1. Immunology and Inflammation
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IL18 signaling promotes homing of mature Tregs into the thymus

  1. Cristina Peligero-Cruz
  2. Tal Givony
  3. Arnau Sebé-Pedrós
  4. Jan Dobeš
  5. Noam Kadouri
  6. Shir Nevo
  7. Francesco Roncato
  8. Ronen Alon
  9. Yael Goldfarb
  10. Jakub Abramson  Is a corresponding author
  1. Department of Immunology, Weizmann Institute of Science, Israel
  2. Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Spain
  3. Universitat Pompeu Fabra (UPF), Spain
Research Article
Cite this article as: eLife 2020;9:e58213 doi: 10.7554/eLife.58213
7 figures, 1 table and 1 additional file

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 Rag.GFP- Tregs from the thymus, Tregs from the spleen, and Rag.GFP+ Tregs from the thymus. Bottom, a heatmap showing the normalized expression of variable genes across samples. Specific genes upregulated in Rag.GFP- Tregs are highlighted. (b) Representative flow cytometry plot showing the expression of IL18R in Tregs from the thymus (up) and the spleen (down). Numbers indicate the percentage of cells within each gate. (c) Frequency of IL18R+ cells among ‘newly produced’ Rag.GFPTregs in the thymus (green dots), ‘mature’ Rag.GFP- Tregs in the thymus (white dots) and Tregs in the spleen (gray triangles) in mice from different ages. Each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data are from one representative experiment with three biological replicates. Significant differences were determined using a 2-way ANOVA, corrected for multiple comparisons by the Bonferroni method and indicated by asterisks ***p<0.001.

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 reporter mice or mice without reporter, respectively. (B) Validation of CD73 staining as a marker for mature Tregs in the thymus. Thymocytes of Rag.GFP Foxp3.RFP mice were stained with fluorescently labeled antibodies. The frequency of Rag.GFP+ or Rag.GFP- Tregs from CD73+ and CD73- Treg populations were analyzed. Numbers indicate the percentage of cells within each gate. Over 85% of CD73+ Tregs are Rag.GFP-, while over 98% of CD73- Tregs are RagGFP+ as previously described by Owen et al., 2019. Representative FACS plots from mice 8–10 weeks old are shown.

Figure 1—figure supplement 2
Mature (Rag.GFPor 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 from the blood (positive control) and from the thymus (CD73- Tregs, CD73+IL18R- Tregs, and CD73+ IL18R+ Tregs). Upper panels correspond to CD45 intravascular stained mouse, while lower panels correspond to the FMO (fluorescence minus one control: mouse without CD45 intravascular staining). (B) Frequency of intravascular stained cells. (C) Frequency of Rag.GFP+ Treg, Rag.GFP-Il18R-Treg, and Rag.GFP-IL18R+Treg populations in the thymus of perfused and non-perfused mice. In B and C, each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data are from experiments with two biological replicates (B) and three biological replicates (C).

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 visualization. (b) A heatmap showing the normalized expression of selected variable genes in Rag.GFP+ Tregs, Rag.GFP-IL18R- Tregs, and Rag.GFP-IL18R+ Tregs from the thymus. (c) Frequencies of different markers analyzed using flow cytometry in Rag.GFP+ Tregs, Rag.GFP-IL18RTregs, and Rag.GFP-IL18R+ Tregs from the thymus. Each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data are from one experiment with three biological replicates. Significant differences were determined using ANOVA, corrected for multiple comparisons by the Bonferroni method and indicated by asterisks *p<0.05, **p<0.01, ***p<0.001.

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-) or mature IL18R+ Tregs (Rag.GFP- IL18R+) is shown. (B) Thymocytes of wild-type mice were stained with fluorescently labeled antibodies. The frequency of intracellular Ki67 marker in newly produced Tregs (CD73-), mature IL18R- Tregs (CD73+IL18R-), or mature IL18R+ Tregs (CD73+IL18R+) is shown. Numbers indicate the percentage of cells within each gate.

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 indicate the percentage of E-cadherin positive cells. FMO (fluorescence minus one) control (left) and sample (right) are shown. (B) Frequency of E-cadherin positive cells in different populations of thymic epithelial cells (CD45-EpCAM+): mTECI (Ly51- MHCIIlo Itgb4+), mTECII (Ly51-MHCIIhi), mTECIII (Ly51-MHCIIlo Itgb4-), mTECIV (Ly51-MHCIIlo L1CAM+), and cTEC (Ly51+). Each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data are from one experiment with three biological replicates.

