Tissue-specific shaping of the TCR repertoire and antigen specificity of iNKT cells
- King’s College London, United Kingdom
- The Francis Crick Institute, United Kingdom
- University of Birmingham, United Kingdom
Figures
Figure 1 with 3 supplements
Different TCRVβ usage and clonal expansion in iNKT cells from several lymphoid tissues.
(A) Flow cytometry plots showing gating strategy (left) and quantification (right) for iNKT cells expressing Vβ7, Vβ8.1/8.2 or Vβother (non-Vβ7 or Vβ8.1/8.2) in the depicted tissues of WT C57BL/6. …
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Figure 1—source data 1
Different TCRVβ usage and clonal expansion in iNKT cells from several lymphoid tissues.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Tissue-dependent bias for TCRVβ usage in iNKT cells.
(A) Flow cytometry profiles showing iNKT cells (TCRβ+CD1d-tet-PBS57+) in the depicted tissues of WT and CD1d-KO mice. Numbers indicate percentage of iNKT cells within TCRβ+ cells. (B) Quantification …
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Figure 1—figure supplement 1—source data 1
Tissue-dependent bias for TCRVβ usage in iNKT cells.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig1-figsupp1-data1-v3.xlsx
Figure 1—figure supplement 2
Frequency of TRBV and TRAV gene usage in iNKT cell TCR sequences.
(A) Total number of TRAV and TRBV sequences and percentages of unique sequences obtained from cells on the indicated tissues. (B) Heat map representation of the frequency of TRAV, TRAJ, TRBV and …
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Figure 1—figure supplement 2—source data 1
Frequency of TRBV and TRAV gene usage in iNKT cell TCR sequences.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig1-figsupp2-data1-v3.xlsx
Figure 1—figure supplement 3
Physicochemical properties of CDR3β sequences.
Median values for length, hydrophobicity, pl, and frequency of polar, aromatic or aliphatic aa in CDR3β sequences identified in the depicted tissues. Data are expressed as box-and-whisker diagrams …
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Figure 1—figure supplement 3—source data 1
Physicochemical properties of CDR3β sequences.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig1-figsupp3-data1-v3.xlsx
Figure 2 with 1 supplement
Distinct TCRVβ usage for iNKT RTE.
(A–B) iNKT RTE were identified as GFP+ cells in the tissues of RAG2GFP mice (6–9 weeks/old). (A) Flow-cytometry (left) and quantification (right) showing the percentage of RAG2GFP+ iNKT cells and …
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Figure 2—source data 1
Distinct TCRVβ usage for iNKT RTE.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
TCRVβ usage for iNKT cell precursors.
(A) iNKT cell precursors were identified by flow-cytometry (left top) as CCR7+PD-1- cells. Q2a expression in iNKT cell precursors (empty profile) and non-precursors (grey, rest) is shown (left …
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Figure 2—figure supplement 1—source data 1
TCRVβ usage for iNKT cell precursors.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig2-figsupp1-data1-v3.xlsx
Figure 3 with 1 supplement
Increased activation and proliferation of LN iNKT cells.
(A) Plots show differentially expressed genes for pairwise comparisons from iNKT cells from the depicted tissues. A fold change cut-off of 1.5 and adjusted p-value cut off of 0.01 were applied to …
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Figure 3—source data 1
Increased activation and proliferation of LN iNKT cells.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Gene expression analyses for iNKT cells from various tissues.
(A) Differentially expressed genes upregulated (red) or downregulated (blue) in iNKT cells from LNs vs. iNKT cells from spleen/thymus. A fold change cut-off of 1.5 and adjusted p-value cut off of …
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Figure 3—figure supplement 1—source data 1
Gene expression analyses for iNKT cells from various tissues.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig3-figsupp1-data1-v3.xlsx
Figure 4 with 1 supplement
The tissue of origin dictates the basal activation and TCRβ repertoire of all iNKT subsets.
(A) Analysis of iNKT cell populations in the tissues of WT C57BL/6 mice, showing flow-cytometry plots (A, left) and frequency (A, right) of NKT1 (RORγt-PLZFloT-bet+), NKT2 (RORγt-PLZFhiT-bet-) and …
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Figure 4—source data 1
The tissue of origin dictates the basal activation and TCRβ repertoire of all iNKT subsets.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
TCRVβ usage for iNKT cell subpopulations.
