Cognate CD4⁺ T cells relocate to mIFRs after vaccination. (a) C57BL/6 mice received naive OT-II T cells and were vaccinated with UV-PR8-OTII. OT-II T cell activation in PLNs was analyzed by flow cytometry (n = 4 mice). *, P < 0.05; ***, P < 0.001. Mean ± SEM. (b) CD11c-eYFP mice received labeled naive OT-II T cells and were vaccinated with UV-PR8-OTII. MPM of a PLN 12 h after vaccination. White arrowheads: LNDC/OT-II contacts. Images are representative of three PLNs; two independent trials. (c) OT-II neighbor analysis of PLNs as shown in b. Each symbol type represents an individual PLN, with four XY planes shown. **, P < 0.005. Horizontal bars indicate mean. (d) Heat map of OT-II neighbor counts as shown in c. OT-II cells were identified from images (left) and displayed with color indicative of the number of corresponding OT-II neighbors (right). B, follicles; M, medulla; T, T cell cortex. (e) CD11c-eYFP recipients received labeled naive OT-II T cells and were vaccinated with UV-PR8-OTII. Fluorescent reconstruction of a PLN 24 h after vaccination is shown. Per, PLN “periphery.” Image is representative of three independent trials; two mice/trial. (f) C57BL/6 mice received OT-II T cells and were vaccinated with UV-PR8-OTII. PLNs were collected at 24 h after vaccination, optically cleared, and imaged. Data are represented with medulla isosurfacing to aid visual interpretation. White arrowheads: mIFR/T cell cluster colocalization. Image is representative of two trials; two mice/trial.