Treatment with PTX and, to a lesser extent, FTY720 impairs IL-7/M25 potency in vivo. (A) Freshly isolated lymphocytes from B6.CD90.1⁺ donor mice were incubated in media alone or in media supplemented with 100 ng/mL PTX for 2 hours at 37°C, washed, labeled with CFSE, and injected intravenously (3 × 10⁶ donor cells per host) into B6.CD90.2⁺ host mice on day 0. FTY720-treated hosts received 3 mg/kg FTY720 intraperitoneally on days −1 and 3. Host mice received intraperitoneal injections of either phosphate-buffered saline (PBS) or rhIL-7/M25 (1.5 μg/7.5 μg per injection) on days 1, 3, and 5. On day 7, host LNs and spleen (SPL) were harvested, and each host tissue was analyzed separately by flow cytometry. CFSE histograms of CD90.1⁺ CD8⁺ splenocytes (left) and total CD90.1⁺ CD8⁺ recovered from host LNs and SPL (right) are representative of at least 2 experiments with 1 to 2 hosts per condition. **P < .005; CRTL, control; ns, not significant. (B) Freshly isolated lymphocytes were treated with PTX or media alone (control) as described in (A) and cultured for 3 days at 37°C in complete RPMI media supplemented with the indicated concentrations of rhIL-7. Samples were stained with CD8-Pacific Blue and propidium iodide (PI), and the percentage of surviving cells (CD8⁺ PI⁻) was determined by flow cytometry. Each data point depicts one sample from an experiment. Results are representative of 3 experiments. (C) Frequency of donor cells recovered on day 7 from the LNs of individual hosts with or without PTX treatment, as described in (A); vertical bars indicate mean frequency for the cohort. Combined data from 3 similar experiments are shown; each point represents the frequency of CD90.1⁺ CD8⁺ lymphocytes for a single host. (D) CFSE-labeled B6.CD90.1⁺ lymphocytes (6 × 10⁶) were injected into B6.CD90.2⁺ hosts treated as in (A) with rhIL-7/M25 and, where indicated, FTY720. On day 7, host spleen (SPL), inguinal (ILN), axillary (ALN), cervical (CLN), and mesenteric (MLN) lymph nodes were harvested separately, and CFSE dilution by CD90.1⁺ CD8⁺ cells was determined by flow cytometry. Results are representative of 2 experiments with 2 separately analyzed hosts per group.