Interaction with APCs in response to FVIII drives CD4⁺ T cells to the T-B border. (A) Multiphoton IVM of inguinal LNs in C57BL/6 CD11c-DTR/GFP mice. Twenty-four hours before microscopy, CD4⁺ T cells were magnetically sorted from OT-II mice, labeled with CTV, and adoptively transferred to the experimental animals. FOVA (5 μg) was injected intradermally in the groin area 1, 5, or 20 hours (n = 2 per time point) before microscopy. LNs were surgically exposed in anesthetized mice and imaged in vivo on an inverted Leica SP8 confocal/multiphoton microscope with a HCX IRAPO L 25×/0.95 water dipping objective. During imaging, the core body temperature of the animals was maintained at 36°C to 37°C with a temperature controller consisting of a rectal probe and a heating pad. Imaging data were collected and processed using Leica LASX software. Sixty-minute time series were captured with Z-stacks collected once every minute for each time point to a depth of ∼100 μm. The full-field views show whole LNs in animals without a FOVA injection (no antigen) and 1 hour after a FOVA (5 μg) injection, with observable CD11c⁺GFP⁺ cells (green), collagen (gray), and CD4⁺CTV⁺ cells (purple). The schematic diagram indicates presumed LN compartments with areas densely populated by bright CD11c⁺GFP⁺ cells (green) representing the T-cell zones and dark round areas representing the B-cell follicles. (B) Optical section and Z-stack projection views of T-cell zone and B-cell follicle areas, 5 hours after a FOVA (5 μg) injection. White triangles indicate an example CD4⁺ T-cell cluster in the T-cell zone around a CD11c⁺GFP⁺ cell interacting with 2 different T cells, 2 minutes apart. (C) Optical section of the T-B border 20 hours after a FOVA (5 μg) injection.