing a functional analysis of transcripts enriched in lineage depleted PBMCs compared to ILCs. Pulled out slices are categories significantly increased in lineage depleted PBMCs. (C) A list of the 10 most abundantly expressed microRNA precursors in ILCs and their expression levels in controls (lineage depleted PBMCs).

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Source: PubMed

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