Mechanisms of allergy/immunology
Optimal identification of human conventional and nonconventional (CRTH2IL7Rα) ILC2s using additional surface markers

https://doi.org/10.1016/j.jaci.2020.01.038Get rights and content

Background

Human type 2 innate lymphoid cells (ILC2s) are identified by coupled detection of CRTH2 and IL7Rα on lineage negative (Lin) cells. Type 2 cytokine production by CRTH2IL7Rα innate lymphoid cells (ILCs) is unknown.

Objective

We sought to identify CRTH2IL7Rα 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 sequencing, quantitative PCR, adoptive transfer to mice, and measurement of airway hyperreactivity by Flexivent.

Results

We found that IL-5 and IL-13 were expressed not only by CRTH2+ but also by CRTH2IL7Rα+ and CRTH2IL7Rα (double-negative [DN]) human blood and lung cells. All 3 ILC populations expressed type 2 genes and induced airway hyperreactivity when adoptively transferred to mice. The frequency of type 2 cytokine–positive IL7Rα and DN ILCs were similar to that of CRTH2 ILCs in the blood and lung. Their frequency was higher in asthmatic patients than in disease controls. Transcriptomic analysis of CRTH2, IL7Rα, and DN ILCs confirmed the expression of mRNA for type 2 transcription factors in all 3 populations. Unexpectedly, the mRNA for GATA3 and IL-5 correlated better with mRNA for CD30, TNFR2, ICOS, CCR4, and CD200R1 than for CRTH2. By 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 sequencing. 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.

Section snippets

Human subjects

We studied blood and bronchoalveolar lavage (BAL) ILC2s from patients with allergic asthma, disease controls, and healthy donors. Asthmatic patients and disease controls were recruited from the outpatient clinics at National Jewish Health during the period from July 2015 through December 2019. Healthy donors were recruited from the blood bank of National Jewish Health. Bronchoscopy and BAL were performed as a part of their clinical work-up for poorly controlled asthma, as defined by the Expert

Type 2 cytokines are expressed by CRTH2 and IL7Rα ILCs

CRTH2 and IL7Rα are commonly used to detect human ILC2s in the Lincell population of blood or other tissues (the Lin antibody cocktail contained antibodies against CD3, CD14, CD16, CD19, CD20, CD56, and FcεRI). We asked whether CRTH2 and IL7Rα captured all type 2 cytokine–positive ILCs. To this goal, we examined type 2 cytokine expression by peripheral blood CRTH2+IL7Rα, CRTH2+IL7Rα+, CRTH2IL7Rα+, and CRTH2IL7Rα ILC populations (Fig 1, A-J) cultured with IL-2/IL-25. The gating strategy is

Discussion

In this article we showed that the commonly used ILC2 surface marker CRTH2 did not identify all type 2 cytokine–positive ILCs. The CRTH2IL7Rα+ and DN (CRTH2IL7Rα) subpopulations also contained type 2 cytokine–positive ILC2s and expressed the type 2 transcription factors. The CRTH2+ ILC population had the highest frequency of type 2 cytokine–positive ILC2s when compared with the IL7Rα+ and DN populations. However, the CRTH2+ population was much smaller in size than the other 2 populations.

References (55)

  • Y. Simoni et al.

    Human innate lymphoid cell subsets possess tissue-type based heterogeneity in phenotype and frequency

    Immunity

    (2018)
  • G. Wei et al.

    Global mapping of H3K4me3 and H3K27me3 reveals specificity and plasticity in lineage fate determination of differentiating CD4+ T cells

    Immunity

    (2009)
  • T.Y. Halim et al.

    Lung natural helper cells are a critical source of Th2 cell-type cytokines in protease allergen-induced airway inflammation

    Immunity

    (2012)
  • T. Polte et al.

    Direct evidence for a critical role of CD30 in the development of allergic asthma

    J Allergy Clin Immunol

    (2006)
  • H. Maazi et al.

    ICOS:ICOS-ligand interaction is required for type 2 innate lymphoid cell function, homeostasis, and induction of airway hyperreactivity

    Immunity

    (2015)
  • K.J. Cavagnero et al.