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- IL18R+ Tregs (in blue). Each symbol represents an individual mouse. Data are from two independent experiments with 21 total mice. The Spearman’s rank correlation coefficient (r) and p-value (p) for the correlations between cell counts and mice age are indicated by asterisks ***p<0.001, ns: non-significant.

Figure 3—figure supplement 1
Frequency and counts of IL18R+ Rag.

GFPTregs 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 (standard error of the mean) is shown. Data are from two experiments with three to six biological replicates. Significant differences were determined using ANOVA, corrected for multiple comparisons by the Bonferroni method and indicated by asterisks (*p<0.05,ns: non-significant).

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 different perturbations: (a) 7 days after 425cGy irradiation and (a) 7–10 days after 20 mg/kg dexamethasone injection. Each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data in (a,b) are from a representative experiment with six biological replicates. Significant differences were determined using 2-way ANOVA, corrected for multiple comparisons by the Bonferroni method and indicated by asterisks **p<0.01, ***p<0.001, ns: non-significant.

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 recipients. (b) The ratio of RFP to GFP injected cells found in the spleen and thymus of recipient mice. (c) Frequency of IL18R+ Tregs from the Foxp3.RFP CD45.2 injected cells found in the spleen and thymus of recipient mice. (d) Schematic representation of the experiment in which Il18r1KO splenocytes were injected together with wild-type (wt) splenocytes into CD45.1 recipients. (e) The ratio of wt/Il18r1KO cells found in the spleen and thymus of recipient mice. (f) Frequency of IL18R+ Tregs from the wt injected cells found in the spleen and thymus of recipient mice. (g) Frequencies (left) and counts (right) of different Treg populations in the thymus of wt mice (solid dots) and Il18r1KO mice (empty dots). (h) Counts of Tregs in the spleen of wt and Il18r1KO mice. Each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data in (b,c) are from three independent experiments with a total of 6 biological replicates. (e,f) are from one experiment with a total of 2 biological replicates. Data in (g,h) are from one representative experiment with 12 total mice. Significant differences were determined in (b,c,e,f) using a paired t-test and in (g,h) using an unpaired t-test and indicated by asterisks *p<0.05, **p<0.01, ***p<0.001, ns: non-significant.

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 B220 APC and CD8 APC (down) (B) Representative FACS plots of post-depletion samples composed of a mix of B220-, CD8-, IL18R- depleted Foxp3.GFP splenocytes and B220-, CD8- depleted Foxp3.RFP splenocytes. Numbers indicate the percentage of cells within each gate.

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 cells within each gate.

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 therefore frequencies of IL18R shown at time 0, also correspond to purity of the sorted cells. Each dot represents an individual mouse. The mean ± SEM (standard error of the mean) is shown. Data are from three independent experiments with a total of 6 biological replicates. Significant differences were determined using a paired t-test and indicated by asterisks (**p<0.01).

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 indicate the percentage of cells within each gate.

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 and served as a positive control. The mean ± SEM (standard error of the mean) is shown. Data are from two independent experiments. The chemotaxis index was calculated as % migrated Tregs in the presence of stimulus/% migrated Tregs in the presence of medium alone (no chemokine). Significant differences were determined using ANOVA, corrected for multiple comparisons by the Bonferroni method and indicated by asterisks **p<0.01, ns: non-significant.

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 <0.1 in both 48 and 96 hr treatment versus control tests. Asterisks indicate adjusted p-values in each test *p<0.1, **p<0.01, ***p<0.001. (b) Representative flow cytometry plots showing CCR6 expression in CD25+FOXP3+Tregs (up) or CD25-FOXP3-CD4 T cells (down) cultured for 96 hr with no stimulation, CD3/CD28 beads, or CD3/CD28 beads plus absence/presence of IL18 (0, 10, 100 ng/mL). Numbers indicate the percentage of cells within each gate. (c) Frequency of CCR6 in CD25+FOXP3+Tregs (black dots) and CD25-FOXP3-CD4 T cells (empty rhombus) cultured for 48 or 96 hr with no stimulation, CD3/CD28 beads, or CD3/CD28 beads plus absence/presence of IL18 (0, 10, 100 ng/mL). Data are from two independent experiments with four biological replicates. Significant differences were determined using ANOVA, corrected for multiple comparisons using the Bonferroni method. For Tregs, differences are indicated by asterisks ***p<0.001, ns:non-significant. For CD4 T cells, ANOVA between conditions in both time-points was not significant.

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 CD3/CD28 beads, stimulated with CD3/CD28 beads and IL18 10 ng/mL, and stimulated with CD3/CD28 beads and IL18 100 ng/mL. Data from two replicates are shown. The mean ± SEM (standard error of the mean) is shown.

