OMIP‐055: Characterization of Human Innate Lymphoid Cells from Neonatal and Peripheral Blood

This OMIP panel was designed to characterize the different subsets of human innate lymphoid cells (ILCs) including ILC1, ILC2, and ILC3 within healthy donors' cord blood (CB) (Table 1) in comparison to peripheral blood (PB). As ILCs represent a rare cell population in blood (0.1–0.5% of lymphocytes), the protocol was designed to rigorously exclude the contaminating cells with a complex lineage-depleting antibody mixture (13 different antibodies) enabling analysis of highly pure ILC subsets by 8-color staining. ILCs can be divided into three different subsets: group 1 comprises natural killer (NK) cells and ILC1s, group 2 ILC2, and group 3 ILC3 1. ILCs have been described to have a lymphoid morphology 2 but lack rearranged antigen receptors and can secrete various cytokines 2. ILCs express CD127, however, the cell surface receptors of most hematopoietic lineages (lin) are absent 3. ILC1s are described as CD45+lin−CD127+CD161+CD117−CRTH2−4, ILC2s as CD45+lin−CD127+CD161+CD117+/-CRTH2+ 5, ILC3s as CD45+lin−CD127+CD161+CD117+CRTH2−6, 7, and NK cells as CD45+lin−CD161+CD94+CD56+ 8. Human ILCs have been identified in various tissues 9 including PB 9 and CB 10. For clarity, the term ILCs will only refer to ILC1, ILC2, and ILC3 not to conventional NK cells. The identification of human ILCs is challenging and depends on the tissue analyzed. Within human tissues, the natural cytotoxicity receptor NKp44 is expressed by ILCs 6, 10, while PB ILCs lack NKp44 expression 10. For instance, NKp44+CD103+ILC1s and NKp44+ ILC3 have been identified within human tonsils 6 and intestinal mucosa 11. The presence or absence of NKp44 has also been associated with different functions within ILC3s. NKp44+ILC3s have been described in gut-10 and tonsils6-secreting IL-22 in the latter 6, while NKp44−ILC3s have been identified within “fetal developing lymph nodes” 6 secreting IL17a 6. So far, no NKp44 expression has been described on ILC2s. Of note, although NKp44 is expressed on in vitro activated peripheral NK cells 12, it is generally not found on ex vivo isolated NK cells 12, and consequently is not essential for the characterization of peripheral ILCs. Depending on the nature of the disease, an accumulation of human ILCs within tissues can be harmful or beneficial to the patient. ILC1s have been described to accumulate within the intestine of Crohn's disease patients secreting IFNγ potentially aggravating the inflammation 4. On the other hand, an increase in human donor (NKp44+) ILC3s was seen in leukemic patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and was associated with reduced susceptibility to graft-versus-host disease (GvHD) 13. Thus, human ILC3 may be of therapeutic relevance for GvHD. For future therapeutic purposes, it will be vital to develop protocols isolating defined ILC populations from blood, which represent an easily accessible cell source in comparison to tissue-resident ILCs. In this regard, CB represents a highly attractive alternative to PB, as CB is a neonatal, virus-free, and ethically uncritical source for ILCs. Moreover, ILCs are significantly more frequent in CB compared to PB 14. Thus, the present staining panel was established for identification, further characterization as well as for cell sorting of human CB- and PB-derived ILCs. An eight-color staining panel was designed, which includes a mixture of FITC-coupled lineage-defining antibodies (lin-FITC) to exclude unwanted cell populations. For the characterization of ILCs from peripheral blood, the following antibodies comprise lin-FITC to exclude: T-cells and natural killer T (NKT) cells (anti-CD3, anti-TCRαβ, and anti-TCRγδ), B-cells (anti-CD19 and anti-CD20), dendritic cells (DCs; anti-CD123, anti-BDCA-2, and anti-CD1a), hematopoietic progenitors (anti-CD34), monocytes (anti-CD14), and mast cells as well as basophils (anti- FcεR1α) 3, 15 (Table 2). For ILC isolation from CB, lin-FITC needs to be adjusted, as CB contains high amounts of erythroid precursors, anti-CD235a (Glycophorin A) should be included. Moreover, to isolate CB ILCs in high purity via sorting, we included anti-CD66b in the lineage panel to exclude granulocytes and their progenitors. In our experience, this measure was necessary to quantitatively avoid CB-derived granulocyte contamination during cell sorting, which is critical for further molecular analysis, for example, RNAseq (see online Supporting Information). As our aim was to characterize NK cells in addition to ILCs within our staining panel, we avoided to add antibodies in the lin-FITC panel that excluded NK cells, such as CD11c and CD16 16, 17 as well as CD94, as described by others 3, 4, 18. Furthermore, within our staining, the cell surface receptors CD45 and CD161 were included. Even though all ILC populations in human PB 18 and CB (see online Supporting Information) highly express CD45, CD45 is mandatory for the unequivocal identification of lymphocytes within tissues 5, 6. Therefore, CD45 is especially useful when comparing human tissue with CB ILCs and was thus included in our staining panel. CD161 was described to be expressed on all human ILC subsets during initial characterization 4, 5, 19. The inclusion of CD161 in the staining panel is useful for optimal ILC subset gating, as CB ILC1s do not express CD161, while ILC2 and ILC3 do. Therefore, CD161 serves as an internal gating control. As mentioned before, NKp44 is not expressed on PB ILCs 10 and not on CB ILCs but should be added to the staining panel when analyzing tissue ILCs (Table 2) or allo-HSCT transplanted leukemic patients 13. Further details are provided within the Supporting Information. In summary, the panel includes the following markers for identification of ILCs in CB and PB: lin-FITC, CD117-BV421™, CRTH2-PE/Dazzle™ 594, CD127-PE/Cy5, CD56-BV650™, CD45-APC/Cy7, and CD161-Alexa Fluor® 700. Additionally, CD94-PE/Cy7 is included to discriminate NK cells from other ILCs (Fig. 1). This panel was intentionally kept at a minimum of eight colors, as this provides additional options for customized staining of ILC subpopulations. This panel was tested on cryopreserved and freshly isolated CB samples. Panel design reagents were tested, titrated, and compared to other antibody conjugates and clones. Details on panel development and optimization are included in the online Supporting Information. Exemplified gating strategy and reagents for panel design are shown in Figure 1 and listed in Table 2. CB samples were obtained from healthy volunteer donors after informed consent. This study was approved by the ethics committee of the Medical Faculty of Heinrich-Heine-University. A staining panel for ILCs has not been described yet. OMIP-007 20, OMIP-029 21, and OMIP-039 22 are dedicated to the characterization of NK cells, which are a subset of group 1 ILCs. The authors thank Katharina Raba (Flow Cytometry Core facility of the ITZ) for instrument maintenance, technical support, and critical discussion about the panel development. The authors thank Prof. Dr. rer. nat. Gesine Kögler (José Carreras stem cell bank at the ITZ) for the provision of CBs. The authors thank the healthy CB donors for providing blood samples. This work was supported by funds from the Düsseldorf School of Oncology (funded by the Comprehensive Cancer Center Düsseldorf/Deutsche Krebshilfe and the Medical Faculty HHU Düsseldorf). This work was supported by the Deutsche Forschungsgemeinschaft DFG SPP 1937 (M.U.). The authors declare no competing financial interests. Table S1 Panel antibodies with individual Dilution Factor Online Table S2: Antibodies tested, but not used in final panel Online Table S3: Instrument configuration Cytoflex Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.