IL18 signaling promotes homing of mature Tregs into the thymus
- Department of Immunology, Weizmann Institute of Science, Israel
- Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Spain
- Universitat Pompeu Fabra (UPF), Spain
Figures
Figure 1 with 2 supplements
A subpopulation of mature Tregs in the thymus expresses IL18R.
(a) Top, a heatmap showing Spearman's correlation coefficient between different samples based on the expression of differentially expressed genes. Samples include four biological replicates each of R…
Figure 1—figure supplement 1
Treg gating strategy.
(A) Representative FACS plots demonstrating the Treg gating strategy used to identify newly produced Tregs (GFP+ or CD73-) and mature Tregs (older) (GFP- or CD73+) in the thymus of Foxp3.RFP Rag.GFP …
Figure 1—figure supplement 2
Mature (Rag.GFP–or CD73+) Tregs are intrathymic (not a contamination from the bloodstream).
(A) Representative FACS plots of intravascular CD45 staining followed by regular staining in blood and thymus. The frequency of CD45 intravascular stain (I.V.) is shown from left to right in Tregs …
Figure 2 with 2 supplements
IL18R- and IL18R+ represent molecularly distinct subsets of mature Tregs in the thymus.
(a) A volcano plot showing differential gene expression between thymic Rag.GFP-IL18R- Tregs and Rag.GFP- IL18R+ Tregs. Genes in red have an adjusted p-value <0.05. Y axis is cut at 1e-10 for …
Figure 2—figure supplement 1
RNA Sequencing protein validation, Representative gating from Figure 2c.
(A) Thymocytes of Rag.GFP Foxp3.RFP mice were stained with fluorescently labeled antibodies. The frequency of different markers in newly produced Tregs (Rag.GFP+), mature IL18R- Tregs (Rag.GFP-IL18R-…
Figure 2—figure supplement 2
E-cadherin (the ligand for CD103) is expressed by thymic epithelial cells.
(A) Representative FACS plots showing the expression of E-cadherin (y-axis) plotted against AF (Autofluorescence, natural fluorescence; x-axis) in total thymic epithelial cells (TEC). Numbers …
Figure 3 with 1 supplement
IL18R+ Tregs are resistant to age-dependent thymus involution.
Thymocyte counts as a function of age in weeks. (a) Rag.GFP+ CD4 conventional T cells, (b) Rag.GFP+ Tregs, (c) Rag.GFP- CD4 conventional T cells, and (d) Rag.GFP- IL18R- Tregs (in gray) and Rag.GFP- …
Figure 3—figure supplement 1
Frequency and counts of IL18R+ Rag.
GFP–Tregs at different ages. Frequency (left) and counts (right) of IL18R+ Rag.GFP– Tregs at different ages in Rag.GFP Foxp3.RFP mice strain. Each dot represents an individual mouse. The mean ± SEM …
Figure 4
IL18R+ Tregs are resistant to stress-dependent thymus involution.
Frequencies (left) and counts (right) of ‘newly produced’ CD73- Tregs, ‘mature’ CD73+IL18R- Tregs and ‘mature’ CD73+IL18R+ Tregs in control mice (solid dots) and treated mice (empty dots) upon …
Figure 5 with 4 supplements
IL18R contributes to maintain mature Tregs in the thymus.
(a) Schematic representation of the experiment in which IL18R depleted splenocytes from Foxp3. GFP mice were injected together with non-IL18R depleted splenocytes from Foxp3.RFP into CD45.1 …
Figure 5—figure supplement 1
Purity of the injected splenocytes in the adoptive transfer experiments shown in Figure 5.
(A) Representative FACS plots of pre-depletion samples, including pooled Foxp3.GFP mice splenocytes stained with B220 APC, CD8 APC, and IL18R APC (up) and Foxp3.RFP mice splenocytes stained with …
Figure 5—figure supplement 2
Representative gating from Figure 5a,b,c.
Representative flow cytometry dot plots showing the gating of the injected CD45.2 cells (GFP and RFP) in the thymus (up) or the spleen (down) of CD45.1 recipients. Numbers indicate the percentage of …
Figure 5—figure supplement 3
IL18R– could give rise to IL18R+ Tregs.
