SLAM/SAP signaling regulates discrete γδ T cell developmental checkpoints and shapes the innate-like γδ TCR repertoire
- Department of Surgery, Larner College of Medicine, University of Vermont, United States
- Department of Pathology and Laboratory Medicine, Larner College of Medicine, University of Vermont Medical Center, United States
Figures
Figure 1 with 3 supplements
SLAM family receptor expression marks transcriptionally distinct developmental stages among E17 γδ T cells.
(A) Schematic workflow depicting the methodology for single-cell RNA sequencing (scRNAseq) library preparation and subsequent data analysis pipeline employed in this study. (B) Uniform manifold …
Figure 1—figure supplement 1
Quality control for single-cell CITE-seq.
(A) Representative gating scheme used for FACS sorting γδ cells from B6 (top) and B6.Sh2d1a-/- (bottom) thymus. (B) Left, Violin plots of B6 E17 thymic γδ T cells. nFeaure_RNA represents number of …
Figure 1—figure supplement 2
TCR repertoire profiling of B6 E17 thymus γδ T cells.
(A) Left, TCR clonotype bubble plot depicting the top 100 TCR clonotypes among B6 E17 γδ T cells (n = 2502 γδ T cells). Each bubble represents a unique TRGV/TRDV clonotype; the bubble size …
Figure 1—figure supplement 3
Identification of a BLKnegMAFnegRORγtpos E17 γδ T cell population expressing CD4 and CD8.
(A) Left, representative dot plot of CD24 and CD25 expression on E17 B6 thymic Vγ4 T cells. Right, histograms depicting BLK, PLZF, and RORγt expression in the gated populations at left. The …
Figure 2 with 1 supplement
Identification of SAP-dependent developmental checkpoints during E17 γδ T cell developmental programming.
(A) Uniform manifold approximation and projection (UMAP) representation of B6 (left) and B6.Sh2d1a-/- (right) γδ T cells from E17 thymi (n = 4 mice per group) is shown at left. Clusters were …
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Figure 2—source data 1
TCR clonotype distribution among E.17 B6 and B6.Sh2d1a-/- gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Expression levels of selected genes among individual B6 and B6.Sh2d1a-/- E17 thymic γδ T cell clusters.
(A) Genes whose expression decreased in B6.Sh2d1a-/- immature γδ T cells. (B) Genes whose expression increased in B6.Sh2d1a-/- immature γδ T cells, or whose expression was highest in the αβT …
Figure 3 with 1 supplement
Increased frequency and number of immature CD4+CD8+c-MAF-RORγt+ γδ T cells in B6.Sh2d1a-/- E17 thymus.
(A) Representative contour plots depicting CD24pos E17 γδ T cells in B6 (top) and B6.Sh2d1a-/- (bottom) mice. Concatenated data from two B6 and three B6.Sh2d1a-/- embryos are shown, and are …
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Figure 3—source data 1
Frequency and number of immature CD4+CD8+ cMaf-RORγt+ E17 γδ T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
SAP-dependent regulation of immature BLK+PLZF+RORγt+ and BLK-PLZF+/-RORγt+ E17 thymic γδ T cells.
(A) Total number of E17 thymic γδ T cells in B6 and B6.Sh2d1a-/- E17 thymus. (B) E17 thymic Vγ4 developmental checkpoints. Left, uniform manifold approximation and projection (UMAP) representation …
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Figure 3—figure supplement 1—source data 1
Frequency and number of uniform manifold approximation and projection (UMAP)/FlowSOM clustered E17 thymic Vγ4 T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig3-figsupp1-data1-v1.xlsx
Figure 4 with 3 supplements
Identification of SAP-dependent developmental checkpoints during neonatal γδ T cell developmental programming.
(A) Uniform manifold approximation and projection (UMAP) representation of B6 (left) and B6.Sh2d1a-/- (right) γδ T cells from D9 neonatal thymi (n = 3 mice per strain). Cluster annotation is based …
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Figure 4—source data 1
Comparison of BLK, RORγt, and cMaf expression among neonatal thymic γδ T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Transcriptional heterogeneity in neonatal thymic γδ T cells.
