Innate Lymphoid Cells: ILCs · 2014. 6. 25. · Innate lymphoid cells (ILCs) are newly described...

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Innate Lymphoid Cells: ILCs Tools for ILCs research Mesenteric Adipocytes Villi Crypt Cross-section of Small Intestine: Anatomical Localization of Innate Lymphoid Cells IEL Dendritic Cells NCR IL3 LTi Cells NK Cells Dendritic Cells NCR ILC3 B Cells LTi Cells Dendritic Cells B Cells LTi Cells Peyer’s Patch LTi cells NK Cells NCR + ILC3 ILC2 Mesentery

Transcript of Innate Lymphoid Cells: ILCs · 2014. 6. 25. · Innate lymphoid cells (ILCs) are newly described...

Page 1: Innate Lymphoid Cells: ILCs · 2014. 6. 25. · Innate lymphoid cells (ILCs) are newly described immune lymphoid cells that are morphologically similar to B cells and T cells but

Innate Lymphoid Cells: ILCsTools for ILCs research

MesentericAdipocytes

Villi

Crypt

Cross-section of Small Intestine: Anatomical Localization of Innate Lymphoid Cells

IEL

Dendritic CellsNCR–IL3LTi Cells

NK CellsDendritic CellsNCR–ILC3B CellsLTi Cells

Dendritic CellsB CellsLTi Cells

Peyer’s PatchLTi cellsNK Cells

NCR+ILC3

ILC2

Mesentery

Page 2: Innate Lymphoid Cells: ILCs · 2014. 6. 25. · Innate lymphoid cells (ILCs) are newly described immune lymphoid cells that are morphologically similar to B cells and T cells but

CLP

ID2Notch

IL-7IL-15

IL-2IL-12

IL-12IL-18

PerforinGranzyme

IFNγTNFαIL-10Il-13

IL-4IL-5IL-9

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IFNγPerforin

Granzyme

IL-17IL-22IFNγ

IL-17IL-22LTα/β

IFNγ

IFNγIFNγIL-22

IL-7

IL-7IL-25IL-33TSLP

IL-12IL-15

IL-12IL-18

IL-23IL-1β

IL-23IL-1β

IL-7

IL-2IL-23

IL-2IL-12

RORαGATA3

GATA3

T-betEOMESE4BP4

T-bet

RORγtAhRT-bet

T-bet

Ikaros

ETS-1PU.1

T-betE4BP4EOMES

CLPLTiP

LTi

ILCP

ILC1

IEL

ILC1

NCR–ILC3

NCR+ILC3

ilLC2

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NKP

iNK

mNK

Cytotoxic NK cell

ImmunoregulatoryNK cell

RORγtAhRRunX1

+

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Interact with this graphic | RnDSystems.com/ILCpathway

Innate Lymphoid Cell Development and CategorizationInnate 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 cytokines 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 epithelium. All ILC populations differentiate from a common lymphoid progenitor (CLP), which is localized to the fetal liver or adult bone marrow, 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 transcription factor 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.

References1. Bendelac, A. et al. (2014) Nature. 508:397.2. Spits, H. et al. (2013) Nat. Rev. Immunol. 13:145.3. Hoyler, T. et al. (2012) Immunity. 37:634.

4. Wong, S.H. et al. (2012) Nat. Immunol. 13:229. 5. Luci, C. et al. (2009) Nat. Immunol. 10:75.

Key:CLP: Common Lymphoid ProgenitorNKP: NK Cell PrecursoriNK: immature NK cellmNK: mature NK celliLCP: innate Lymphoid Cell ProgenitorLTiP: Lymphoid Tissue inducer ProgenitorLTi: Lymphoid Tissue inducer cellNCR: Natural Cytotoxicity Triggering

ReceptorIEL: Intra Epithelial LymphocyteiILC2: immature ILC2

Transcription FactorsInducing CytokinesSecreted Cytokines

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.

