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• W2918489132 abstract "Innate lymphoid cells (ILCs) are a functionally and phenotypically heterologous group of small lymphocytes that lack conventional linage markers but can become potent effector cells upon appropriate stimulations thereby producing large amounts of cytokines. Some examples of ILCs include natural killer (NK) cells, lymphoid tissue-inducer cells, and helper-like ILCs. This study focuses on the helper-like ILCs. Helper-like ILCs are currently divided into 3 different subsets according to their distinct patterns of cytokine production and master transcription factors, which resemble helper T-cell subsets. In general, type 1 innate lymphoid cells (ILC1s) resemble TH1 cells and have been implicated in immunity against intracellular pathogens, type 2 innate lymphoid cells (ILC2s) resemble TH2 cells and have been implicated in immunity against helminths, and type 3 innate lymphoid cells (ILC3s) resemble TH17 and TH22 cells and have been implicated in immunity against extracellular bacteria. Interestingly, unlike T-helper subsets, recent studies revealed that ILCs are highly plastic cells that enable them to respond promptly to environmental changes.1Morita H. Moro K. Koyasu S. Innate lymphoid cells in allergic and nonallergic inflammation.J Allergy Clin Immunol. 2016; 138: 1253-1264Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar In addition to being functionally diverse and interconvertible, the developmental pathways of ILCs are not well understood. Although gene expression profiles of ILCs have been studied in tissues, such as human tonsils,2Björklund Å. Forkel M. Picelli S. Konya V. Theorell J. Friberg D. et al.The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.Nat Immunol. 2016; 17: 451-460Crossref PubMed Scopus (325) Google Scholar only limited information is available on human circulating ILCs.3Boyd A. Ribeiro J.M. Nutman T.B. Human CD117 (cKit)+ innate lymphoid cells have a discrete transcriptional profile at homeostasis and are expanded during filarial infection.PLoS One. 2014; 9: e108649Crossref PubMed Scopus (39) Google Scholar Tissue-resident ILCs could be quite different from the ILCs in blood. To understand the distinct functions of the type 1, 2 and 3 ILC subsets, it is necessary to delineate both the overlapping and unique functional signatures of these cells. We present here one of the first whole-genome gene expression studies of the 3 ILC subsets from peripheral blood of healthy donors. The study was approved by the Cantonal Ethics Committee of Zurich. This study aims to establish the human ILC transcriptome reference data set in healthy subjects for comparison in disease settings, with enhanced utility because in most clinical situations blood samples but not tissue biopsy specimens are readily available. A robust “2-round sorting” procedure was used to obtain highly pure ILC subsets. In brief, ILC subsets were isolated by using flow cytometry according to a published protocol by the Mjösberg group2Björklund Å. Forkel M. Picelli S. Konya V. Theorell J. Friberg D. et al.The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.Nat Immunol. 2016; 17: 451-460Crossref PubMed Scopus (325) Google Scholar with minor modifications: ILC1s as CD45+Lin−CD127+CD161+CRTH2−c-Kit−, ILC2s as CD45+Lin−CD127+CD161+CRTH2+c-Kit−, and ILC3s as CD45+Lin−CD127+CD161+CRTH2−c-Kit+. The purity of ILCs during this first-round sort was approximately 90% for ILC1s, 94% for ILC2s, and 58% for ILC3s. The ILCs were then resorted, collecting 1000 cells per subset by using more stringent gating directly into lysis buffer. Additional sorting experiments confirmed that the final ILC purity was high (86% to 96%), and more importantly, there was no detectable contamination of lineage-positive cells after second sorting (see Fig E1 in this article's Online Repository at www.jacionline.org). The samples (3 ILC subsets per donor and 6 different male healthy donors aged 25–40 years; ie, 18 samples in total) were analyzed by using RNA sequencing (RNAseq; raw data are available at the Gene Expression Omnibus as GSE112591, token yhmbgicchdkbncv). A total of 13,297 genes were detected as being expressed. Principal component analysis revealed 3 clearly distinct clusters of each ILC subset (Fig 1, A), demonstrating significantly distinct gene expression profiles of ILCs. The key ILC gene signatures were expressed as expected, such as high IL7R and KLRB1 expression (encoding CD127 and CD161, respectively) in all ILC subsets, high PTGDR2 (encoding CRTH2) and GATA3 (encoding GATA-binding factor 3) expression in ILC2s, high KIT (encoding c-Kit) and AHR (encoding Aryl hydrocarbon receptor) expression in ILC3s, and high EOMES (encoding eomesodermin homolog) expression