Tables

Appendix 1—key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Antibodyanti-CD3 APC
(rat monoclonal)
Biolegend
Cat#100236
FACS (1uL/test)
Antibodyanti-CD3 PerCP/Cy5.5
(rat monoclonal)
Biolegend
Cat#100218
FACS (1,5uL/test)
Antibodyanti-CD4 PB (rat monoclonal)Biolegend
Cat#100428
FACS (1uL/test)
Antibodyanti-CD4 PECy7 (rat monoclonal)Biolegend
Cat#100422
FACS (0,5uL/test)
Antibodyanti-CD8a PB (rat monoclonal)Biolegend
Cat#100725
FACS (0,5uL/test)
Antibodyanti-CD8a APC (rat monoclonal)Biolegend
Cat#100712
FACS (0,5uL/test)
Antibodyanti-CD25 PE (rat monoclonal)Biolegend
Cat#101904
FACS (2uL/test)
Antibodyanti-CD25 BV711 (rat monoclonal)
Biolegend
Cat#102049
FACS (0,5uL/test)
Antibodyanti-CD45 PerCP/Cy5.5 (rat monoclonal)Biolegend
Cat#103132
8μg in 200μL PBS iv/mouse
Antibodyanti-CD45 unconjugated (rat monoclonal)Biolegend
Cat#103101
1.25μg in 2mL Macs/organ
Antibodyanti-CD45.1 APC/Cy7 (mouse monoclonal)
Biolegend
Cat#110715
FACS (2uL/test)
Antibodyanti-CD45.2 PerCP/Cy5.5 (mouse monoclonal)
Biolegend
Cat#109827
FACS (1uL/test)
Antibodyanti-CD45.2 APC (mouse monoclonal)
Biolegend
Cat#109814
FACS (0,5uL/test)
Antibodyanti-CD45R(B220) APC (rat monoclonal)
Biolegend
Cat#103212
FACS (1uL/test)
or 6μL (in total600μL)
/spleen for depletion
Antibodyanti-CD62L APC/Cy7 (rat monoclonal)
Biolegend
Cat#104427
FACS (1uL/test)
Antibodyanti-CD69 APC/Cy7 (Armenian Hamster monoclonal)Biolegend
Cat#104525
FACS (1uL/test)
Antibodyanti-CD73 APC (rat monoclonal)Biolegend
Cat#127210
FACS (0,3uL/test)
Antibodyanti-CD73 PE (rat monoclonal)Biolegend
Cat#127206
FACS (0,3uL/test)
Antibodyanti-CD103 APC (Armenian Hamster monoclonal)
Biolegend
Cat#121413
FACS (1uL/test)
Antibodyanti-CD184 (CXCR4) APC (rat monoclonal)
Biolegend
Cat#146507
FACS (0,5uL/test)
Antibodyanti-CD196 (CCR6) BV605 (Armenian Hamster monoclonal)
Biolegend
Cat#129819
FACS (2uL/test)
Antibodyanti-CD218a (IL18Ra) PerCPF710 (rat monoclonal)
eBiosciencesCat#465183-80
FACS (1uL/test)
Antibodyanti-CD218a (IL18Ra) APC (rat monoclonal)
eBiosciencesCat#175183-82
FACS (1uL/test)
Antibodyanti-CD279 (PD-1) PECy7 (rat monoclonal)
Biolegend
Cat#135215
FACS (2uL/test)
Antibodyanti-Foxp3 AF488
(rat monoclonal)
Biolegend
Cat#126406
FACS (1uL/test)
Antibodyanti-Ki67 BV605
(rat monoclonal)
Biolegend
Cat#652413
FACS (1uL/test)
Antibodyanti-Ly6-G/Ly6-C APC (rat monoclonal)
Biolegend
Cat#108412
FACS (2uL/test)
OtherFixable viability dye e506
eBioscience
Cat#65-0866-18
FACS (0,1uL/test)
OtherDapi
Merck
Cat#10236276001
1:1000 RT, 5min, no wash for sorting
alive cells
Chemical compound, drugDexamethasone
Sigma
Cat#4902
Peptide, recombinant proteinCXCL12 (SDF-1α)
R&D Systems
Cat#460-SD
Peptide, recombinant proteinIL18
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, algorithmFlow Jo
Treestar
www.flowjo.com
Software, algorithmGraphpad Prism
Graphpad
www.graphpad.com
Othercounting beads
Spherotech
Cat#ACBP-100-10
Other6.5mm transwells, 5µm pore inserts
Corning
Cat#3421

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