Fig. 2: CD8⁺ T cells from MS patients display high sensitivity to natalizumab in vitro exposure.

A Schematic representation of the HCI pipeline applied to pretreatment PBMCs from MS patients. PBMCs were exposed or not to natalizumab, seeded on VCAM-1 coated plates, stained for CD4, CD8, CD14, or CD19, 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⁺, CD8⁺, CD14⁺, and CD19⁺ subpopulations for further analysis. Created in BioRender. Chaves, B. (2025) https:// BioRender.com/3pcr603. B Representative 20x confocal images of PBMCs from a MS patient unexposed (top) or exposed to natalizumab (bottom), seeded on VCAM-1 and stained for F-actin (green), pSLP76 (red) and nuclei (blue). Scale bar: 20 µm. Individual CD4⁺ and CD8⁺ T cells, CD19⁺ B cells and CD14⁺ monocytes are indicated with a white line. Images have been composed from different images to gather representative examples of each cell subset. C–F PCA plots indicative of the effect of natalizumab on CD14⁺ monocytes (C), CD19⁺ B cells (D), CD4⁺ T cells (E), and CD8⁺ T cells (F) based on parameters pertaining to adhesion, morphology, actin, and pSLP76 intensity. Dots correspond to each of the 40 patients with data stemming from the mean of three independent experiments. Gray dots indicate untreated samples, while red dots represent those treated with natalizumab. Data were obtained from 56 to 9776 CD4⁺ T cells, 22 to 8421 CD8⁺ T cells, 9 to 8050 CD19⁺ B cells, and 3 to 321 CD14⁺ monocytes. G Values for individual parameters are shown for CD8⁺ T cells in unexposed (gray dots) and natalizumab-exposed (red dots) samples. Parameters include number of adherent cells, cell area, width to length ratio, pSLP76 mean intensity, and F-actin mean intensity. Dots correspond to each of the 40 patients with data stemming from the mean of three independent experiments. Data were obtained from 56 to 9776 CD4⁺ T cells, 22 to 8421 CD8⁺ T cells, 9 to 8050 CD19⁺ B cells, and 3 to 321 CD14⁺ monocytes. p values were calculated by a two-sided paired t-test used for statistical evaluation. Source data for all panels are provided as a Source Data file.