Human Innate Lymphoid Cells: crosstalk with CD4 + regulatory T cells and role in Type 1 Diabetes
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1 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
2 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
3 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)
4 ILCs in Type 1 Diabetes ILC2s are among the first lymphoid populations to enter into the pancreas of mice. p < 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
5 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.
6 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)
7 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?
8 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
9 Effect of Tregs on ILC3 cytokine production in vitro
10 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
11 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 CD4 CD IL-22 IL-22
12 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 CD4 CD IL-22 IL-22 IL-22
13 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 CD4 CD ILC3 alone ILC3 + MTreg 5 IL % IL-22 % IL-17 %IFN-g ILC3s alone ILC3s + CD45R0 + Tregs IL
14 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 CD4 CD4 CD4 MFI MFI MFI MFI 10,144
15 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)
16 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 CD IL-22
17 TCRab Effect of Tregs on ILC3 cytokine production in vitro TCRab TCRab Co-culture ILC3s alone ILC3s + CD45RO + Tregs ILC3s + CD45RO - Tregs CD117 CD117 CD IL-22 IL-22
18 TCRab Effect of Tregs on ILC3 cytokine production in vitro TCRab TCRab Co-culture ILC3s alone ILC3s + CD45RO + Tconv ILC3s + CD45RO - Tconv CD117 CD117 CD IL-22 IL-22
19 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 CD IL-22 IL-22
20 Effect of Tregs on ILC3 cytokine production in vitro %+ ILC3s IL-22+ ILC3s with beads wo beads wo pma/iono ILC3s alone ILC3s w/ CD45RO+ Tconv ILC3s w/cd45ro- Tconv ILC3s alone ILC3s w/ CD45RO+ Tconv ILC3s w/cd45ro- Tconv
21 Effect of Tregs on ILC3 cytokine production in vitro % IL-22+ ILC3s alone w/ CD45RO+ Treg w/ CD45RO+ Tconv alone w/ Treg CD45RO+ % IL-22+ ILC3s w/ Treg CD45RO- w/ Tconv CD45RO+ w/ Tconv CD45RO- % + ILC3s % + ILC3s alone w/ CD45RO+ Treg w/ CD45RO+ Tconv alone w/ Treg CD45RO+ w/ Treg CD45RO- w/ Tconv CD45RO+ w/ Tconv CD45RO-
22 Intracellular expression of in ILC3s? (surface) CD45RO+ Tconv ILC3s (intracellular) Look at and TCR RNA expression levels by qpcr.
23 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.
24 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.
25 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.
26 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 CD Easy Sep depletion kit (Stem Cell) vs MACS depletion kit (Miltenyi)? In collaboration with Steve Ziegler s lab.
27 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 SSC CD45 ILC3 (88) ILC1 (4) BV510 CD127 CRTH2 CD4 In collaboration with Steve Ziegler s lab.
28 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.
29 Many thanks to: Bluestone Lab Ziegler Lab: Steven Ziegler Florence Roan
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