in ILC1s (Fig 1, B). This provides validation of the cellular identity for each ILC subset and demonstrates the effectiveness of our sorting strategy. NCR2 (encoding NKp44) is detected at low levels across all samples, which is not surprising given that we sorted for NKp44− ILC3s. First, we examined the top 200 most abundantly expressed genes in each ILC subset. Fig 1, C, provides a Venn diagram of these genes, which are 246 in total, with 194 differentially expressed (heat map is provided in Fig E2 in this article's Online Repository at www.jacionline.org). Of the genes with high expression across all subsets, several are related to antigen presentation, including HLA-A, HLA-B, HLA-C, and HLA-E. IL2RG (encoding CD132, the cytokine receptor common gamma chain) is also expressed at high levels by all ILC subsets. It has been reported that ILCs were absent in the blood of patients with severe combined immunodeficiency (which is caused by common γ chain deficiency).4Vely F. Barlogis V. Vallentin B. Neven B. Piperoglou C. Ebbo M. et al.Evidence of innate lymphoid cell redundancy in humans.Nat Immunol. 2016; 17: 1291-1299Crossref PubMed Scopus (227) Google Scholar This suggests that CD132 plays an important role in sustaining the existence of ILCs. The expression of HLA-E and IL2RG (Fig 1, D) has not been previously reported in tissue-derived ILCs and is a new finding. FXYD5 and CD164 are expressed at higher levels by ILC3s (Fig 1, D), which were not previously reported in tissue-derived ILCs. FXYD5 (FXYD domain containing ion transport regulator 5) belongs to a family of ion transporter regulators and was known to have a proinflammatory role, including upregulation of chemokine expression.5Brazee P.L. Soni P.N. Tokhtaeva E. Magnani N. Yemelyanov A. Perlman H.R. et al.FXYD5 is an essential mediator of the inflammatory response during lung injury.Front Immunol. 2017; 8: 623Crossref PubMed Scopus (19) Google Scholar CD164 is a molecule that might be implicated in adhesion and migration of hematopoietic progenitor cells. How these molecules contribute to ILC function warrants future investigation. We analyzed CD164 protein expression using flow cytometry and confirmed increased CD164 expression by ILC3s (Fig 1, E), opening opportunities for further study of this potentially new ILC marker. Of the other differentially expressed genes within the top 200 group, some are well-known molecules implicated in immune cell functions, such as PDCD4 (encoding programmed cell death protein 4), FCER1G (encoding receptor I for the Fc fragment of IgE), CXCR4, and CCL5 (Fig E2). The top 200 genes were further analyzed for protein-protein interactions by using the STRING Web resource to obtain an overview of their cellular functions. We identified a cluster of genes that are related to ribosomal functions, another cluster related to mitochondria, and a third cluster related to HLA class I antigen presentation functions (see Fig E3 in this article's Online Repository at www.jacionline.org). We next examined the entire data set, focusing on differences between the 3 ILC subsets. The overall differential expression profiles are shown by using a heat map with 529, 323, and 669 group-specific genes for ILC1s, ILC2s, and ILC3s, respectively (Fig 2, A), and volcano plots (Fig 2, B). Table E1 in this article's Online Repository at www.jacionline.org provides a comprehensive list of these genes, and Table E2 in this article's Online Repository at www.jacionline.org provides Gene Ontology IDs for the differentially expressed genes. Of the differentially expressed genes, many are related to immune functions, such as the gene groups depicted in Fig 2, C, including cytokines and chemokines, antigen presentation, and innate immunity, as well as signal transduction (see Fig E4 in this article's Online Repository at www.jacionline.org). It has been reported that the ILC1s are a fictional cell subset composed of various contaminating cell populations.6Simoni Y. Fehlings M. Kloverpris H.N. McGovern N. Koo S.L. Loh C.Y. et al.Human innate lymphoid cell subsets possess tissue-type based heterogeneity in phenotype and frequency.Immunity. 2017; 46: 148-161Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar In our data ILC1s expressed a higher level of PRF1 (encoding perforin) and several granzymes, including GZMA, GZMB, GZMH, GZMM, and GZMK. We analyzed intracellular expression of the GZMB protein by using flow cytometry. In contrast to NK cells, in which 29.8% expressed GZMB, few ILC1s (2.4%) and none of the ILC2s or ILC3s were found to express this molecule; ILC1s also did not express CD56 (see Fig E6 in this article's Online Repository at www.jacionline.org). Although we cannot absolutely exclude the possibility of contaminating NK cells, these results make it unlikely that the GZMB-positive cells expressing ILC1 markers are themselves NK cells. ILC2 expression of molecules with neuronal regulation functions has been recently reported in mice.7Klose C.S.N. Mahlakoiv T. Moeller J.B. Rankin L.C. Flamar A.L. Kabata H. et al.The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation.Nature. 