Androgen withdrawal increases CCL25 production from UEA⁺ medullary TEC; CCL25 production is critical for thymic expansion after castration. Thymi were removed from 3 mice per group and TEC digestion performed to isolate epithelial populations for mRNA analysis. Whole thymus lysates from 7 mice in each group was used for protein studies. (A) RT-PCR demonstrated increased CCL25 production in castrated mice 2 weeks after castration (data representative of 3 separate experiments with confirmation of similar TEC enrichment by flow cytometry). (B) Western blot confirmed enhanced CCL25 protein production after castration in whole thymus lysates using Keratin 8 to normalize epithelial cell numbers (graph includes normalized data from 3 mice in each group with standard error bars; CCL25 protein was also increased compared with actin, data not shown; data are representative of 2 separate experiments, 6-7 thymi per group total). These images were developed with a Kodak GelLogic 2200 camera using Kodak Molecular Imaging Software 4.5.1SE. (C) Sorted epithelial populations (from 6 thymi per group) confirmed the greatest increase in CCL25 production was in the UEA⁺ TEC of the castrated cohort (data representative of 2 sorted experiments) using quantitative RT-PCR. (D) Figure displays the timing of CCL25 antibody (TECK) or control IgG antibody with respect to the timing of surgery (day 0) or euthanasia (day 8) of the 3 experimental groups (early, late, and near-continuous). Small boxes (in the early group) represent the smaller (50 μg) dose, larger boxes represent the larger (100 μg) dose and may overlap days to reflect the anticipated increase in circulating antibody levels over this time frame. Thymocyte (E) and DP (F) increase after castration is abrogated in the presence of neutralizing antibody to CCL25 (summary of 4-5 mice/group with SE bars, mice received 100 μg of control IgG or CCL25 antibody (TECK) on the day of surgery, and 50 μg of antibody 3 and 5 days after surgery; killed 1 week after castration). (G) DN 2-4 increases after castration were abrogated in the presence of TECK antibody after administration of 100 μg on days 4 and 6 after surgery compared with IgG antibody with castration (summary of 5-6 mice/group with SE bars killed 1 week after castration). (H) Summary of the effect of the timing of CCL25 blockade with respect to the timing of androgen withdrawal.