Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: 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 log 2 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 log 2 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 log 2 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 (αT) or an isotype-matched control (iso) are shown, as indicated. All scale bars = 5 µm. Source data are provided as a Source Data file.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Staining, Control, Incubation

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: 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/μm 2 ), and either CD111 or CD155 (400 molecules/μm 2 ) 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.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Ligation, Expressing, Fluorescence, Microscopy, Control, Imaging, Confocal Microscopy

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: 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/μm 2 ), CD111 or CD155 (400 molecules/μm 2 ) and fluorescently labelled OKT3 (100 molecules/μm 2 ; 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.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Ligation, Expressing, Isolation, Microscopy

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting individual point mutations introduced into TIGIT-SNAP. b Representative confocal microscopy images showing WT and mutant forms of TIGIT-SNAP (green) on the surface of Jurkat T cells conjugated for 20 mins with different Raji B cell populations (either CD111- or CD155-expressing; stained via V5 and shown in magenta). A merged fluorescence-BF image is also provided. c Mean log 2 fold change (±S.D., n = 3 independent experiments) in synaptic TIGIT enrichment in Jurkat T cells, from the conjugates shown in b . Adjusted P values from a one-way ANOVA with Šídák’s multiple comparisons are displayed, with differences from the WT-111 condition displayed in black and the WT-155 condition displayed in grey. d Representative TIRF microscopy images of WT and mutant forms of TIGIT-SNAP at the IS of Jurkat cells that have interacted with PLBs loaded with ICAM-1, and CD111 or CD155 for 20 mins, as in Fig. . Intensities have been scaled equally, and colour scales provided. e Mean degree of TIGIT clustering measured from the images shown in d (±S.D., n = 3–4 independent experiments, as indicated). Adjusted P values from a one-way ANOVA with Dunnett’s multiple comparisons are displayed, and coloured as in c . f ELISA data showing the relative amount of IL-2 released from either parental or different forms of TIGIT-SNAP-expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells. Data is shown as the mean log 2 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 mixed-effects analysis with a Dunnett’s multiple comparison test displayed). Differences from the parental condition are displayed above in black and from the WT condition displayed below in grey. g Western blot analysis of TIGIT using either Phos-tag SDS-PAGE (left) or standard SDS-PAGE to examine TIGIT phosphorylation in Raji-Jurkat conjugates, as labelled above. Data are representative of 3 independent experiments. h Representative TIRF microscopy images of different forms of TIGIT (SNAP labelled; magenta) and the TCR (OKT3 in PLB; green) in Jurkat cells upon interaction with PLBs containing ICAM-1, either CD111 or CD155, and fluorescently labelled OKT3 (100 molecules/μm 2 ), for 10 mins. i Mean Pearson’s correlation coefficient (±S.D; n = ≧50 cells from 2 independent experiments) between TIGIT and OKT3 from the images shown in h . Adjusted P values from a Kruskal-Wallis test with Dunn’s multiple comparisons are shown, and coloured as in c . All scale bars = 5 μm. Source data are provided as a Source Data file.

Article Snippet: Entry clones for the nectin ligands CD111 and CD155 were generated by cloning PCR amplifications (without their STOP codons) from cDNA ORF clones (HG11611-M & HG10109-UT; Sino Biological) into the pDONR/Zeo vector (generating pENTR/Zeo-CD111-NoSTOP and pENTR/Zeo-CD155-NoSTOP, respectively).

Techniques: Confocal Microscopy, Mutagenesis, Expressing, Staining, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Western Blot, SDS Page