(Top) Analysis of iNKT cell populations in the tissues of WT C57BL/6 mice, showing flow-cytometry plots (left) and frequency (right) of CD4+NK1.1-, NK1.1+CD4-, CD4+NK1.1+ or CD4-NK1.1- iNKT cells. …
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Figure 4—figure supplement 1—source data 1
TCRVβ usage for iNKT cell subpopulations.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig4-figsupp1-data1-v3.xlsx
Figure 5 with 1 supplement
Differential lipid antigen recognition for iNKT cells from various lymphoid tissues.
(A–D) iNKT cells from the depicted tissues were co-stained with CD1d-tet-PBS57 and CD1d-tet-αGlcCer or CD1d-tet-OCH. (A) Flow-cytometry profiles. (B) Quantification of αGlcCer- (left) or OCH- …
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Figure 5—source data 1
Differential lipid antigen recognition for iNKT cells from various lymphoid tissues.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Differential lipid antigen recognition for iNKT cells from non-lymphoid tissues .
(A–D) iNKT cells from liver, lung or SI-LP were co-stained with CD1d-tet-PBS57 and CD1d-tet-αGlcCer or CD1d-tet-OCH. (A) Flow-cytometry profiles. (B) Quantification of αGlcCer- (left) or OCH- …
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Figure 5—figure supplement 1—source data 1
Differential lipid antigen recognition for iNKT cells from non-lymphoid tissues.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig5-figsupp1-data1-v3.xlsx
Figure 6 with 3 supplements
TCR repertoire of iNKT cells changes following immunisation and environmental challenges.
(A–D) Mice were injected with OCH (or PBS as control, c) and iNKT cells from spleen and lymph nodes were analysed 3 or 13 days later. (A) Flow-cytometry profiles showing Ki-67 expression in all iNKT …
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Figure 6—source data 1
TCR repertoire of iNKT cells changes following immunisation and environmental challenges.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Changes in the iNKT cell TCR repertoire following immunisation with αGalCer.
Mice were injected with αGalCer (or PBS as control) and iNKT cells from spleen and lymph nodes were analysed 3 days later. (A) Flow-cytometry profiles showing Ki-67 expression in all iNKT cells …
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Figure 6—figure supplement 1—source data 1
Changes in the iNKT cell TCR repertoire following immunisation with αGalCer.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig6-figsupp1-data1-v3.xlsx
Figure 6—figure supplement 2
Cytokine secretion by iNKT cells relates to TCRVβ usage.
Splenocytes were stimulated in vitro with αGalCer (left, in vitro) or WT mice were injected in vivo with αGalCer (right, in vivo) and cytokine production was measured by intracellular staining 2 hr …
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Figure 6—figure supplement 2—source data 1
Cytokine secretion by iNKT cells relates to TCRVβ usage.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig6-figsupp2-data1-v3.xlsx
Figure 6—figure supplement 3
Frequency of iNKT cells after antibiotic treatment.
Frequency of iNKT cells in the tissues of mice (Thymus = grey; Spleen = yellow; mLN = blue; iLN = red) treated with antibiotics in the drinking water vs control mice. n = 10 mice from two …
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Figure 6—figure supplement 3—source data 1
Frequency of iNKT cells after antibiotic treatment.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig6-figsupp3-data1-v3.xlsx
Figure 7
Distinct phenotype and TCR repertoire for human iNKT cells from different anatomical locations.
(A) Mean percentage of iNKT cells (CD1d-tet-PBS57+CD3+) from total CD3+B220-CD14- cells in blood or tonsils. (B–C) Flow-cytometry (left) and quantification (right) showing the percentage of CD4+, …
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Figure 7—source data 1
Distinct phenotype and TCR repertoire for human iNKT cells from different anatomical locations.
