Fig 2: TIGIT clusters are proximal to T-cell receptor (TCR) clusters.a Schematic depicting the model system employed to visualise TIGIT and the TCR at the Immune Synapse (IS) of T cells upon co-ligation. Both Jurkat T cells expressing TIGIT-SNAP, and peripheral blood-isolated primary T cells that express TIGIT endogenously interact with PLBs containing nectin ligands (CD111 or CD155), ICAM-1 and the directly labelled, mono-biotinylated stimulatory TCR antibody OKT3 and imaged with TIRF microscopy. b Video stills of Jurkat T cells expressing TIGIT-SNAP and labelled with dye (magenta) interacting with PLBs containing ICAM-1 (100 molecules/µm2), CD111 or CD155 (400 molecules/µm2) and fluorescently labelled OKT3 (100 molecules/µm2; green), using live TIRF microscopy. Acquisition times are indicated at the top right of each column of images (mins:secs). Brightfield images are shown above. The data are representative of 3 independent experiments. c Kymographs showing a single spatial position, as indicated by the dashed yellow line in b, over time. d Representative TIRF microscopy images showing the relative localisation of TIGIT (antibody labelled; magenta) and the TCR (green) upon interaction with PLBs, as in b, in fixed primary CD4+ and CD8 + T cells at the indicated times. Throughout, scale bars = 5 µm. The data are representative of 3 independent donors. Pearson correlation coefficients (r) are displayed on merged images.

Fig 3: TIGIT assembles into dense, dynamic clusters at the Immune Synapse (IS) in a ligand-dependant manner.a Schematic depicting the model system employed to visualise TIGIT at the IS of T cells upon ligation. TIGIT expressing T cells interact with Planar Lipid Bilayers (PLB) containing laterally mobile ligands and imaged with Total Internal Reflection Fluorescence (TIRF) microscopy. b TIRF microscopy images showing TIGIT-GFP at the IS of Jurkat cells that have interacted with PLBs loaded with ICAM-1 (100 molecules/µm2), and either CD111 or CD155 (400 molecules/µm2) for 20 mins. Cells preincubated with an antagonistic TIGIT antibody or an isotype-matched control are shown, as indicated. c Mean degree of TIGIT clustering measured from the images shown in b (±S.D.; n = 3 independent experiments with adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons shown; ns = not significant). d Representative TIRF microscopy images showing the spatial distribution of TIGIT at the IS of primary CD4+ and CD8 + T cells that have interacted with PLBs loaded with ICAM-1, and the ligands CD111 or CD155 for 20 mins, as in b. In both b and d the fluorescent intensities have been scaled equally, and the colour scales provided. e Mean degree of TIGIT clustering measured from the images shown in d (±S.D., n = 3 individual donors). Adjusted P values from a paired T-test with Holm-Šídák’s multiple corrections are displayed. f Video stills from live TIRF microscopy imaging of Jurkat T cells expressing TIGIT-SNAP interacting with PLBs containing ICAM-1 and either CD111 or CD155 (as in b). Acquisition times are indicated at the top left (mins). g Kymographs showing a single spatial position, as indicated by the dashed yellow line in f, over time. h Zoomed video stills from Jurkat TIGIT-SNAP on PLBs containing ICAM-1 and CD155, from f, displaying occurrences where TIGIT clusters appear to split (top row, yellow arrow) or fuse (bottom row, magenta arrow). Arrows mark specific xy locations, and time intervals are displayed above. i Confocal microscopy images of a FRAP experiment showing the recovery of both CD155-AF647 within the PLB and TIGIT-GFP on the surface of Jurkat cells within clusters. PLBs contain both ICAM-1 and CD155-AF647. Images were taken before photobleaching (Pre-bleach), and at the indicated times (in seconds) following photobleaching (Post-bleach). j FRAP profiles of both CD155-AF647 and TIGIT-GFP from cells measured as shown in i. Data is presented as the mean ±S.D. (n = 11 cells from 2 independent experiments). Scale bars = 5 µm (b, d, f) and 1 µm (h, i). Source data are provided as a Source Data file.

