Human Innate Lymphoid Cells: crosstalk with CD4 + regulatory T cells and role in Type 1 Diabetes

Similar documents
Supplemental Figure 1. Signature gene expression in in vitro differentiated Th0, Th1, Th2, Th17 and Treg cells. (A) Naïve CD4 + T cells were cultured

Optimizing Intracellular Flow Cytometry:

Application Information Bulletin: Human NK Cells Phenotypic characterizing of human Natural Killer (NK) cell populations in peripheral blood

CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads (Miltenyi Biotec) for depletion of CD25 +

Measuring Dendritic Cells

Supplemental Figure 1. IL-3 blockade with Fab CSL362 depletes plasmacytoid dendritic cells (pdcs), but not basophils, at higher doses.

ILC2 Cells in the Holsapple Laboratory CRIS Annual Meeting

Optimizing Intracellular Flow Cytometry:

Effector T Cells and

well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±

Supplementary Figure 1. Characterization of basophils after reconstitution of SCID mice

SUPPLEMENTARY INFORMATION

DURACLONE IF BE CERTAIN ABOUT THE RESPONSE. l res. a il n c n. For Research Use Only - Not for use in Diagnostic procedures

Innate immune regulation of T-helper (Th) cell homeostasis in the intestine

Natural Killer Cells: Development, Diversity, and Applications to Human Disease Dr. Michael A. Caligiuri

Generation of ST2-GFP reporter mice and characterization of ILC1 cells following infection

Tolerance, autoimmunity and the pathogenesis of immunemediated inflammatory diseases. Abul K. Abbas UCSF

NMED-A65251A. Supplementary Figures.

Adaptive immune responses: T cell-mediated immunity

SUPPLEMENTARY INFORMATION

New insights into CD8+ T cell function and regulation. Pam Ohashi Princess Margaret Cancer Centre

DNA vaccine, peripheral T-cell tolerance modulation 185

Shiv Pillai Ragon Institute, Massachusetts General Hospital Harvard Medical School

ILC1 and ILC3 isolation and culture Following cell sorting, we confirmed that the recovered cells belonged to the ILC1, ILC2 and

BD Flow Cytometry Reagents Multicolor Panels Designed for Optimal Resolution with the BD LSRFortessa X-20 Cell Analyzer

Type 1 Diabetes: Islet expressing GAD65 (green) with DAPI (Blue) Islet expressing Insulin (red) in 3D confocal imaging

Regulation of anti-tumor immunity through migration of immune cell subsets within the tumor microenvironment Thomas F. Gajewski, M.D., Ph.D.

Autoimmunity. Autoimmunity arises because of defects in central or peripheral tolerance of lymphocytes to selfantigens

Blocking antibodies and peptides. Rat anti-mouse PD-1 (29F.1A12, rat IgG2a, k), PD-

CELLULAR AND MOLECULAR REQUIREMENTS FOR REJECTION OF B16 MELANOMA IN THE SETTING OF REGULATORY T CELL DEPLETION AND HOMEOSTATIC PROLIFERATION

In vitro human regulatory T cell expansion

Targeting tumour associated macrophages in anti-cancer therapies. Annamaria Gal Seminar Series on Drug Discovery Budapest 5 January 2018

Eosinophils are required. for the maintenance of plasma cells in the bone marrow

Immunological alterations in mice irradiated with low doses

Primary Adult Naïve CD4+ CD45RA+ Cells. Prepared by: David Randolph at University of Alabama, Birmingham

In vitro human regulatory T cell expansion

Alessandra Franco, M.D., Ph.D. UCSD School of Medicine Department of Pediatrics Division of Allergy, Immunology and Rheumatology

Supplemental Table I.

The broadest portfolio for your innate lymphoid cell research

D CD8 T cell number (x10 6 )

HD1 (FLU) HD2 (EBV) HD2 (FLU)

Supplemental Figure 1

Relevant Disclosures

Frontiers of Science Foundation Scholar. Toby Bothwell. Freshman, University of Arkansas. final research manuscripts

Monocyte subsets in health and disease. Marion Frankenberger

Cover Page. The handle holds various files of this Leiden University dissertation.