2017; 549: 282-286Crossref PubMed Scopus (322) Google Scholar, 8Cardoso V. Chesne J. Ribeiro H. Garcia-Cassani B. Carvalho T. Bouchery T. et al.Neuronal regulation of type 2 innate lymphoid cells via neuromedin U.Nature. 2017; 549: 277-281Crossref PubMed Scopus (336) Google Scholar In our data NMU (encoding neuromedin U) was downregulated in ILC2s compared with ILC1s; NMUR1 was downregulated in ILC2s compared with ILC3s. It is unclear whether this might be attributed to genetic differences between mice and human subjects. Molecules important for ILC development and function have been previously described for tissue-derived ILCs.1Morita H. Moro K. Koyasu S. Innate lymphoid cells in allergic and nonallergic inflammation.J Allergy Clin Immunol. 2016; 138: 1253-1264Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar Circulating ILCs also had these gene signatures: ILC1s expressed greater levels of TBX21 (encoding T-bet), GNLY and IL23R; ILC3s expressed greater levels of TCF7, NOTCH1, and TOX, and RORA is highly expressed by ILC1s and ILC2s compared with ILC3s, for example. Using commercially available mAbs, we were able to validate protein expression for certain molecules (see Fig E5 in this article's Online Repository at www.jacionline.org). CD200R1 encodes for the cell-surface transmembrane glycoprotein CD200 receptor 1, which is usually expressed by myeloid cells and CD4+ T cells and plays a role in regulating the expression of proinflammatory molecules, such as TNF and interferon family molecules. ILC2s had a greater level of surface expression of CD200R1 compared with ILC1s and ILC3s, which is consistent with the immune regulatory roles of ILC2s. Discrepancies between RNA and protein expression by ILCs had been reported previously,2Björklund Å. Forkel M. Picelli S. Konya V. Theorell J. Friberg D. et al.The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.Nat Immunol. 2016; 17: 451-460Crossref PubMed Scopus (325) Google Scholar and this was also observed in our data. Expressed by NK cells, NKG2A and CD94 form the receptor for HLA-E, which negatively regulates NK cell activation. NKG2A protein expression by ILC1s can reflect NK cell contamination. However, because cells expressing CD94 and CD16 linage markers were gated out, our data might also suggest overlapping phenotypes between ILC1s and NK cells. The myeloid cell-surface antigen CD33 is widely used as a diagnostic marker for acute myeloid leukemia. CD33 RNA was expressed at greater levels by ILC3s compared with ILC1s and ILC2s, and flow cytometric analysis confirmed surface protein expression. It would be interesting to investigate the role of ILC3s in leukemia in future studies. Our current data also provided new insights into the expression of inflammatory cytokine receptors, such as the TNF and TNF receptor superfamilies, in circulating ILC subsets. TNFRSF4 encodes CD134, which is an important molecule implicated in the function of effector T cells, as well as regulatory T cells.9Govindaraj C. Scalzo-Inguanti K. Scholzen A. Li S. Plebanski M. TNFR2 expression on CD25(hi)FOXP3(+) T cells induced upon TCR stimulation of CD4 T cells identifies maximal cytokine-producing effectors.Front Immunol. 2013; 4: 233Crossref PubMed Scopus (21) Google Scholar ILC3s expressed high transcript levels of TNFRSF4, and protein expression is also detectable, which warrants further investigation if and how TNFRSF4 and its protein might be implicated in ILC3 functions. EVA1 (encoding epithelial V-like antigen 1) is expressed in thymus epithelium and implicated in thymocyte development. TNFRSF19 (encoding TNF receptor superfamily 19) is highly expressed during embryonic development. Both of these molecules were expressed at greater levels in ILC2s compared with ILC1s and ILC3s, suggesting potential developmental roles for ILC2s during early life. Table E3 in this article's Online Repository at www.jacionline.org provides a list of the top 50 functional pathways identified by means of Metacore analysis (Clarivate Analytics or Thomson Reuters). A better understanding of the key gene expression signatures will help to decipher the functional properties of these potent immune effort cells; for example, ILCs might be preferentially poised to respond functionally to changes in inflammatory cytokine levels in the microenvironments to which they home and exert their function. Inspecting the entire data set, we noted that