Frequency of IL18R in Foxp3.GFP Tregs at the time of injection to the recipient (time 0) and 12 days after the injection (end point). Foxp3.GFP Tregs were IL18R depleted before injection, and …
Figure 5—figure supplement 4
Representative gating from Figure 5d,e,f.
Representative flow cytometry dot plots showing the gating of the injected CD45.2 cells (Il18r1 KO) and CD45.1/2 cells (WT) in the thymus (up) or the spleen (down) of CD45.1 recipients. Numbers …
Figure 6
IL18 does not chemoattract Tregs.
In vitro transwell chemotaxis assay showing migration of splenic Tregs in the presence of IL18 added to the lower wells at indicated doses. SDF-1α (CXCL12) was added at a concentration of 100 ng/mL …
Figure 7 with 1 supplement
IL18/IL18R signaling upregulates CCR6.
(a) A heatmap showing normalized expression of variable genes in splenic Tregs cultured with CD3/CD28 beads and absence/presence of IL18 (0, 10, 100 ng/mL). Selected genes have an adjusted p-value <0…
Figure 7—figure supplement 1
CCR6 upregulation upon IL18 is dependent on IL18R signaling.
Frequency of CCR6 expression in Tregs from Il18r1 KO mice cultured in vitro for 48 hr (left plot) or 96 hr (right plot) at different conditions (from left to right): not stimulated, stimulated with …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-CD3 APC (rat monoclonal) | Biolegend | Cat#100236 | FACS (1uL/test) |
| Antibody | anti-CD3 PerCP/Cy5.5 (rat monoclonal) | Biolegend | Cat#100218 | FACS (1,5uL/test) |
| Antibody | anti-CD4 PB (rat monoclonal) | Biolegend | Cat#100428 | FACS (1uL/test) |
| Antibody | anti-CD4 PECy7 (rat monoclonal) | Biolegend | Cat#100422 | FACS (0,5uL/test) |
| Antibody | anti-CD8a PB (rat monoclonal) | Biolegend | Cat#100725 | FACS (0,5uL/test) |
| Antibody | anti-CD8a APC (rat monoclonal) | Biolegend | Cat#100712 | FACS (0,5uL/test) |
| Antibody | anti-CD25 PE (rat monoclonal) | Biolegend | Cat#101904 | FACS (2uL/test) |
| Antibody | anti-CD25 BV711 (rat monoclonal) | Biolegend | Cat#102049 | FACS (0,5uL/test) |
| Antibody | anti-CD45 PerCP/Cy5.5 (rat monoclonal) | Biolegend | Cat#103132 | 8μg in 200μL PBS iv/mouse |
| Antibody | anti-CD45 unconjugated (rat monoclonal) | Biolegend | Cat#103101 | 1.25μg in 2mL Macs/organ |
| Antibody | anti-CD45.1 APC/Cy7 (mouse monoclonal) | Biolegend | Cat#110715 | FACS (2uL/test) |
| Antibody | anti-CD45.2 PerCP/Cy5.5 (mouse monoclonal) | Biolegend | Cat#109827 | FACS (1uL/test) |
| Antibody | anti-CD45.2 APC (mouse monoclonal) | Biolegend | Cat#109814 | FACS (0,5uL/test) |
| Antibody | anti-CD45R(B220) APC (rat monoclonal) | Biolegend | Cat#103212 | FACS (1uL/test) or 6μL (in total600μL) /spleen for depletion |
| Antibody | anti-CD62L APC/Cy7 (rat monoclonal) | Biolegend | Cat#104427 | FACS (1uL/test) |
| Antibody | anti-CD69 APC/Cy7 (Armenian Hamster monoclonal) | Biolegend | Cat#104525 | FACS (1uL/test) |
| Antibody | anti-CD73 APC (rat monoclonal) | Biolegend | Cat#127210 | FACS (0,3uL/test) |
