Innate Lymphoid Cells: ILCs Tools for ILCs research

NK Cells Dendritic Cells Crypt NCR–ILC3 B Cells LTi Cells Dendritic Cells B Cells

LTi Cells + NCR ILC3 Peyer’s Patch LTi cells IEL NK Cells Mesenteric Adipocytes

Villi

ILC2

Dendritic Cells NCR–IL3 LTi Cells

Mesentery

Cross-section of Small Intestine: Anatomical Localization of Innate Lymphoid Cells Innate Lymphoid Cell Development and Categorization Innate lymphoid cells (ILCs) are newly described immune lymphoid cells that are morphologically similar to B cells and T cells but lack rearranged antigen receptors. ILCs secrete high concentrations of and are implicated in innate immunity, inflammation, lymphoid tissue formation, and tissue remodeling. Consistent with their role in immune surveillance and their involvement in early detection of pathogens, ILCs are localized to mucosal surfaces and respond to secreted molecules from the . All ILC populations differentiate from a common lymphoid progenitor (CLP), which is localized to the fetal liver or adult , in response to the expression of specific transcription factors.1 Because ILCs share developmental and functional similarities with helper T (Th) cells, nomenclature for ILCs has been established based on Th cell classification.2 ILCs are categorized into three groups according to the transcription factors mediating their development and the cytokines they secrete. Group-1 ILCs are under the control of the T-bet and include natural killer (NK) cells and ILC1 cells. They secrete type-1 cytokines such as IFN-γ and TNF-α in response to intracellular pathogens. Group-2 ILCs rely on the GATA3 and RORα transcription factors and produce type-2 cytokines (IL-5, IL-9, IL-13) in response to extracellular parasite infections.3,4 Finally, Group-3 ILCs, including Lymphoid Tissues inducer cells (LTi) and ILC3 cells, are under the control of the RORγt transcription factor and produce IL-17 and/or IL-22.5 LTi cells are required for the development of lymphoid tissues, while ILC3 cells mediate the balance between intestinal symbiotic microbiota and immunity. References 1. Bendelac, A. et al. (2014) Nature. 508:397. 4. Wong, S.H. et al. (2012) Nat. Immunol. 13:229. 2. Spits, H. et al. (2013) Nat. Rev. Immunol. 13:145. 5. Luci, C. et al. (2009) Nat. Immunol. 10:75. 3. Hoyler, T. et al. (2012) Immunity. 37:634.

Key: CLP CLP: Common Lymphoid Progenitor Ikaros NKP: NK Cell Precursor iNK: immature NK cell IL-7 mNK: mature NK cell IL-15 iLCP: innate Lymphoid Cell Progenitor ID2 RORγt Notch LTiP: Lymphoid Tissue inducer Progenitor AhR LTi: Lymphoid Tissue inducer cell CLP RunX1 NCR: Natural Triggering NKP IL-7 LTiP Receptor IEL: Intra Epithelial LTi IL-23 iILC2: immature ILC2 IL-1β Transcription Factors IL-2 IL-12 Inducing Cytokines IL-17 Secreted Cytokines IL-22 ILCP LTα/β GATA3 iNK IL-12 ETS-1 IL-7 IL-18 PU.1 ilLC2 ILC1 T-bet

RORα GATA3 IL-12 IL-12 IL-18 IL-23 IL-7 IL-15 IL-25 IL-1β IFNγ IL-33 TSLP mNK T-bet E4BP4 IEL T-bet EOMES EOMES – ILC2 IL-17 NCR ILC3 RORγt E4BP4 IL-22 AhR γ IFN T-bet

IL-4 IFNγ IL-5 — — IL-9 IL-13 T-bet T-bet Cytotoxic Immunoregulatory + IL-2 IL-2 + NK cell NK cell IL-12 IL-23

IFNγ Perforin TNFα Granzyme IL-10 ILC1 NCR+ILC3 Il-13 IFNγ γ IFN IL-22

Innate Lymphoid Cells Lineage-specific pathway.The illustration depicts a model of the hierarchy of innate lymphoid cell differentiation and should be considered neither comprehensive nor definitive. Interact with this graphic | RnDSystems.com/ILCpathway Innate Lymphoid Cell Gating Strategies for Flow Cytometry Group 1 ILCs Flow cytometry is essential for the study of Natural killer cells innate lymphoid cells. It allows Mouse CD3− CD49b+ CD161+ NKp46 + CD27high/low multiparameter analysis of cell populations Human CD3− CD57+ CD56dim/bright CD16dim/− based on the expression of cell surface and/ ILC1 or intracellular molecules. Mouse/Human CD34− CD127+ NKp44−CD117− Consensus gating strategies were IEL established in collaboration with researchers 1,2 Mouse CD45+ CD3ε− CD19− CD161+ NKp46+ CD160+ in the ILC field. Human CD45+ CD3ε− CD19− NKp44+ NKp46+ CD103+ References Group 2 ILCs 1. Fuchs, A. et al. (2013) Immunity. 38:769. 2. Molofsky, A.B. et al. (2013) J. Exp. Med. ILC2 210:535. Mouse/Human CD45+ CD3ε− CD4− CD8− CD19− CD11b− CD161− CD90.2+ T1/ST2+ GATA3high Group 3 ILCs LTi cells Mouse/Human CD4+/− CD127+ Integrin α4β7+ RORγt+ NKp46− CCR6+ NCR− ILC3 Mouse CD45+ CD3− CD19− RORγt+ NKp46− CD161−T-bet+/− Human CD45+ CD3− CD19− CD127+ NKp44+ CCR6− T-bet+/− NCR+ ILC3 Mouse CD45+ CD3− CD19− RORγt+ NKp46+ CD161low/− T-bethigh Human CD45+ CD3− CD19− RORγt+ CD127+ NKp44+ CCR6+T-bethigh

