, Volume 69, Issue 7, pp 439–450 | Cite as

Identification of innate lymphoid cells in single-cell RNA-Seq data

  • Madeleine Suffiotti
  • Santiago J. Carmona
  • Camilla Jandus
  • David GfellerEmail author
Original Article


Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.


Innate lymphoid cells Single-cell RNA-Seq Immune cell type evolution Immunogenomics Cell type predictions 



D.G. is supported by the Ludwig Institute for Cancer Research (LICR) and the Center for Advanced Modeling Science (CADMOS); S.J.C. is supported by SystemsX (MelamonX grant). C.J is supported by the Swiss National Science Foundation (Ambizione Fellowship PZOOP3_161459).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

251_2017_1002_MOESM1_ESM.pdf (160 kb)
ESM 1 (PDF 159 kb)


  1. Akula S, Thorpe M, Boinapally V, Hellman L (2015) Granule associated serine proteases of hematopoietic cells—an analysis of their appearance and diversification during vertebrate evolution. PLoS One 10:e0143091. doi: 10.1371/journal.pone.0143091 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Alabyev BY, Guselnikov SV, Najakshin AM et al (2000) CD3epsilon homologues in the chondrostean fish Acipenser ruthenus. Immunogenetics 51:1012–1020CrossRefPubMedGoogle Scholar
  3. Altenhoff AM, Škunca N, Glover N et al (2015) The OMA orthology database in 2015: function predictions, better plant support, synteny view and other improvements. Nucleic Acids Res 43:D240–D249. doi: 10.1093/nar/gku1158 CrossRefPubMedGoogle Scholar
  4. Artis D, Spits H (2015) The biology of innate lymphoid cells. Nature 517:293–301. doi: 10.1038/nature14189 CrossRefPubMedGoogle Scholar
  5. Bernink JH, Krabbendam L, Germar K et al (2015) Interleukin-12 and -23 control plasticity of CD127(+) group 1 and group 3 innate lymphoid cells in the intestinal lamina propria. Immunity 43:146–160. doi: 10.1016/j.immuni.2015.06.019 CrossRefPubMedGoogle Scholar
  6. Björklund AK, Forkel M, Picelli S et al (2016) The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing. Nat Immunol. doi: 10.1038/ni.3368
  7. Boehm T (2012) Evolution of vertebrate immunity. Curr Biol 22:R722–R732. doi: 10.1016/j.cub.2012.07.003 CrossRefPubMedGoogle Scholar
  8. Brocker C, Thompson D, Matsumoto A et al (2010) Evolutionary divergence and functions of the human interleukin (IL) gene family. Hum Genomics 5:30–55. doi: 10.1186/1479-7364-5-1-30 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Carmona SJ, Teichmann SA, Ferreira L et al (2017) Single-cell transcriptome analysis of fish immune cells provides insight into the evolution of vertebrate immune cell types. Genome Res 27(3):451–461. doi: 10.1101/gr.207704.116
  10. Chea S, Schmutz S, Berthault C et al (2016) Single-cell gene expression analyses reveal heterogeneous responsiveness of fetal innate lymphoid progenitors to notch signaling. Cell Rep 14:1500–1516. doi: 10.1016/j.celrep.2016.01.015 CrossRefPubMedGoogle Scholar
  11. Constantinides MG, McDonald BD, Verhoef PA, Bendelac A (2014) A committed precursor to innate lymphoid cells. Nature 508:397–401. doi: 10.1038/nature13047 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Delconte RB, Shi W, Sathe P et al (2016) The helix-loop-helix protein ID2 governs NK cell fate by tuning their sensitivity to interleukin-15. Immunity 44:103–115. doi: 10.1016/j.immuni.2015.12.007 CrossRefPubMedGoogle Scholar
  13. Diefenbach A, Colonna M, Koyasu S (2014) Development, differentiation, and diversity of innate lymphoid cells. Immunity 41:354–365. doi: 10.1016/j.immuni.2014.09.005 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Eberl G, Colonna M, Di Santo JP, McKenzie ANJ (2015) Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology. Science 348:aaa6566–aaa6566. doi: 10.1126/science.aaa6566 CrossRefGoogle Scholar
  15. Friedman J, Hastie T, Tibshirani R (2010) Regularization paths for generalized linear models via coordinate descent. J Stat Softw 33:1–22. doi: 10.1109/TPAMI.2005.127 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Grün D, Lyubimova A, Kester L et al (2015) Single-cell messenger RNA sequencing reveals rare intestinal cell types. Nature 525:251–255. doi: 10.1038/nature14966 CrossRefPubMedGoogle Scholar
