Defective migration of BATF KO CD4⁺ T cells into the intestine. (A) WT and BATF KO CD4⁺ T cells were activated with RA for 5 d and then added to the upper chamber in a Transwell assay. 3 µg/ml CCL25 was added to the lower chamber, and the cells in the lower chamber were counted after 3 h. The data are shown as percent net migration after normalization to input cells and subtraction of the background migration rates. (B) WT and BATF KO CD4⁺ T cells were activated for 5–6 d with concanavalin A, IL-2, and RA and were then differentially labeled with CFSE and TRITC. The cells were mixed in a 1:1 ratio and injected i.v. into WT mice. (B and C) Numbers of cells in the indicated organs were assessed by flow cytometry (B) or confocal microscopy (C) after 20 h. Bars, 50 µm. (B) The homing index indicates the frequency of KO cells relative to WT cells. (D) The tissues were examined also with multiphoton microscopy. Pooled data from three experiments are shown in A and B. Pooled data from multiple images (3–12) obtained from two independent experiments are shown in C and D. All error bars are SEM obtained from pooled data. Significant differences between WT and BATF KO T cells are shown (*, P < 0.05).