(A) Representative FACS plots for the expression of Tnfrsf4 on CD4⁺Foxp3-GFP⁺ T cells (Tregs), CD4⁺Foxp3^– T cells, CD8⁺ CTLs, L-Gr-1⁺ cells, and LSCs in the BM of Foxp3^DTR CML mice. One representative out of 9 plots is depicted. Staining, black; isotype control, gray. (B) Frequency of CD4⁺Foxp3-GFP⁺ T cells, CD4⁺Foxp3^– T cells, CD8⁺ CTLs, L-Gr-1⁺ cells, and LSCs in the BM of CML mice expressing Tnfrsf4 (n = 9 mice/cell subset). (C) LSCs from the BM of Foxp3^DTR CML mice were cultured with BM CD8⁺ T cells and/or BM Tregs of the same mice pretreated for 2 hours with a Tnfrsf4 antibody (clone OX-86, 30 μg/mL) or respective control antibody at a ratio of 1:1:1 in triplicate followed by plating in methylcellulose. Colonies were enumerated 7 days later; 1-way ANOVA followed by Tukey’s post hoc test. (D and E) BL/6 CML mice were randomized to control IgG or anti-Tnfrsf4 antibody treatment (OX-86, 200 μg/mouse, i.p, for 6 times every second day, starting at day 12), and leukemia development and survival was monitored. (D) Number of L-Gr-1⁺ cells in the blood of IgG-treated and Tnfrsf4 antibody–treated BL/6 CML mice; 2-way ANOVA followed by Sidak’s multiple comparison test (n = 5 mice/group). (E) Kaplan-Meier survival curves of control IgG– and TNFRSF4 Ab–treated CML mice (IgG, n = 5; Tnfrsf4, n = 5); log-rank test. (F–J) CD8/Treg ratio in the BM, Treg numbers in the BM, spleen weight, numbers of L-Gr-1⁺ cells, and LSCs in the BM of control IgG– and TNFRSF4 Ab–treated CML mice (IgG, n = 5; Tnfrsf4, n = 4) 18 days after CML transplantation. t test. Data are displayed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. One representative out of 2 independent experiments is shown.