(A) Dot plot showing scaled expression level of selected genes in B6 D9 thymic γδ T cells. Level of normalized expression is shown using a color scale ranging from low (yellow) to high (purple). Dot …
Figure 4—figure supplement 2
Flow cytometric analysis of D9 thymic γδ T cells.
(A) SLAMF6 and SLAMF7 expression defines γδNKT cell subsets. Left, representative dot plot of PLZF and T-bet expression in B6 D9 thymic Vγ1Vδ6.3 γδNKT cells. Representative contour plots of ICOS and …
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Figure 4—figure supplement 2—source data 1
Phenotypic characterization of neonatal thymic γδ T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig4-figsupp2-data1-v1.xlsx
Figure 4—figure supplement 3
scRNAseq with TCR repertoire profiling of B6 and B6.Sh2d1a-/- adult thymic γδ T ells.
(A) Uniform manifold approximation and projection (UMAP) representation of B6 and B6.Sh2d1a-/- adult thymus (6 weeks old) γδ T cells, n = 3 mice per strain. (B) Feature plots illustrating the cell …
Figure 5
SAP shapes the neonatal γδ TCR repertoire.
(Α) TCR clonotype bubble plots depicting the top 100 B6 and B6.Sh2d1a-/- TCR clonotypes among D9 thymic γδ T cells. Each bubble represents a unique clonotype, is sized according to its frequency as …
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Figure 5—source data 1
TCR clonotype distribution among neonatal B6 and B6.Sh2d1a-/- gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig5-data1-v1.xlsx
Figure 6 with 1 supplement
Restricted TCR repertoire in peripheral innate-like γδT1 and γδT17 subsets.
(A) Uniform manifold approximation and projection (UMAP) representation of B6 adult lung γδ T cells. Clusters are annotated based on gene expression and TCR profiling data. (B) Feature plot …
Figure 6—figure supplement 1
Lung Vγ4 exhibit a high usage of TRDV7 chains with a diverse CDR3.
(A) TRDV usage among FACS-sorted lung Vγ4 γδ Τ cells from seven male and six female mice. (B) Left, amino acid length distribution of top lung TRDV5+ CDR3δ clonotypes in the lung Vγ4 γδ T cell …
Figure 7 with 1 supplement
Specific decrease in Vγ4/Vδ7 γδT1 cells in the lungs of SAP-deficient mice.
(A) Uniform manifold approximation and projection (UMAP) representation of B6 (left) and B6.Sh2d1a-/- (right) lung γδ T cells, pooled from 3 mice per strain. (B) SAP-dependent variation in lung γδ T …
Figure 7—figure supplement 1
Significant and specific reduction of peripheral TRGV4/TRDV7+ γδT1 in B6.Sh2d1a-/- mice.
(A) The relative expression of the specified TRDV transcripts in adult lung and spleen CD45+ leukocytes (normalized to total TRDC transcript levels) in B6 and B6.Sh2d1a-/- mice, **p < 0.01, ****p < …
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Figure 7—figure supplement 1—source data 1
Characterization of Vγ4 γδT1 in B6 and B6.Sh2d1a-/- periphery.