A B C

150 150 150

120 120 120

90 90 90

60 60 60 Relative Cell Number Relative Cell Number Relative Cell Number

30 30 30

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CCR6 CXCR5 NKp46

D E Detection of CCR6, CXCR5, NKp46, Il-17RA and ST2 on Mouse Innate Lymphoid Cells by Flow Cytometry. Single 150 150 cell suspensions were obtained from mouse intestinal epithelium.1. Cells negative for CD3, CD5, B220 were gated on by flow cytometry and stained for expression of 120 120 different ILC markers (filled histograms). Antibodies used include: A) PE-conjugated Rat Anti-Mouse CCR6 90 90 Monoclonal Antibody (Catalog # FAB590P); B) PE-conjugated Rat Anti-Mouse CXCR5 Monoclonal Antibody (Catalog # FAB6198P); C) PE-conjugated Rat 60 60 Anti-Mouse NKp46/NCR1 Monoclonal Antibody (Catalog # Relative Cell Number Relative Cell Number FAB22252P); D) PE-conjugated Goat Anti-Mouse IL-17RA/ IL-17R Antigen Affinity-purified Polyclonal Antibody 30 30 (Catalog # FAB448P); E) PE-conjugated Rat Anti-Mouse ST2/IL-1R4 Monoclonal Antibody (Catalog # FAB10041P). Cells stained with isotype controls are shown for 0 0 comparison (open histograms). 100 101 102 103 104 100 101 102 103 104 IL17RA ST2 1.Protocol adapted from: Hepworth, MR. et al. (2013) Nature. 498:113.

learn more | RnDSystems.com/ILCidentification Detection of Intracellular Cytokines for Flow Cytometry

Fluorochrome-conjugated Antibodies for Flow Cytometry A 30 Molecule Species Fluores- Alexa Fluor APC PE PerCP cein 488 700 25 Group 1 ILCs: NK, ILC1, IEL 20 IFN-γ Human IC285A IC285F IC285P IC285C IC285G Mouse IC485A IC485F IC485P IC485C IC485N 15 TNF-a Human IC210F IC210P

Mouse IC410F IC410P 10

IL-10 Human IC2172A IC2172F IC2172P IC2172C Relative Cell Number

Mouse 5 Group 2 ILCs: ILC2 IL-4 Human IC304A IC304F IC304P IC304C 0 100 101 102 103 104 Mouse NKP46/NCR1 IL-5 Human IC605F IC605P Mouse IC405P B 30 IL-9 Human IC209A Mouse 25 IL-13 Human IC2131A IC2131F IC2131P

Mouse 20 Group 3 ILCs: LTi, NCR+ILC3, NCR−ILC3 IL-17/IL-17A Human IC3171A IC3171P IC3171C IC317G IC317N 15 Mouse IC421F IC421P IC421C IL-17E/IL-25 Human IC1258A IC1258F IC1258P 10 Relative Cell Number Mouse IC13991P 5 IL-22 Human IC7821A IC7821F IC7821P Mouse IC582A IC582F IC582P IC582C 0 -α/ Human IC2111F 100 101 102 103 104 TNF-β CD161 Mouse Detection of NKp46/NKR1 and CD161 on Innate Lymphoid Cells Isolated from Human Peripheral Blood A 104 B 104 Mononuclear Cells (PBMCs). T cells, B cells and monocytes were removed from human PBMCs using the MagCellect Human NK Cell Isolation Kit (Catalog # MAGH109). Cells negative for CD3, CD14, CD19 and 3 3 10 10 CD56 were gated on by flow cytometry and stained for expression of different ILC makers (filled histograms).

2A Antibodies used include: A) PE-conjugated Mouse Anti-Human NKp46/NCR1 Monoclonal Antibody (Catalog 102 102 # FAB1850P); B) PE-conjugated Mouse Anti-Human

CRTH-2 CD161 Monoclonal Antibody (Catalog # FAB7448P). Cells Mouse IgG stained with isotype controls are shown for comparison (open histograms). 101 101

100 100 100 101 102 103 104 100 101 102 103 104 CD3, CD14, CD19, CD56 CD3, CD14, CD19, CD56 Detection of CRTH-2 on Innate Lymphoid Cells isolated from Human Peripheral Blood Mononuclear Cells (PBMCs). T cells, B cells and monocytes were removed from human PBMCs using the MagCellect Human NK Cell Isolation Kit (Catalog # MAGH109). Cells were stained with CD3, CD14, CD19 and CD56 APC and either A) PE-conjugated Mouse

IgG2A Isotype Control (Catalog # IC003P) or a B) PE-conjugated Mouse Anti-Human CRTH-2 Monoclonal Antibody (Catalog # FAB33381P). Quadrants were set based on isotype controls.

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