Supplementary Figure 1. mrna expression of chitinase and chitinase-like protein in splenic immune cells. Each splenic immune cell population was

Supplementary Information:

Supporting Information

Naive, memory and regulatory T lymphocytes populations analysis

Supplemental Information. Aryl Hydrocarbon Receptor Controls. Monocyte Differentiation. into Dendritic Cells versus Macrophages

IMMUNOLOGY. Source, Isolate, Culture, And Analyze Immune Cells. Scientists Helping Scientists

ECM1 controls T H 2 cell egress from lymph nodes through re-expression of S1P 1

Fluorochrome Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 CTLA-4 CTLA-4 CD15 CD3 FITC. Bio) PD-1 (MIH4, BD) ICOS (C398.4A, Biolegend) PD-L1 (MIH1, BD)

MATERIALS AND METHODS. Neutralizing antibodies specific to mouse Dll1, Dll4, J1 and J2 were prepared as described. 1,2 All

SUPPLEMENTARY INFORMATION

Supporting Information

Characterization of Il-22-Producing Cells in the Human Thymus and the Mechanism by Which Il-22 Expression Is Modulated by Thymic Epithelial Cells.

Chronic variable stress activates hematopoietic stem cells

Direct ex vivo characterization of human antigen-specific CD154 + CD4 + T cells Rapid antigen-reactive T cell enrichment (Rapid ARTE)

Horizon 2020 Programme. SFS-01b Tackling losses from terrestrial animal diseases

SUPPLEMENTARY METHODS

ankylosing spondylitis Department of Clinical Immunology, Xijing Hospital, The Fourth Military

CyTOF analyses in rheumatoid arthritis. Deepak Rao, MD PhD Rheumatology, Immunology, Allergy, BWH

Tolerance 2. Regulatory T cells; why tolerance fails. Abul K. Abbas UCSF. FOCiS

Commercially available HLA Class II tetramers (Beckman Coulter) conjugated to

Supplementary Figure 1.

Cell-mediated Immunity

Cell isolation. Spleen and lymph nodes (axillary, inguinal) were removed from mice

Supplementary Table 1 Clinicopathological characteristics of 35 patients with CRCs

In vitro human regulatory T cell suppression assay

Rapid antigen-specific T cell enrichment (Rapid ARTE)

Dendritic cell subsets and CD4 T cell immunity in Melanoma. Ben Wylie 1 st year PhD Candidate

Chapter 1. Chapter 1 Concepts. MCMP422 Immunology and Biologics Immunology is important personally and professionally!

Complete Transcriptome Analysis of Latently Infected CD4 + T Cells

SUPPORTING INFORMATIONS

Vasoactive intestinal peptide inhibits IFN-α secretion and modulates the immune function of plasmacytoid dendritic cells

pro-b large pre-b small pre-b CCCP (µm) Rag1 -/- ;33.C9HCki

Comprehensive evaluation of human immune system reconstitution in NSG. and NSG -SGM3 mouse models toward the development of a novel ONCO-HU

IMMUNOTOOLS: EFFECT OF NOTCH-DEFICIENT MACROPHAGES TO AUTOIMMUNE DISEASE WIPAWEE WONGCHANA

X P. Supplementary Figure 1. Nature Medicine: doi: /nm Nilotinib LSK LT-HSC. Cytoplasm. Cytoplasm. Nucleus. Nucleus

Supplementary Figures

Supplementary Figure S1. Flow cytometric analysis of the expression of Thy1 in NH cells. Flow cytometric analysis of the expression of T1/ST2 and

Chitin Activates Parallel Immune Modules that Direct Distinct Inflammatory Responses via Innate Lymphoid Type 2 and T Cells

Control of intestinal inflammation by regulatory T cells

Therapeutic PD L1 and LAG 3 blockade rapidly clears established blood stage Plasmodium infection

The Skinny of the Immune System

Designing An)gen- specific Immunotherapy for Treatment of Type 1 Diabetes.

Nature Immunology: doi: /ni Supplementary Figure 1. Production of cytokines and chemokines after vaginal HSV-2 infection.

