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Fine-tuning T cell activation through nanopore diameter variation. (a) Schematic illustrating a T cell spreading on a nanoporous surface with microvilli extending into individual nanopores. (b) Orthogonal view of a naive primary human T cell on a nanoporous surface with the plasma membrane stained using Wheat Germ Agglutinin (WGA, green), imaged by Airyscan microscopy. The x-y view shows the first z-slice within the nanoporous surface, revealing microvilli cross sections as dot-like structures inside individual pores. The red arrow in the x-z view highlights a microvillus protruding into a nanopore. (c) SEM image of a nanoporous anodic aluminum oxide substrate with a 239 nm median pore diameter, referred to as a Medium nanoporous substrate. (d) Violin plot showing the distributions of nanopore diameters in anodic aluminum oxide substrates with varying pore sizes. The median pore diameter values are indicated above the violin plots. (e) Schematic illustrating the experimental setup for T cell activation using nanoporous substrates. Substrates are placed in a multiwell plate, and T cells are seeded on top of the substrates in T cell culture medium. (f) Activation induced in naive primary human T cells (CD8 + and CD4+), shown as the percentage of CD69 + T cells 24 h after seeding on the indicated substrates, measured by flow cytometry. Bars indicate the mean ± SD across donors (D; n _D = 4–9). Symbols represent mean values of technical replicates per donor. Statistical analysis was performed on per-donor mean values from donors present in both conditions using a paired t test with Benjamini–Hochberg (FDR) adjustment for multiple comparisons. Significance symbols represent adjusted p-values: ns: p ≥ 0.05, **: p < 0.01, and ****: p < 0.0001. (g) Cross section (x-z) of Airyscan microscopy images of representative T cells on Anodisc nanoporous substrates with small (107 nm) and medium-sized (229 nm) median nanopore diameters. CD45 (magenta) is visualized via antibody staining, and the membrane is stained using WGA (green). The dotted line marks the beginning of the nanopores, also marked by the arrow on the side. The white arrows indicate microvilli extending into nanopores. (b, c, g) Scale bar = 1 μm. Plots are isotropic in the x, y, and z directions.