a number of T-cell receptor (TCR) genes were expressed. The constant regions were either abundantly (TRAC, TRBC1, TRBC2, and TRDC were within the top 200 group; Fig 1, C) or differentially (TRAC, TRBC2, TRDC, and TRGC) expressed among the ILC subsets, with ILC3s expressing the highest level of TRDC (Fig 2, D, and see Table E1). Human type I NK T cells are known to express TRBV25 and TRAV10-TRAJ18, and mucosa-associated invariant T cells express TRBV20 and a wide range of other TCR genes; however, these cell types are usually linage positive. In our data CD4 was not detectable, CD8 was expressed at very low levels, and only ILC1s expressed CD3 (Fig 2, D). T-cell contamination of ILC1s is possible, which is consistent with the literature.7Klose C.S.N. Mahlakoiv T. Moeller J.B. Rankin L.C. Flamar A.L. Kabata H. et al.The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation.Nature. 2017; 549: 282-286Crossref PubMed Scopus (322) Google Scholar However, the above data argue against conventional T-cell contamination for ILC2s and ILC3s, although it is not possible to exclude this possibility in absolute terms. A number of TRBJ, TRDJ, and TRGJ genes are also among the top 200 group, but expression of TCR variable region transcripts were generally low (see Figs E7-E9 in this article's Online Repository at www.jacionline.org). TCR expression by tissue-derived ILCs has been suggested previously,2Björklund Å. Forkel M. Picelli S. Konya V. Theorell J. Friberg D. et al.The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.Nat Immunol. 2016; 17: 451-460Crossref PubMed Scopus (325) Google Scholar but follow-up studies have not confirmed this. The reanalysis of published single-cell RNAseq data from human tonsils by the Mjösberg group indicate that although TRBJ transcripts were expressed by a moderate number of ILCs, TRBV transcripts were expressed by only a small number of ILC1s and few ILC2s and ILC3s (see Fig E10, A, in this article's Online Repository at www.jacionline.org). These findings are consistent with our bulk ILC RNAseq data, suggesting expression of TCR transcripts in ILCs rather than contamination by T cells. Interestingly, TRBV28 was detected in 8 ILC3s in the above work by the Mjösberg group,2Björklund Å. Forkel M. Picelli S. Konya V. Theorell J. Friberg D. et al.The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing.Nat Immunol. 2016; 17: 451-460Crossref PubMed Scopus (325) Google Scholar a slightly higher number compared with ILC1s and ILC2s. We examined TCR protein expression using flow cytometry and indeed detected TRBV28 surface expression on circulating ILC3s (see Fig E10, B). Interestingly, although the TRBJ1 and TRBJ2 genes are expressed at moderate levels, only TRBD1 was expressed by 3 ILC subsets (see Fig E5, A). On chromosome 7, TRBD2 is adjacent to TRBJ2, and TRBD1 is approximately 300 kb upstream (see Fig E5, B), and it would be quite unusual if TRBJ2 only rearranged with TRBD1. One possibility could be that ILCs are developmentally blocked in the TCR gene rearrangement, have other defects in TCR expression, or both. Such a scenario might explain why ILCs mimic T cells in cytokine production profiles. In the future, it will be interesting to study the details of TCR gene rearrangement and expression pathways in ILCs. Taken together, in this study we identified novel gene signatures of the 3 different ILC subsets, which might be further developed into ILC biomarkers. We also provide evidence for abnormal TCR expression in ILCs, which suggests a potentially new developmental link between ILCs and T cells. We thank Christian Doerig for support and strategic advice, Andrew Farmer for helpful discussions and technical advices on TCR analysis, Avidan Neumann for helpful discussions, and Mark Sleeman and Hans Netter for general support. Download .docx (.02 MB) Help with docx files Online Repository text Download .pdf (.86 MB) Help with pdf files Fig E6 Download .pdf (.07 MB) Help with pdf files Fig E7 Download .pdf (.1 MB) Help with pdf files Fig E8 Download .pdf (.07 MB) Help with pdf files Fig E9 Download .pdf (2.33 MB) Help with pdf files Fig E10 Download .xlsx (.26 MB) Help with xlsx files Table E1 Download .xlsx (.55 MB) Help with xlsx files Table E2 Download .xlsx (.03 MB) Help with xlsx files Table E3 Download .pdf (.9 MB) Help with pdf files Fig E1 Download .pdf (.06 MB) Help with pdf files Fig E2 Download .pdf (.52 MB) Help with pdf files Fig E3 Download .pdf (.05 MB) Help with pdf files Fig E4 Download .pdf (.48 MB) Help with pdf files Fig E5" @default.
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• W2918489132 title "Gene expression signatures of circulating human type 1, 2, and 3 innate lymphoid cells" @default.
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