Page 3: Innate Lymphoid Cells: ILCs · 2014. 6. 25. · Innate lymphoid cells (ILCs) are newly described immune lymphoid cells that are morphologically similar to B cells and T cells but

learn more | RnDSystems.com/ILCidentification

Flow cytometry is essential for the study of innate lymphoid cells. It allows multiparameter analysis of cell populations based on the expression of cell surface and/or intracellular molecules.

Consensus gating strategies were established in collaboration with researchers in the ILC field.1,2

References1. Fuchs, A. et al. (2013) Immunity. 38:769.2. Molofsky, A.B. et al. (2013) J. Exp. Med. 210:535.

Innate Lymphoid Cell Gating Strategies for Flow CytometryGroup 1 ILCs

Natural killer cells

Mouse CD3− CD49b+ CD161+ NKp46 + CD27high/low

Human CD3− CD57+ CD56dim/bright CD16dim/−

ILC1

Mouse/Human CD34− CD127+ NKp44−CD117−

IEL

Mouse CD45+ CD3ε− CD19− CD161+ NKp46+ CD160+

Human CD45+ CD3ε− CD19− NKp44+ NKp46+ CD103+

Group 2 ILCs

ILC2

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

Detection of CCR6, CXCR5, NKp46, Il-17RA and ST2 on Mouse Innate Lymphoid Cells by Flow Cytometry. Single 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 different ILC markers (filled histograms). Antibodies used include: A) PE-conjugated Rat Anti-Mouse CCR6 Monoclonal Antibody (Catalog # FAB590P); B) PE-conjugated Rat Anti-Mouse CXCR5 Monoclonal Antibody (Catalog # FAB6198P); C) PE-conjugated Rat Anti-Mouse NKp46/NCR1 Monoclonal Antibody (Catalog # FAB22252P); D) PE-conjugated Goat Anti-Mouse IL-17RA/IL-17R Antigen Affinity-purified Polyclonal Antibody (Catalog # FAB448P); E) PE-conjugated Rat Anti-Mouse ST2/IL-1R4 Monoclonal Antibody (Catalog # FAB10041P). Cells stained with isotype controls are shown for comparison (open histograms).1.Protocol adapted from: Hepworth, MR. et al. (2013) Nature. 498:113.

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Detection of NKp46/NKR1 and CD161 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 negative for CD3, CD14, CD19 and CD56 were gated on by flow cytometry and stained for expression of different ILC makers (filled histograms). Antibodies used include: A) PE-conjugated Mouse Anti-Human NKp46/NCR1 Monoclonal Antibody (Catalog # FAB1850P); B) PE-conjugated Mouse Anti-Human CD161 Monoclonal Antibody (Catalog # FAB7448P). Cells stained with isotype controls are shown for comparison (open histograms).

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.

Detection of Intracellular Cytokines for Flow CytometryA

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Molecule Species

Fluorochrome-conjugated Antibodies for Flow Cytometry

APC Fluores-cein PE PerCP

Alexa Fluor

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Group 1 ILCs: NK, ILC1, IEL

IFN-γ Human IC285A IC285F IC285P IC285C IC285G

Mouse IC485A IC485F IC485P IC485C IC485N

TNF-α Human IC210F IC210P

Mouse IC410F IC410P

IL-10 Human IC2172A IC2172F IC2172P IC2172C

Mouse

Group 2 ILCs: ILC2

IL-4 Human IC304A IC304F IC304P IC304C

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IL-5 Human IC605F IC605P

Mouse IC405P

IL-9 Human IC209A

Mouse

IL-13 Human IC2131A IC2131F IC2131P

Mouse

Group 3 ILCs: LTi, NCR+ILC3, NCR−ILC3

IL-17/IL-17A Human IC3171A IC3171P IC3171C IC317G IC317N

Mouse IC421F IC421P IC421C

IL-17E/IL-25 Human IC1258A IC1258F IC1258P

Mouse IC13991P

IL-22 Human IC7821A IC7821F IC7821P

Mouse IC582A IC582F IC582P IC582C

Lymphotoxin-α/TNF-β

Human IC2111F

Mouse