- https://cdn.elifesciences.org/articles/97229/elife-97229-fig7-figsupp1-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6J | Jackson Laboratory | Cat#: 000664 | |
| Strain, strain background (Mus musculus) | B6.129S6-Sh2d1atm1Pls/J | Jackson Laboratory | Cat#: 025754 | |
| Biological sample (Mus musculus) | Thymus, spleen, lung | This paper | Freshly isolated tissue | |
| Antibody | Anti-mouse CD16/32, Fc block (rat monoclonal) | Biolegend | Cat#: 101301 | (1:200) |
| Antibody | Anti-CD4-BUV395 (rat monoclonal) | Thermo Fisher | Cat#: 363-0042-82 | (1:500) |
| Antibody | Normal Rat Serum (IgG) | Stem Cell Technologies | Cat#: 13551 | (1:200) |
| Antibody | Anti-CD11b-Alexa Fluor 647 (rat monoclonal) | Biolegend | Cat#: 101220 | (1:1000) |
| Antibody | Anti-CD11c-Alexa Fluor 647 (rat monoclonal) | Biolegend | Cat#: 117314 | (1:200) |
| Antibody | Annexin V Conjugate | Thermo Fisher | Cat#: 35111 | (1:20) |
| Antibody | Anti-CD19-Alexa Fluor 647 (rat monoclonal) | Biolegend | Cat#: 115525 | (1:500) |
| Antibody | Anti-CD24-SB600 (rat monoclonal) | Thermo Fisher | Cat#: 63-0242-82 | (1:200) |
| Antibody | Anti-CD24-BV605 (rat monoclonal) | Biolegend | Cat#: 101827 | (1:400) |
| Antibody | Anti-CD25-PE-Dazzle594 (rat monoclonal) | Biolegend | Cat#: 102048 | (1:500) |
| Antibody | Anti-CD25-Alexa Fluor 488 (rat monoclonal) | Biolegend | Cat#: 102017 | (1:800) |
| Antibody | Anti-CD27-PE-Dazzle594 (hamster monoclonal) | Biolegend | Cat#: 124228 | (1:400) |
| Antibody | Anti-CD44-BV510 (rat monoclonal) | Biolegend | Cat#: 103044 | (1:400) |
| Antibody | Anti-CD45RB-APC-Cy7 (rat monoclonal) | Biolegend | Cat#: 103310 | (1:800) |
| Antibody | Anti-CD73-APC-F750 (rat monoclonal) | Biolegend | Cat#: 127222 | (1:400) |
| Antibody | Anti-CD150-BV650 (rat monoclonal) | Biolegend | Cat#: 115931 | (1:400) |
| Antibody | Anti-CD196-BV785 (hamster monoclonal) | Biolegend | Cat#: 129823 | (1:200) |
| Antibody | Anti-CD319-APC (rat monoclonal) | Biolegend | Cat#: 152004 | (1:800) |
| Antibody | Anti-cMAF-Alexa488 (mouse monoclonal) | Thermo Fisher | Cat#: 53-9855-82 | (1:100) |
| Antibody | Anti-IFNγ-PE (rat monoclonal) | Biolegend | Cat#: 505808 | (1:200) |
| Antibody | Anti-IL17a-BV605 (rat monoclonal) | Biolegend | Cat#: 506927 | (1:200) |
| Antibody | Anti-IL4-PE-Cy7 (rat monoclonal) | Biolegend | Cat#: 504118 | (1:100) |
| Antibody | Anti-PLZF-PE (hamster monoclonal) | Biolegend | Cat#: 145804 | (1:100) |
| Antibody | Anti-T-bet-PE-Dazzle-594 (mouse monoclonal) | Biolegend | Cat#: 644828 | (1:200) |
| Antibody | Anti-TCRb-Alexa647 (hamster monoclonal) | Biolegend | Cat#: 109218 | (1:800) |
| Antibody | Anti-Vγ5-FITC (hamster monoclonal) | BD Biosciences | Cat#: 553229 | (1:300) |
| Antibody | Anti-Vδ6.3-R718 (hamster monoclonal) | BD Biosciences | Cat#: 752197 | (1:250) |
| Antibody | Anti-RORγt-PE-CF594 (mouse monoclonal) | BD Biosciences | Cat#: 562684 | (1:200) |
| Antibody | Anti-TCRδ-BB700 (hamster monoclonal) | BD Biosciences | Cat#: 745818 | (1:100) |
| Antibody | Anti-Vγ1-BV421 (hamster monoclonal) | Biolegend | Cat#: 141116 | (1:300) |
| Antibody | Anti-CD8α-Alexa700 (rat monoclonal) | Thermo Fisher | Cat#: 56-0081-82 | (1:400) |
| Antibody | Anti-CD45-eFluor506 (rat monoclonal) | Thermo Fisher | Cat#: 69-0451-82 | (1:400) |
| Antibody | Anti-CD200-PerCP-eF710 (rat monoclonal) | Thermo Fisher | Cat#: 46-5200-82 | (1:500) |
