Fig. 5: CD8⁺ T cells from non-responder patients are resistant to natalizumab inhibition of cell spreading in an actin polymerization-dependent manner.

A Schematic representation of the functional tests used to assess the differences between responder and non-responder patients. CD8⁺ T cells from responder and non-responder patients were purified and submitted to two distinct functional tests: under-agarose migration and exposure to F-actin inhibitory drugs. Created in BioRender. Chaves, B. (2025) https://BioRender.com/56lb3fy. B Representative snapshots of migrating CD8⁺ T cells from one responder and one non-responder MS patient under-agarose in the presence of VCAM-1. Blue indicates non-responder cells, and red indicates responder cells. Scale bars represent 40 µm. To the right of each image, a representative zoom of cells exposed or not to natalizumab. C Ratio of responder/non-responder cells per homotypic aggregation cluster. Bar represents the average of four responder cells and four non-responder cells exposed to natalizumab after 1 h of migration. Data stems from the mean of two independent experiments per donor. D–F Cell area, cell velocity, and cell straightness of CD8⁺ T cells from four responders and four non-responder MS patients migrating under-agarose in the presence of VCAM-1. Data were obtained from 816 to 1417 CD8⁺ T cells per patient. Bars represent mean values along 2 h of migration. Clinical response to natalizumab treatment is indicated by colors: red dots and bars indicate responders, while blue represents non-responders. Data stems from the mean of two independent experiments. p values were calculated by a Tukey’s multiple comparisons test. G, H Width to length ratio (G) and ARPC1B symmetry 12 (F) features of CD8⁺ T cells from two responder and two non-responder MS patients exposed to natalizumab, stimulated on VCAM-1 and treated with the actin drugs latrunculin B (LAT) or jasplakinolide (JASP) for 10 min where indicated and stained for F-actin (green) and ARPC1B (gold). Red violin plots indicate responders, while blue represents non-responders. Data were obtained from 16 to 1067 cells per condition with two replicate wells. Full lines represent median values, dashed lines represent upper and lower quartiles. p values were calculated by a Dunn’s multiple comparisons test of responder and non-responder samples in the conditions without drug and LAT. Schematic drawings of cell ratio, width to length and ARPC1B symmetry 12 are represented on the side of each corresponding graph. Confocal images were acquired with a 63x objective. Scale bar: 5 µm. Source data for all panels are provided as a Source Data file.