- https://cdn.elifesciences.org/articles/51663/elife-51663-fig7-data1-v3.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Rag2-GFP: Tg(Rag2-EGFP)1Mnz | PMID: 10458165 | MGI:3784416 | |
| Strain, strain background (Mus musculus) | Nur77-GFP: C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J | PMID: 21606508 | MGI:5007644 | |
| Strain, strain background (Mus musculus) | CD1d-KO: Del(3Cd1d2-Cd1d1)1SbpJ | PMID: 14632651 | MGI:5582477 | |
| Antibody | PBS57-loaded CD1d-tetramer (mouse) | NIH Tetramer Core Facility | https://tetramer.yerkes.emory.edu | (1:1000) |
| Antibody | anti-mouse B220 (rat monoclonal) | BioLegend | 103224 | (1:200) |
| Antibody | anti-mouse CD8α (rat monoclonal) | BioLegend | 100714 | (1:200) |
| Antibody | anti-mouse CD11b (rat monoclonal) | BioLegend | 101226 | (1:200) |
| Antibody | anti-mouse CD11c (armenian hamster monoclonal) | BioLegend | 117323 | (1:200) |
| Antibody | anti-mouse PLZF (armenian hamster monoclonal) | BioLegend | 145807 | (1:200) |
| Antibody | anti-mouse RORγt (mouse monoclonal) | BD Biosciences | 564722 | (1:200) |
| Antibody | anti-mouse T-bet (mouse monoclonal) | BioLegend | 644823 | (1:200) |
| Antibody | anti-mouse TCRβ (armenian hamster monoclonal) | BioLegend | 109233 | (1:200) |
| Antibody | anti-mouse Vβ7 (rat monoclonal) | BioLegend | 118306 | (1:200) |
| Antibody | anti-mouse Vβ8.1/8.2 (rat monoclonal) | eBiosciences | 46-5813-80 | (1:200) |
| Antibody | anti-mouse CCR7 (rat monoclonal) | BioLegend | 120105 | (1:100) |
| Antibody | anti-mouse PD1 (rat monoclonal) | BioLegend | 135219 | (1:200) |
| Antibody | anti-mouse Qa2 (mouse monoclonal) | BD Biosciences | 743312 | (1:200) |
| Antibody | anti-mouse CD25 (rat monoclonal) | BioLegend | 102015 | (1:200) |
| Antibody | anti-mouse Ki-67 (rat monoclonal) | BioLegend | 652425 | (1:200) |
| Antibody | anti-mouse ICOS (rat monoclonal) | BioLegend | 117405 | (1:200) |
| Antibody | anti-mouse CD4 (rat monoclonal) | BioLegend | 100433 | (1:200) |
| Antibody | anti-mouse NK1.1 (mouse monoclonal) | eBiosciences | 11-5941-85 | (1:200) |
| Antibody | anti-mouse CD27 (armenian hamster monoclonal) | BioLegend | 124215 | (1:200) |
| Antibody | anti-mouse CCR6 (armenian hamster monoclonal) | BioLegend | 129809 | (1:200) |
| Antibody | anti-mouse CD45.1 (mouse monoclonal) | BioLegend | 110731 | (1:200) |
| Antibody | anti-mouse CD45.2 (mouse monoclonal) | BioLegend | 109805 | (1:200) |
| Antibody | anti-mouse CCR8 (rat monoclonal) | BioLegend | 150320 | (1:200) |
| Antibody | anti-mouse IL-4 (rat monoclonal) | BioLegend | 504111 | (1:200) |
| Antibody | anti-mouse IFN-γ (rat monoclonal) | BioLegend | 505810 | (1:200) |
| Antibody | PBS57-loaded CD1d-tetramer (human) | NIH Tetramer Core Facility | https://tetramer.yerkes.emory.edu | (1:500) |
| Antibody | anti-human CD3 (mouse monoclonal) | BioLegend | 300418 | (1:200) |
| Antibody | anti-human CD4 (rat monoclonal) | BioLegend | 357415 | (1:200) |
| Antibody | anti-human CD8a (mouse monoclonal) | BioLegend | 300913 | (1:200) |
| Antibody | anti-human CD25 (mouse monoclonal) | BioLegend | 302613 | (1:200) |
| Antibody | anti-human CD69 (mouse monoclonal) | BioLegend | 310921 | (1:200) |
| Antibody | anti-human Vα24 (mouse monoclonal) | BioLegend | 360003 | (1:200) |
| Antibody | anti-human Vβ11 (human monoclonal) | Miltenyi Biotech | 130-108-799 | (1:200) |
| Antibody | anti-human CD19 (mouse monoclonal) | BioLegend | 302223 | (1:200) |
| Antibody | anti-human CD14 (mouse monoclonal) | BioLegend | 325615 | (1:200) |
| Commercial assay or kit | Zombie (fixable viability dye) | BioLegend | 423105 | |
| Commercial assay or kit | Dynabeads Biotin binder | Invitrogen | 11047 | |
| Commercial assay or kit | Click-iT Plus EdU Flow-cytometry assay kit | Invitrogen | C10418 | |
| Chemical compound, drug | αGalCer (α-Galactosylceramide, KRN7000) | Enzo Life Sciences | ALX-306–027 | |
| Chemical compound, drug | OCH | Enzo Life Sciences | ALX-306–029 | |
| Software, algorithm | MiXCR | Bolotin et al. (2013) | https://mixcr.readthedocs.io/en/master/ | |
| Software, algorithm | Weblogo | Crooks et al. (2004) | https://weblogo.berkeley.edu | |
| Software, algorithm | Brepertoire | Margreitter et al. (2018) | http://mabra.biomed.kcl.ac.uk/BRepertoire_5/? |