| Antibody | Anti-CD278-SB645 (rat monoclonal) | Thermo Fisher | Cat#: 64-9942-82 | (1:1000) |
| Antibody | Anti-Ki67-PE-eFluor610 (rat monoclonal) | Thermo Fisher | Cat#: 61-5698-82 | (1:1000) |
| Antibody | Anti-Ly108-BUV615 (13G3 monoclonal) | BD Bioscience | Cat#: 751341 | (1:400) |
| Antibody | Anti-TCR-Vγ4-PE-Cy7 (hamster monoclonal) | Thermo Fisher | Cat#: 25-5828-82 | (1:400) |
| Antibody | Anti-S1P1 (rat monoclonal) | R&D Systems | Cat#: FAB7089P | (1:100) |
| Antibody | Anti-Blk (rabbit polyclonal) | Cell Signaling Technologies | Cat#: 3262S | (1:200) |
| Antibody | Anti-Vdelta1 (17D1) (rat monoclonal) | Willi Born; National Jewish Health, CO, USA | Hybridoma | |
| Sequence-based reagent | GDC1F | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | GC1R1 | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | GC1R2 | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | DC1R1 | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | GDC2F | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | GC2R1 | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | GC2R2 | This paper | Sequence is in Materials and methods | |
| Sequence-based reagent | DC2R1 | This paper | Sequence is in Materials and methods | |
| Commercial assay or kit | Fixable UV LIVE/DEAD Kit | Thermo Fisher Scientific | Cat#: L23105 | (1:400) |
| Commercial assay or kit | BD Horizon Brilliant Staining Buffer | BD Biosciences | Cat#: 566349 | |
| Commercial assay or kit | Invitrogen eBioscience Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent | Thermo Fisher Scientific | Cat#: 50-112-9060 | |
| Commercial assay or kit | BrdU Flow Kit | BD Biosciences | Cat#: 559619 | |
| Commercial assay or kit | Fixable Viability Dye eFluor 780 | Thermo Fisher Scientific | Cat#: 65-0865-14 | (1:1000) |
| Commercial assay or kit | Binding Buffer for Annexin V (10×) | Thermo Fisher Scientific | Cat#: 00-0055-43 | |
| Chemical compound, drug | 5-bromo-deoxyuridine (BrdU) | Sigma-Aldrich | Cat#: 19-160 | |
| Software, algorithm | FlowJo | BD Biosciences | ||
| Software, algorithm | R | R Foundation | RRID:SCR_001905 |
Additional files
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Supplementary file 1
Cluster-specific gene markers for thymic gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-supp1-v1.xlsx
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Supplementary file 2
TCR clonotypes in B6 and B6.Sh2d1a-/- thymic gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-supp2-v1.xlsx
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Supplementary file 3
Pseudobulk analysis of differential gene expression among immature B6 and B6.Sh2d1a-/- thymic gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-supp3-v1.xlsx
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Supplementary file 4
Bulk RNAseq differential gene expression between neonatal immature B6 and B6.Sh2d1a-/- gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-supp4-v1.xlsx
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Supplementary file 5
Cluster-specific gene markers for adult lung gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-supp5-v1.xlsx
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Supplementary file 6
TCR clonotypes in B6 and B6.Sh2d1a-/- lung gamma delta T cells.
- https://cdn.elifesciences.org/articles/97229/elife-97229-supp6-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/97229/elife-97229-mdarchecklist1-v1.pdf
