Fig. 1: Automated cell imaging of healthy donor peripheral blood T cells upon in vitro natalizumab exposure.

A Schematic representation of the HCI pipeline applied to PBMCs from healthy donors. PBMCs were exposed or not to natalizumab, seeded on VCAM-1 coated plates, stained for CD4 or CD8, F-actin and pSLP76 and imaged with an automated confocal microscope. Parameters pertaining to cell adhesion, cell shape, F-actin and pSLP76 were extracted separately from CD4⁺ and CD8⁺ subpopulations for further analysis. Created in BioRender. Chaves, B. (2025) https://BioRender.com/3pcr603. B, C Representative 40x confocal images of CD4⁺ (B) and CD8⁺ (C) T cells, exposed or not to natalizumab, seeded on VCAM-1 and stained for F-actin (green), pSLP76 (red), and nuclei (blue). Scale bar: 5 µm. PCA plots indicative of the effect of natalizumab on CD4⁺ (B) and CD8⁺ (C) T cells, based on parameters pertaining to adhesion, morphology, actin and pSLP76 intensity. Dots correspond to ten individual donors with data stemming from the mean of two independent experiments, each with two replicate wells. Gray dots indicate untreated samples, while red dots represent those treated with natalizumab. Data were obtained from 20 to 189 CD4⁺ T cells and 14 to 520 CD8⁺ T cells per donor. D, E Values for individual parameters are shown for CD4⁺ T cells (D) and CD8⁺ T cells (E) in unexposed (gray dots) and natalizumab-exposed (red dots) samples. Parameters include the number of adherent cells, cell area, width-to-length ratio, pSLP76 mean intensity, and F-actin mean intensity. Dots correspond to ten individual donors with data stemming from the mean of two independent experiments, each with two replicate wells. Data were obtained from 20 to 189 CD4⁺ T cells and 14 to 520 CD8⁺ T cells per donor. p values were calculated by a two-sided paired t- test. Source data for all panels are provided as a Source Data file.