Effect of EGFL7 on immune reconstitution and graft-versus-leukemia effect. (A-C) Splenocytes and thymocytes were collected from PBS-treated mice as they reached their endpoints (day +28 to 35) and from EGFL7-treated mice (day +30 to 35 post-BMT). (A) Absolute counts of TCR⁺, CD4⁺, and CD8⁺ lymphocytes in the spleen of Syn, GVHD + rEGFL7, and GVHD + PBS treated (B6→B6D2F1) mice. Data were pooled from 3 experiments with 4 to 7 mice per group. Gating was performed on CD45.1⁺ and CD45.2⁺ cells. (B) Absolute counts of BM-derived thymocytes (CD45.2⁺) in Syn, GVHD + rEGFL7, and GVHD + PBS treated (B6→B6D2F1) mice. Data were pooled from 3 experiments with 4 to 8 mice per group. Results show mean ± SEM. Statistical analysis compared GVHD + rEGFL7 vs GVHD + PBS treated (B6→B6D2F1) mice, and P values were determined by a Mann-Whitney U test. (C) Dot-plot analysis of thymocytes (derived from the BM cells [CD45.2⁺CD45.1⁻]), based on the expression of CD4, CD8, and CD45.2 antigens, in the thymus of SYN control, GVHD + rEGFL7, and GVHD + PBS mice. These data are representative of 3 or more experiments with 6 to 8 mice per group. Histogram showed mean ± SEM. Statistical analysis compared GVHD + rEGFL7 vs GVHD + PBS treated (B6→B6D2F1) mice, and P values were determined by a Mann-Whitney U test. Transplant for GVL was performed in BALB/c mice as described in “Methods.” (D-E) Whole-body bioluminescent signal intensity of recipient mice (n = 4-5 per cohort). Mice were imaged on indicated days. Average radiance expressed as mean ± SEM. One representative transplant experiment of 2 is shown. (F) Splenocytes were isolated at the time when mice reached their endpoints, days 28 to 33 post-BMT for PBS vehicle and days 30 to 35 post-BMT in rEGFL7-treated mice. Percentage GFP positivity representing P815 leukemic cell infiltration in the spleen. Each dot represents a single mouse. (G) Representative flow cytometric contour plots. Allo. spl., allogeneic splenocytes; GFP, green fluorescent protein; Min, minimum; Max, maximum; SSC-A, side-scatter-area; SYN, syngeneic control; TCD-BM, T cell depleted bone marrow. ***P < .001.