Fig 4: TIGIT accumulates at the Immune Synapse (IS) in a ligand-dependent manner and inhibits T-cell intrinsic activation.a Schematic depicting the model system employed to visualise TIGIT on the surface of T cells when interacting with Raji B cells expressing different nectin ligands. b Flow cytometry analysis showing the expression of TIGIT in Jurkat cells (above) and CD111 and CD155 in Raji cells (below), in both the parental and expression lines together with isotype-matched controls. c Confocal microscopy images showing TIGIT-GFP (green) on the surface of Jurkat cells (T) conjugated for 20 mins with different Raji cell (B) populations, as indicated to the left of the panel. CD19 (yellow) is used to mark Raji cells and a V5 stain labels expressed nectins (magenta). Respective brightfield images (BF) are also provided. The bottom two rows show Jurkat T cells that have been preincubated with either an antagonistic TIGIT antibody or an isotype-matched control. d Mean log2 fold change in synaptic TIGIT enrichment in Jurkat cells, from the conjugates shown in c (±S.D.; n = 3 independent experiments; adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are given; ns = not significant). e Representative confocal microscopy images showing TIGIT (green) on the surface of primary T cells conjugated with different Raji B cell populations, as indicated to the left. CD4 and CD8 (yellow) were stained to mark T cell subsets, and BF provided. f Mean log2 fold change (±S.D., n = 3 independent donors matched by colour) in synaptic TIGIT enrichment in primary T cells, from the conjugates shown in e. Adjusted P values from a paired T-test are given (Holm-Šídák method). g Schematic depicting the model system employed to test the inhibitory effect of TIGIT on the surface of Jurkat T cells when interacting with cells expressing different nectin ligands. Staphylococcal Enterotoxin E (SEE) was used to stimulate Jurkat cells. h Relative amounts of IL-2 released from either parental or TIGIT-SNAP-expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells for 6 h. Data is shown as the mean log2 fold changes between Raji-CD155 conjugates compared to Raji-CD111 conjugates (±S.D., n = 5 independent experiments with adjusted P values from a one-way ANOVA with Holm-Šídák’s multiple comparisons displayed). Cells pre-incubated with an antagonistic TIGIT antibody (aT) or an isotype-matched control (iso) are shown, as indicated. All scale bars = 5 µm. Source data are provided as a Source Data file.

Fig 5: Mass cytometry analysis of peripheral blood and dissociated tumour cell (DTC) samples from renal and lung cancer patients shows limited co-expression of CD226 and TIGIT on T cells.a Frequency (left) and expression level (right) of TIGIT on CD45RA- CD4+ Tregs, CD4 + T cells, or CD8 + T cells in blood (blue) or tumour (red) samples. Relative TIGIT expression intensity was determined by median metal intensity (MMI) of TIGIT+ cells within each subset. Boxes depict the 25th-75th percentile with a line showing the median. Whiskers display minimum to maximum values. n = 14 matched patient samples. b Co-expression of TIGIT and CD226 was investigated on CD45RA- CD4+ Tregs, CD4+ T cells (non-Tregs), and CD8+ T cells from the blood and tumour. Plots display the frequency of singly or coexpressing populations on concatenated files of the matched blood or tumour samples. c Mean frequency of cells co-expressing TIGIT and CD226 in each CD45RA- T cell subset from blood and tumour. n = 14 matched samples (parent populations with <50 events excluded). d UMAP analysis performed on T cells from matched blood and tumour samples (n = 14 of each). Samples with greater than 3,000 T cell events (n = 19) were downsampled to 3,000 events prior to concatenation. UMAP projections show concatenated T cells from the blood (top; n = 35,691 events) or tumour (bottom; n = 42,000 events), highlighting contour (left) or median metal intensity (MMI) of TIGIT, CD226, and CD96 (right). Arbitrary dashed lines are used to highlight visual examples of discrete and co-expression patterns of TIGIT and CD226. e FlowSOM metaclusters were created on T cells concatenated from all matched samples and projected onto the same UMAP as in d. f Expression intensity heatmap of the indicated markers for each of the 12 FlowSOM metaclusters in e. Color scale indicates row-adjusted z-score expression for each marker. g Mean frequency (±SEM) of T cells in each metacluster for each individual matched sample (n = 14). Gray bars represent the frequency of TIGIT+ events (per biaxial gating) within each metacluster (blood and tumour samples are combined; n = 28, except where <50 events in parent population; statistical differences between blood and tumour are listed in Supplementary Table 3). h Plots displaying the correlation of the frequency of CD155 expression on myeloid antigen-presenting cells (mAPC) with the frequency of CD226+ (left), CD96+ (middle), and TIGIT+ (right) cells from CD4+ Tregs (red circles), CD4+ T cells (black circles), or CD8+ T cells (gray squares) in the tumour microenvironment (n = 44). Linear regression lines and Spearman’s Rho (rs) are shown for correlations that were significant (Two-tailed; P < 0.05, as indicated). Non-parametric matched-pairs two-tailed Wilcoxon tests were used to determine differences between PBMCs and DTCs. Source data are provided as a Source Data file.