Determinants of Immunogenicity and Tolerance. Abul K. Abbas, MD Department of Pathology University of California San Francisco

Hua Tang, Weiping Cao, Sudhir Pai Kasturi, Rajesh Ravindran, Helder I Nakaya, Kousik

Supplementary Figure 1

Mononuclear Phagocytes - Origins, Fates & Functions - Dr. Steffen Jung

T H 1, T H 2 and T H 17 polarization of naïve CD4 + mouse T cells

Antibodies for human plasmacytoïd dendritic cells studies Dendritics SAS, 60 avenue Rockefeller, F Lyon

Supplementary Figure 1. Enhanced detection of CTLA-4 on the surface of HIV-specific

Title of file for HTML: Supplementary Information Description: Supplementary Figures and Supplementary Table

DuraClone IM. Standardized phenotyping panels for studies of the human immune system

n BASIC-TRANSLATIONAL REVIEW

Part III Innate and Adaptive Immune Cells: General Introduction

Transcription:

Joint Lab Meeting 03/03/2015 Human Innate Lymphoid Cells: crosstalk with CD4 + regulatory T cells and role in Type 1 Diabetes Caroline Raffin Bluestone Lab

ILCs: Definition - Common lymphoid progenitor (CLP) - Lymphoid morphology but do not express TCR. - Absence of typical lineage markers on their surface (Lin - ) - 3 groups of ILCs: (including NK cells) Responsive to IL-12 and IL-15 Can be separated into CD4 + and CD4 - subsets Responsive to IL-33 and IL-25 Can be separated into C-kit + and C-kit - subsets Responsive to IL-1β and IL-23 Can be separated into NKp44 + and NKp44 - subsets ILCs: Innate Lymphoid Cells

ILCs: Role in immunity Host defence against infectious agents Tissue homeostasis Preservation of epithelial integrity Development of chronic inflammation: Crohn s disease, atopic dermatitis, asthma, chronic rhinosinusitis, psoriasis. Tumour development: melanoma, human colorectal cancer. Autoimmune disorders: Mutiple Sclerosis (Pery et al., 2012)

ILCs in Type 1 Diabetes ILC2s are among the first lymphoid populations to enter into the pancreas of mice. p < 0.0001 The number of ILC2s in the pancreas of NOD mice in lower than in control mice (C57BL/6) ILC1s ILC2s ILC3s ns ILC2s and ILC3s are altered in the blood of young T1D patients (20-29 years old ; Steve Ziegler s data). ILCs may play a role in T1D onset and/or progression

ILCs in Type 1 Diabetes A defect in Tregs may contribute to the development of T1D. Both Treg and ILCs seem to be altered in NOD mice and young T1D patients.

ILCs: Interaction with CD4 + T cells Cytokine-mediated functional interactions / Competition for limited ligands: responsiveness to common cytokines. (Hughes et al, Immunity 2010 ; Mortha et al. Science 2014) Direct cell-cell contact - ILC2s and ILC3s express MHCII, both in mouse and humans. - ILC2s function in a contact-dependent manner and favor T H 2 polarization while inhibiting T H 1 polarization in mice. (Mirchandani et al, JI, 2014 ; Drake et al, Allergy, 2014) - ILC3s promote intestinal homeostasis and modulate CD4 + T cell function either through direct MHCII-dependent interactions in mice. (Hepworth et al, 2013, Nature) - ILC3s elicite cognate CD4+ T-cell immune responses (CMH-II, co-stim). (von Burg et al, PNAS, 2014) - ICOS/ICOSL interactions - OX40/OX40L interactions Indirectly by interacting with APCs - ILC3s promote intestinal homeostasis and modulate CD4 + T cell function indirectly by acting on dendritic cell (DC) cytokine secretion profile. (Mortha et al., Science, 2014)

Specific Aims A defect in Tregs may contribute to the development of T1D. Both Treg and ILCs seem to be altered in NOD mice and young T1D patients. Is there a crosstalk between ILCs and Tregs in humans? Are ILCs involved in T1D pathogenesis?