| Antibody | anti-CD73 PE (rat monoclonal) | Biolegend | Cat#127206 | FACS (0,3uL/test) |
| Antibody | anti-CD103 APC (Armenian Hamster monoclonal) | Biolegend | Cat#121413 | FACS (1uL/test) |
| Antibody | anti-CD184 (CXCR4) APC (rat monoclonal) | Biolegend | Cat#146507 | FACS (0,5uL/test) |
| Antibody | anti-CD196 (CCR6) BV605 (Armenian Hamster monoclonal) | Biolegend | Cat#129819 | FACS (2uL/test) |
| Antibody | anti-CD218a (IL18Ra) PerCPF710 (rat monoclonal) | eBiosciences | Cat#465183-80 | FACS (1uL/test) |
| Antibody | anti-CD218a (IL18Ra) APC (rat monoclonal) | eBiosciences | Cat#175183-82 | FACS (1uL/test) |
| Antibody | anti-CD279 (PD-1) PECy7 (rat monoclonal) | Biolegend | Cat#135215 | FACS (2uL/test) |
| Antibody | anti-Foxp3 AF488 (rat monoclonal) | Biolegend | Cat#126406 | FACS (1uL/test) |
| Antibody | anti-Ki67 BV605 (rat monoclonal) | Biolegend | Cat#652413 | FACS (1uL/test) |
| Antibody | anti-Ly6-G/Ly6-C APC (rat monoclonal) | Biolegend | Cat#108412 | FACS (2uL/test) |
| Other | Fixable viability dye e506 | eBioscience | Cat#65-0866-18 | FACS (0,1uL/test) |
| Other | Dapi | Merck | Cat#10236276001 | 1:1000 RT, 5min, no wash for sorting alive cells |
| Chemical compound, drug | Dexamethasone | Sigma | Cat#4902 | |
| Peptide, recombinant protein | CXCL12 (SDF-1α) | R&D Systems | Cat#460-SD | |
| Peptide, recombinant protein | IL18 | Biolegend | Cat#767002 | |
| Commercial assay or kit | Truenuclear Buffer kit | Biolegend | Cat#424401 | |
| Commercial assay or kit | CD8a Ly-2 Microbeads | Miltenyi | Cat#130-117-044 | |
| Commercial assay or kit | CD4 L3t4 Microbeads | Miltenyi | Cat#130-117-043 | |
| Commercial assay or kit | anti-APC Microbeads | Miltenyi | Cat#130-090-855 | |
| Commercial assay or kit | LD columns | Miltenyi | Cat#130-042-901 | |
| Commercial assay or kit | Lysis/Binding buffer | Invitrogen | Cat#61012 | |
| Commercial assay or kit | Dynabeads | Invitrogen | Cat#61012 | |
| Commercial assay or kit | CD3/CD28 beads | Miltenyi | Cat#130-095-925 | |
| Strain, strain background (M. musculus) | C57/BL6 mice (C57/BL6JOlaHsd) | Envigo-Israel | ||
| Strain, strain background (M. musculus) | Rag.GFP mice (FVB- Tg (Rag2- egfp)1Mnz/J) | The Jackson laboratory | JAX stock 005688 | |
| Strain, strain background (M. musculus) | Foxp3.RFP mice (C57BL/6-Foxp3tm1Flv/J) | The Jackson laboratory Y.Merbl | JAX stock 008374 | |
| Strain, strain background (M. musculus) | Foxp3.GFP mice | G. Shakhar Bettelli et al., 2006 | ||
| Strain, strain background (M. musculus) | CD45.1 mice (B6.SJL-Ptprca Pepcb/BoyJ) | The Jackson laboratory I. Schachar | JAX stock 002014 | |
| Strain, strain background (M. musculus) | Il18r1 KO mice (B6.129P2- Il18r1tm1Aki/J) | The Jackson laboratory | JAX stock 004131 | |
| Software, algorithm | Flow Jo | Treestar | www.flowjo.com | |
| Software, algorithm | Graphpad Prism | Graphpad | www.graphpad.com | |
| Other | counting beads | Spherotech | Cat#ACBP-100-10 | |
| Other | 6.5mm transwells, 5µm pore inserts | Corning | Cat#3421 |