  17. Haugland GT, Jordal A-EO, Wergeland HI (2012) Characterization of small, mononuclear blood cells from salmon having high phagocytic capacity and ability to differentiate into dendritic like cells. PLoS One 7:e49260. doi: 10.1371/journal.pone.0049260 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Heng TSP, Painter MW (2008) The immunological genome project: networks of gene expression in immune cells. Nat Immunol 9:1091–1094. doi: 10.1038/ni1008-1091 CrossRefPubMedGoogle Scholar
  19. Ishizuka IE, Chea S, Gudjonson H et al (2016) Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage. Nat Immunol 17:269–276. doi: 10.1038/ni.3344 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Jiang W, Chai NR, Maric D, Bielekova B (2011) Unexpected role for granzyme K in CD56bright NK cell-mediated immunoregulation of multiple sclerosis. J Immunol 187:781–790. doi: 10.4049/jimmunol.1100789 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Kaiser P, Rothwell L, Avery S, Balu S (2004) Evolution of the interleukins. Dev Comp Immunol 28:375–394. doi: 10.1016/j.dci.2003.09.004 CrossRefPubMedGoogle Scholar
  22. Kløverpris HN, Kazer SW, Mjösberg J et al (2016) Innate lymphoid cells are depleted irreversibly during acute HIV-1 infection in the absence of viral suppression. Immunity 44:391–405. doi: 10.1016/j.immuni.2016.01.006 CrossRefPubMedGoogle Scholar
  23. Konya V, Mjösberg J (2016) Lipid mediators as regulators of human ILC2 function in allergic diseases. Immunol Lett. doi: 10.1016/j.imlet.2016.07.006
  24. Koppang EO, Fischer U, Moore L et al (2010) Salmonid T cells assemble in the thymus, spleen and in novel interbranchial lymphoid tissue. J Anat 217:728–739. doi: 10.1111/j.1469-7580.2010.01305.x CrossRefPubMedPubMedCentralGoogle Scholar
  25. Koues OI, Collins PL, Cella M et al (2016) Distinct gene regulatory pathways for human innate versus adaptive lymphoid cells. Cell 165:1134–1146. doi: 10.1016/j.cell.2016.04.014 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Li B, Dewey CN (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinf 12:323. doi: 10.1186/1471-2105-12-323 CrossRefGoogle Scholar
  27. Lim AI, Menegatti S, Bustamante J et al (2016) IL-12 drives functional plasticity of human group 2 innate lymphoid cells. J Exp Med 213:569–583. doi: 10.1084/jem.20151750 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Macosko EZ, Basu A, Satija R et al (2015) Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161:1202–1214. doi: 10.1016/j.cell.2015.05.002 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Moltke von J, O’Leary CE, Barrett NA et al (2017) Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s. J Exp Med 214:27–37. doi: 10.1084/jem.20161274 CrossRefGoogle Scholar
  30. Moore FE, Garcia EG, Lobbardi R et al (2016) Single-cell transcriptional analysis of normal, aberrant, and malignant hematopoiesis in zebrafish. J Exp Med 213:979–992. doi: 10.1084/jem.20152013 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Nagasawa T, Nakayasu C, Rieger AM et al (2014) Phagocytosis by thrombocytes is a conserved innate immune mechanism in lower vertebrates. Front Immunol 5:445. doi: 10.3389/fimmu.2014.00445 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Neu KE, Tang Q, Wilson PC, Khan AA (2017) Single-cell genomics: approaches and utility in immunology. Trends Immunol 38:140–149. doi: 10.1016/ CrossRefPubMedGoogle Scholar
  33. Parker HS, Leek JT, Favorov AV et al (2014) Preserving biological heterogeneity with a permuted surrogate variable analysis for genomics batch correction. Bioinformatics 30:2757–2763. doi: 10.1093/bioinformatics/btu375 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Proserpio V, Lönnberg T (2016) Single-cell technologies are revolutionizing the approach to rare cells. Immunol Cell Biol 94:225–229. doi: 10.1038/icb.2015.106 CrossRefPubMedGoogle Scholar