ILCs: sorting protocol FSC C-kit Lin C-kit Lin Lineage markers CD1a CD11c CD14 SSC CD127 CD19 4 Blood (less than 0,1% of the cells) Spleen (around 0,5% of the cells) CD123 FcɛRIα ILC3 (5.2) ILC2 (77) ILC3 (84.1) ILC2 (3.4) BDCA2 CD94 TCRαβ TCRγδ ILC1 (18) CRTH2 ILC1 (2.7) CRTH2

Effect of Tregs on ILC3 cytokine production in vitro

C-kit Effect of Tregs on ILC3 cytokine production in vitro Thawed spleen sample Staining for ILCs - Viability ef780 - Lineage FITC - BV510 - CD127 BV650 - CRTH2 PE - CD117 V450 Staining for CD4 + T cells - Viability ef780 - PE - CD8 BV510 - CD45RO PB - CD25 FITC - CD127 BV510 ILC3s CD4 - fraction CD4 + fraction CD45R0 + Treg Co-culture for 1.5 days at ratio 1:1 (with IL-2, IL-7 and IL-15 + beads ratio 1:10) CRTH2

Effect of Tregs on ILC3 cytokine production in vitro IL-17 IL-17 Cytokine production assessment: stimulation with IL-2, IL-1β, IL-23, IL-15 and IL-7 during 7 hours with add of BFA/Monensin after 2 hours. ILC3s alone CD45RO + Tregs alone ILC3s + CD45RO + Tregs 28 69 CD4 CD4 5.6 12.6 IL-22 IL-22

Effect of Tregs on ILC3 cytokine production in vitro IL-17 IL-17 Cytokine production assessment: stimulation with IL-2, IL-1β, IL-23, IL-15 and IL-7 during 7 hours with add of BFA/Monensin after 2 hours. ILC3s alone CD45RO + Tregs alone ILC3s + CD45RO + Tregs 28 69 CD4 CD4 5.6 0.6 12.6 IL-22 IL-22 IL-22

Effect of Tregs on ILC3 cytokine production in vitro IL-17 IL-17 Cytokine production assessment: stimulation with IL-2, IL-1β, IL-23, IL-15 and IL-7 during 7 hours with add of BFA/Monensin after 2 hours. ILC3s alone CD45RO + Tregs alone ILC3s + CD45RO + Tregs 28 69 CD4 CD4 15 10 ILC3 alone ILC3 + MTreg 5 IL-22 5.6 0 % IL-22 % IL-17 %IFN-g ILC3s alone ILC3s + CD45R0 + Tregs IL-22 12.6

Effect of Tregs on ILC3 cytokine production in vitro Count Cytokine production assessment: stimulation with IL-2, IL-1β, IL-23, IL-15 and IL-7 during 7 hours with add of BFA/Monensin after 2 hours. ILC3s alone CD45RO + Tregs alone ILC3s + CD45RO + Tregs 28 69 CD4 CD4 CD4 MFI 611 0.2 MFI 2412 40.8 96.3 MFI 6260 92.5 MFI 10,144

Effect of Tregs on ILC3 cytokine production in vitro Thawed spleen sample Anti-CD4 magnetic microbeads Staining for ILCs - Viability ef780 - Lineage FITC - BV510 - CD127 BV650 - CRTH2 PE - CD117 V450 Staining for CD4 + T cells - Viability ef780 - PE - CD8 BV510 - CD45RO PB - CD25 FITC - CD127 BV510 ILC3s CD4 - fraction CD4 + fraction CD4+ subsets Co-culture for 2.5 days ratio 1:1 (with IL-2, IL-7 and IL-15 + beads ratio 1:10)

Effect of Tregs on ILC3 cytokine production in vitro TCRab Cytokine production assessment: stimulation with IL-2, IL-1β, IL-23 and pma/ionomycin during 7 hours with add of BFA/Monensin after 2 hours. ILC3s alone CD45RO-Tregs alone CD45RO-Tconv alone CD45RO+Tconv alone CD117 0 0.1 96.5 0.1 98.8 0.1 96.5 0.7 65 34.7 3.4 0.0 1.1 0.0 2.8 0.0 IL-22

TCRab Effect of Tregs on ILC3 cytokine production in vitro TCRab TCRab Co-culture ILC3s alone ILC3s + CD45RO + Tregs ILC3s + CD45RO - Tregs CD117 CD117 CD117 0 0.1 31.2 5.3 77.5 0.2 34.4 7.9 91.3 0.1 65 34.7 49.3 14.2 22.1 0.2 43.7 14 8.4 1.0 IL-22 IL-22

TCRab Effect of Tregs on ILC3 cytokine production in vitro TCRab TCRab Co-culture ILC3s alone ILC3s + CD45RO + Tconv ILC3s + CD45RO - Tconv CD117 CD117 CD117 0 0.1 32.6 9.8 89.6 0.6 16.5 7.3 93.2 0.0 65 34.7 42.3 15.3 9.6 0.2 53.2 23.0 6.7 0.0 IL-22 IL-22