    Unconventional ST2- and CD127-negative lung ILC2 populations are induced by the fungal allergen Alternaria alternate

    JACI

    (2019)
  • C.S. Klose et al.

    Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis

    Nat Immunol

    (2016)
  • J.M. Mjosberg et al.

    Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161

    Nat Immunol

    (2011)
  • A.K. Bjorklund et al.

    The heterogeneity of human CD127(+) innate lymphoid cells revealed by single-cell RNA sequencing

    Nat Immunol

    (2016)
  • M. Salimi et al.

    A role for IL-25 and IL-33-driven type 2 innate lymphoid cells in atopic dermatitis

    J Exp Med

    (2013)
  • C.L. Kirkham et al.

    Complexity and diversity of the NKR-P1:Clr (Klrb1:Clec2) recognition systems

    Front Immunol

    (2014)
  • K. Nagata et al.

    Selective expression of a novel surface molecule by human Th2 cells in vivo

    J Immunol

    (1999)
  • L. Cosmi et al.

    CRTH2 is the most reliable marker for the detection of circulating human type 2 Th and type 2 T cytotoxic cells in health and disease

    Eur J Immunol

    (2000)
  • D.J. Jackson et al.

    IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo

    Am J Respir Crit Care Med

    (2014)
  • M.L. Robinette et al.

    IL-15 sustains IL-7R-independent ILC2 and ILC3 development

    Nat Commun

    (2017)
  • L.M. Mora-Velandia et al.

    A human Lin(-) CD123(+) CD127(low) population endowed with ILC features and migratory capabilities contributes to immunopathological hallmarks of psoriasis

    Front Immunol

    (2017)
  • J.G. Quatromoni et al.

    An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells

    J Leukoc Biol

    (2015)
  • Cited by (25)

    • Group 2 innate lymphoid cells in human asthma

      2023, Allergology International
      Citation Excerpt :

      However, CRTH2 might be less useful as a marker for ILC2s because some ILC2-like cells lack CRTH2 expression in peripheral blood.25–27 For example, it has been reported that CRTH2– ILCs expressing CD30 and TNFR2 express GATA3 and produce type 2 cytokines.26 It has also been reported that peripheral blood CD117+ CRTH2– ILCs that express another ILC2-related marker, KLRG1, could upregulate CRTH2 expression in vitro upon IL-7 stimulation,27 indicating that CD117+ CRTH2– ILCs are precursors or immature status of ILC2s.

    • Roles of innate lymphoid cells (ILCs) in allergic diseases: The 10-year anniversary for ILC2s

      2021, Journal of Allergy and Clinical Immunology
      Citation Excerpt :

      Functionally, CCR10− ILC2s showed robust production of type 2 cytokines, whereas CCR10+ ILC2s displayed an ILC1-like phenotype with robust IFN-γ production and little to no IL-5 or IL-13.106 Liu et al107 used IL-5 expression as a functional surrogate for ILC2s and fully characterized ILC2s for their expression of CD127 (IL-7R) and CRTH2 and effector functions in subjects with asthma. CRTH2+CD127+ ILC2s produced type 2 cytokines alone, whereas CRTH2−CD127+ ILC2s and CRTH2−CD127− ILC2s produced IL-17 and IFN-γ, respectively, in addition to type 2 cytokines.

    • Group 2 innate lymphoid cells in nasal polyposis

      2021, Annals of Allergy, Asthma and Immunology
      Citation Excerpt :

      In contrast, other common ILC2 markers such as GATA3, IL-7Ra, and TSLP receptor (cytokine receptor-like factor 2) were expressed more uniformly across all tissues.14 Liu et al15 recently found that some ILC2 subsets in patients with asthma lack cell surface expression of CD127 or CRTH2 but produce type 2 cytokines. Although the heterogeneity of human ILC2s has become clearer, there are no reports about this variability within CRS.

    View all citing articles on Scopus

    Supported by National Institutes of Health grants AI102943, AI137970, and HL126895; grants from the Cohen Family Foundation, the NBL Fellowship Foundation, and Colorado Technology; and the Department of Medicine of National Jewish Health.

    Disclosure of potential conflict of interest: The authors declare that they have no relevant conflicts of interest.

    These authors contributed equally to this work as first authors.

    View full text