Journal: bioRxiv

Article Title: T-cell signaling relies on partial CD45-exclusion at sub-micron sized cellular contacts

doi: 10.1101/2025.08.29.673015

Figure Lengend Snippet: a, b . Epifluorescence-based widefield imaging of CD8 + T-cells interacting with a monolayer of Lo U-2 OS WT (a) and Lo U-2 OS ICAM-1 (b) cells presenting N-Med ImmTACs. White arrowheads indicate the first detectable close-contacts marked by accumulation of fluorescent CD58-HaloTag expressed by the U-2 OS cells. Images are displayed as maximum-intensity projections. Scale bars, 5 µm. c . Fraction of CD8 + T-cells forming close contacts with N-Med ImmTAC-presenting U-2 OS cells after 5’ (n=3 videos). Error bars show SD. Data were compared using a Welch t test. d . Killing of U-2 OS cells by CD8 + T-cells over time measured using Incucyte imaging. Killing assays were performed using CD8 + T-cells from three donors, in triplicate. Error bars show SEM. Data for Lo U-2 OS WT and Lo U-2 OS ICAM-1 cells at selected time points (t = 3, 6, 9 h) were compared using a Wilcoxon signed-rank test. e . N-Med EC 50 values measured in co-cultures of CD8 + T-cells with U-2 OS WT and U-2 OS ICAM-1 cells. EC 50 values were determined as in Fig. 1; see also Fig. S4. Error bars correspond to the 95% CI. EC 50 values were compared using an F-test of normalised data. Three co-cultures were performed as biological repeats, with measurements in triplicate. Data were collected using CD8 + T-cells from at least two donors. f-n . Image-based analysis of CD8 + T-cells interacting with Lo U-2 OS WT and Lo U-2 OS ICAM-1 cells [n=6 videos each with >20 interacting T cells; comparisons are made with T cells interacting with non ImmTAC-presenting U-2 OS cells (Fig. 2e-g)]. f, g . Average number of close contacts formed per T cell versus time (f) and 5’ after detecting the first close contact (g). h, i . Average summed area of close contacts per T cell versus time (h) and 5’ after detecting the first close contact (i). j, k . Average areas (j) and diameters (k) of all close contacts l . Average area of individual close contacts versus time. m, n . Average cumulative area of the target-cell surface scanned by a single T cell versus time (m) and 5’ after detecting the first close contact (n). Data were compared using a Kruskal-Wallis test and post-hoc Dunn’s test for multiple comparisons. Violin plots indicate median and quartiles. Imaging experiments were performed using CD8 + T-cells from at least two donors.

Article Snippet: Data analysis and processing was carried out using Incucyte GUI software (Sartorius).

Techniques: Imaging

Journal: bioRxiv

Article Title: T-cell signaling relies on partial CD45-exclusion at sub-micron sized cellular contacts

doi: 10.1101/2025.08.29.673015

Figure Lengend Snippet: a . UMAP visualisation of signal intensities for CD8 + T cells isolated from two donors left unstimulated or stimulated with N-Med-presenting A375 cells, PMA/ionomycin, or pervanadate for 1, 5, 15, and 30 min. Intensities were measured by mass cytometry and analysed using CyGNAL. All cells are shown in grey, with overlays colored by treatment and timepoint. Four replicates for each treatment/timepoint condition. Co-cultures were performed as two biological repeats in duplicate. b . Levels of selected signaling effectors in (a). Error bars indicate SEM. c . Proportion of CD69 + T-cells following co-culture with N-Med-presenting A375 cells. n=3; error bars correspond to SEM. Co-culture assays were performed as three biological repeats in triplicate. Data were collected using CD8 + T-cells isolated from three donors. Data was compared using a Mann-Whitney test. d . Killing of A375 cells by CD8 + T-cells over time measured using Incucyte imaging. Killing assays were performed as three biological repeats in triplicate. Data were collected using CD8 + T-cells isolated from three donors.

Article Snippet: Data analysis and processing was carried out using Incucyte GUI software (Sartorius).

Techniques: Isolation, Mass Cytometry, Co-Culture Assay, MANN-WHITNEY, Imaging