TCRab Effect of Tregs on ILC3 cytokine production in vitro TCRab TCRab ILC3s alone without beads ILC3s + CD45RO + Tconv Co-culture without beads ILC3s + CD45RO - Tconv CD117 CD117 CD117 0.2 15.9 5.3 96.4 0.4 0.7 0.2 99.3 0.0 32.3 53.6 25.3 3.2 0.0 73 26.1 0.7 0.0 IL-22 IL-22

Effect of Tregs on ILC3 cytokine production in vitro %+ ILC3s IL-22+ ILC3s with beads 50 45 40 35 30 25 20 15 10 5 0 wo beads wo pma/iono ILC3s alone ILC3s w/ CD45RO+ Tconv ILC3s w/cd45ro- Tconv 40 35 30 25 20 15 10 5 0 ILC3s alone ILC3s w/ CD45RO+ Tconv ILC3s w/cd45ro- Tconv

Effect of Tregs on ILC3 cytokine production in vitro 35 30 25 20 15 10 5 0 % IL-22+ ILC3s alone w/ CD45RO+ Treg w/ CD45RO+ Tconv 40 35 30 25 20 15 10 5 0 alone w/ Treg CD45RO+ % IL-22+ ILC3s w/ Treg CD45RO- w/ Tconv CD45RO+ w/ Tconv CD45RO- % + ILC3s 80 70 60 50 40 30 20 10 0 % + ILC3s alone w/ CD45RO+ Treg w/ CD45RO+ Tconv 45 40 35 30 25 20 15 10 5 0 alone w/ Treg CD45RO+ w/ Treg CD45RO- w/ Tconv CD45RO+ w/ Tconv CD45RO-

Intracellular expression of in ILC3s? (surface) CD45RO+ Tconv ILC3s 0.6 96.6 100 0.1 2.7 (intracellular) Look at and TCR RNA expression levels by qpcr.

ILCs characterization and comparison by RNA Sequencing Healthy Donors Blood vs ILC1 vs CD4 + vs CD4 - vs Spleen ILC2 C-kit + vs C-kit - T1D Donors vs vs (at different stages of the disease) Pancreatic LN ILC3 NKp44 + vs NKp44 - In collaboration with Steve Ziegler s lab.

ILCs characterization and comparison by RNA Sequencing Thawing Lin + cell depletion Sorting by flow cyometry RNA extraction RT RNASeq Core In collaboration with Steve Ziegler s lab.

ILCs characterization and comparison by RNA Sequencing C-kit Thawing Lin + cell depletion Sorting by flow cyometry RNA extraction RT RNASeq Core - Comparison fresh versus frozen samples (PBMCs) Fresh Frozen CRTH2 - Resting O/N or not? In collaboration with Steve Ziegler s lab.

ILCs characterization and comparison by RNA Sequencing Lin Thawing Lin + cell depletion Sorting by flow cyometry RNA extraction RT RNASeq Core Lin staining for MACS depletion PBMCs Spleen 1.2 CD127 8.6 Easy Sep depletion kit (Stem Cell) vs MACS depletion kit (Miltenyi)? In collaboration with Steve Ziegler s lab.

ILCs characterization and comparison by RNA Sequencing FSC Lineage Viability Dye C-kit Lineage CD56 Thawing Lin + cell depletion Sorting by flow cyometry RNA extraction RT RNASeq Core 12.3 84.7 71.9 SSC CD45 ILC3 (88) ILC1 (4) BV510 CD127 CRTH2 CD4 In collaboration with Steve Ziegler s lab.

ILCs characterization and comparison by RNA Sequencing Thawing Lin + cell depletion Sorting by flow cyometry RNA extraction RT RNASeq Core SMARTer Ultra Low Input RNA Kit for Sequencing - v3 cdna Synthesis for Single-Cell Transcriptome Analysis Clontech (low cell number 500 to 1000 cells) In collaboration with Steve Ziegler s lab.

Many thanks to: Bluestone Lab Ziegler Lab: Steven Ziegler Florence Roan

2024 © healthdocbox.com Privacy Policy | Terms of Service | Feedback