Figure 1. 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 approximation and projection (UMAP) visualization displaying 11 distinct clusters of E17 B6 thymic γδ T cells, n = 4 individual mice. Clusters are annotated based on comprehensive protein and gene expression data. (C) Feature plots illustrating the cell surface protein expression profiles of CD24, CD73, CD44, CD45RB, SLAMF1, and SLAMF6 on B6 E17 thymic γδ T cells. Each data point represents a cell, color-coded to indicate varying protein expression levels (high: dark blue, low: yellow). (D) Feature plot illustrating the gene expression profiles of signature genes among individual B6 E17 thymic γδ T cells. Each data point represents a cell, color-coded based on gene expression levels (high: purple, low: yellow). (E) Dot plot demonstrating the scaled expression levels of selected genes in E17 B6 thymic γδ T cells. Normalized expression levels are depicted using a color scale ranging from low (yellow) to high (purple). Dot size corresponds to the fraction of cells within each cluster expressing the specific marker. (F) UMAP representation of E17 B6 thymic γδ T cells indicating the expression of selected TRGV (TCRγ; left) and TRDV (TCRδ; right) chain V-segment usage (in blue) across individual cells. (G) Violin plots illustrating the expression patterns of selected genes among E17 B6 thymic γδ T cell clusters. (H) Visualization of single-cell trajectories using PAGA (partition-based graph abstraction) with single-cell embedding (left) showing connectivity between individual nodes (middle). Weighted edges represent statistical measures of interconnectivity. The diffusion pseudotime plot (right) delineates inferred pseudotime progression of cells along developmental trajectories using cluster 5 (C5) as the root, highlighting their developmental order (from purple to yellow).

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 genes detected in each cell. nCount_RNA represents total number of molecules detected within a cell, percent.mt represents the frequency of mitochondrial genes expressed in each cell, and percent.ribo represents the frequency of ribosomal genes expressed in each cell. Middle, ridge plots demonstrating successful enrichment for selected hashtags in E17 thymus γδ T cells following demultiplexing. Right, uniform manifold approximation and projection (UMAP) representations showing distribution of demultiplexed γδ Τ cells among individual B6 E17 thymus samples. (C) Violin plots depicting distribution of cell surface protein oligo-conjugated antibody derived tags (ADTs) among different B6 E17 thymus γδ T cells clusters. (D) Violin plots depicting expression levels of selected genes among individual E17 B6 thymic γδ T cells clusters. (E) Representative dot plots of CD24 and SLAMF7 staining on neonatal thymus γδ T cells. The position of CD44⁺CD45RB⁺ cells is shown in blue. Data are representative of two independent experiments, n = 10 mice.

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 corresponds to the clonotype frequency, and colors correspond to specific TRGV chains utilized. The four most frequent clonotypes are numbered and their corresponding CDR3γ and CDR3δ sequences are displayed below. Right, uniform manifold approximation and projection (UMAP) representations depicting the location of the four most frequent clonotypes E17 thymic γδT cell clusters. (B) Diversity of the E17 γδ TCR repertoire, represented by the Inverse Simpson index. (C) Amino acid length distributions of E17 thymic γδ T cell CDR3γ (top) and CDR3δ (bottom) in immature Blk⁺, Etv5⁺, Maf⁺ C2 and C7 (left), immature RORγt^high C1 (middle), and mature RORγt^high c8 (right) clusters. The top 10 clonotypes are color-coded, all other clonotypes are shown in gray.

Figure 1—figure supplement 3. Identification of a BLK^negMAF^negRORγt^pos 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 geometric mean fluorescence intensity of individual populations is shown. (B) Feature plots showing cell surface protein expression of Vγ1 (top) and Vγ4 (bottom) on B6 E17 thymus γδ T cells. Each point represents a cell, color-coded based on the protein expression level (high: dark blue, low: yellow). (C) Top left, uniform manifold approximation and projection (UMAP) representation of E17 B6 γδ T cell flow cytometry data with selected FlowSOM clusters indicated by color. All other cells are shown in gray. Right, individual dot plots overlaid with the selected FlowSOM clusters from the UMAP plot. Data represent concatenated data from five B6 TCRβ^negTCRδ^posγδ T cells and are representative of two independent experiments. (D) Increasing BLK^negRORγt^pos frequency as thymocytes progress to DN4 stage. Representative contour plots of E17 DN thymocytes with selected gates shown (left) and BLK and RORγt expression associated with each gated population (right). Numbers indicate the percentage of cells in the gated population. (E) Immature Vγ1Vδ1 T cells comprise the major IL17-producing Vγ1 T cell population in E17 thymus. Left, representative contour plot of Vγ1 and Vδ1 (17D1) expression among E17 thymic γδT cells. Middle, Dot plot of CD24 and SSC expression on E17 thymic γδT cells. The position of Vγ1⁺Vδ1⁺ E17 T cells is shown in red. Right, dot plots of IL-17 and IFN-γ expression in E17 thymic Vγ1⁺17D1⁺ and Vγ1⁺17D1⁻ cells after PMA/ionomycin stimulation.