TCR affinity governs OT-I T cell localization during expansion. (A–E) LNs were isolated 24 or 84 h after transfer of 10⁴ GFP⁺ OT-I T cells into Prf1-deficient mice containing N4-, Q4-, or T4-pulsed DCs and analyzed by SPIM. (A) 3D volume rendering of the HEV network (gray) and GFP⁺ OT-I T cells (green) at 24 h (only for N4) and 84 h in LNs containing N4-, Q4-, or T4-pulsed DCs. Higher magnification images show OT-I T cell localization in the T cell zone (24-h LNs) or IFRs (84-h LNs; red arrowheads). Bars, 300 µm. Data are representative of three independent experiments with four mice at 84 h and one experiment at 24 h after T cell transfer. (B) Total OT-I T cell numbers per LN 84 h after T cell transfer. (C) 3D quantification scheme of SPIM-generated datasets. Cortical position (P_c), medullary position (P_m), and cortical-medullary position (P_cm), together with the polar angle θ fixed at 20°, were used to subdivide 3D LN renderings into microenvironments as described in Fig. S1. (D) The percentage of total OT-I T cells that was detected within medulla, cortex, T cell zone, and IFRs. (E) Medulla/cortex ratio of OT-I T cells. (B–E) Data are pooled from three independent experiments with total of five to six LNs isolated from total of four mice per condition. (B, D, and E) Each dot represents one LN. Statistical significance was analyzed by Kruskal-Wallis tests with Dunn’s posttest. *, P < 0.05; **, P < 0.01.