  35. Ranson T, Vosshenrich CAJ, Corcuff E et al (2003) IL-15 is an essential mediator of peripheral NK-cell homeostasis. Blood 101:4887–4893. doi: 10.1182/blood-2002-11-3392 CrossRefPubMedGoogle Scholar
  36. Ritchie ME, Phipson B, Wu D et al (2015) Limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 43:e47–e47. doi: 10.1093/nar/gkv007 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Robinette ML, Fuchs A, Cortez VS et al (2015) Transcriptional programs define molecular characteristics of innate lymphoid cell classes and subsets. Nat Immunol 16:306–317. doi: 10.1038/ni.3094 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Šedý J, Bekiaris V, Ware CF (2015) Tumor necrosis factor superfamily in innate immunity and inflammation. Cold Spring Harb Perspect Biol 7:a016279. doi: 10.1101/cshperspect.a016279 CrossRefPubMedCentralGoogle Scholar
  39. Seehus CR, Aliahmad P, la Torre de B et al (2015) The development of innate lymphoid cells requires TOX-dependent generation of a common innate lymphoid cell progenitor. Nat Immunol 16:599–608. doi: 10.1038/ni.3168 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Shih H-Y, Sciumè G, Mikami Y et al (2016) Developmental acquisition of regulomes underlies innate lymphoid cell functionality. Cell 165:1120–1133. doi: 10.1016/j.cell.2016.04.029 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Silver JS, Kearley J, Copenhaver AM et al (2016) Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs. Nat Immunol 17:626–635. doi: 10.1038/ni.3443 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Sonnenberg GF, Artis D (2015) Innate lymphoid cells in the initiation, regulation and resolution of inflammation. Nat Med 21:698–708. doi: 10.1038/nm.3892 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Spits H, Bernink JH, Lanier L (2016) NK cells and type 1 innate lymphoid cells: partners in host defense. Nat Immunol 17:758–764. doi: 10.1038/ni.3482 CrossRefPubMedGoogle Scholar
  44. Tian Z, van Velkinburgh JC, Wu Y, Ni B (2016) Innate lymphoid cells involve in tumorigenesis. Int J Cancer 138:22–29. doi: 10.1002/ijc.29443 CrossRefPubMedGoogle Scholar
  45. Tirosh I, Izar B, Prakadan SM et al (2016) Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352:189–196. doi: 10.1126/science.aad0501 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Trabanelli S, Curti A, Lecciso M et al (2015) CD127+ innate lymphoid cells are dysregulated in treatment naïve acute myeloid leukemia patients at diagnosis. Haematologica 100:e257–e260. doi: 10.3324/haematol.2014.119602 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111. doi: 10.1093/bioinformatics/btp120 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Vallentin B, Barlogis V, Piperoglou C et al (2015) Innate lymphoid cells in cancer. Cancer Immunol Res 3:1109–1114. doi: 10.1158/2326-6066.CIR-15-0222 CrossRefPubMedGoogle Scholar
  49. Van der Maaten L, Hinton GE (2008) Visualizing high-dimensional data using t-SNE. J Mach Learn Res 9:2579Google Scholar
  50. Vivier E, van de Pavert SA, Cooper MD, Belz GT (2016) The evolution of innate lymphoid cells. Nat Immunol 17:790–794. doi: 10.1038/ni.3459 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Wang T, Holland JW, Carrington A et al (2007) Molecular and functional characterization of IL-15 in rainbow trout Oncorhynchus mykiss: a potent inducer of IFN-gamma expression in spleen leukocytes. J Immunol 179:1475–1488CrossRefPubMedGoogle Scholar
  52. Zheng GXY, Terry JM, Belgrader P et al (2017) Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049. doi: 10.1038/ncomms14049

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Madeleine Suffiotti
    • 1
    • 2
  • Santiago J. Carmona
    • 1
    • 2
  • Camilla Jandus
    • 1
  • David Gfeller
    • 1
    • 2
    Email author
  1. 1.Ludwig Centre for Cancer ResearchUniversity of LausanneEpalingesSwitzerland
  2. 2.Swiss Institute of Bioinformatics (SIB)LausanneSwitzerland

Personalised recommendations