CXCR63 is required for efficient signal integration from successive DC encounters. (A–C) GFP⁺ CXCR3^+/+ or CXCR3^−/− OT-I T cells (10⁴/mouse) were transferred into Prf1-deficient mice that contained N4-pulsed DCs. At 84 h after T cell transfer, LNs were isolated and analyzed by SPIM. (A) 3D volume rendering of LYVE-1⁺ lymphatic vessel network (red) and GFP⁺ CXCR3^+/+ or CXCR3^−/− OT-I T cells (green). Higher magnification images show the indicated IFRs (red arrowheads). Bars, 300 µm. Data are representative of two independent experiments with two mice. (B and C) Medulla/cortex ratio (B) and percentage of CXCR3^+/+ and CXCR3^−/− OT-I T cells in IFRs over total cell number (C). Data are pooled from at least two independent experiments with total of 7–10 LNs isolated from four to six mice per condition. CXCR3^+/+ OT-I data are taken from Fig. 4 (D and E). (D) Percentage of FSC^high cells on day 6 after transfer. (E and F) Percentage of CD25⁺ OT-I T cells (E) and CD25 MFI levels (normalized to unpulsed DC-primed CXCR3^+/+ OT-I T cell values; F). (G) Percentage of OT-I T cells on day 6 after a second transfer of unpulsed or N4-pulsed DCs 60 h after T cell transfer as in Fig. 5. (D–G) Data are pooled from three independent experiments with total of 8–12 LNs isolated from four to six mice per condition. (B–G) Bars show the mean, and dots represent data from individual LNs. Statistical significance was analyzed by Student’s t tests. *, P < 0.05; **, P < 0.01.