Gal-9 KO mice were more resistant to AML. (A-B) B6 WT or gal-9 KO mice (10 mice/group) were injected intravenously with 10⁶ C1498FFDsR. (A) Whole-body imaging was performed on 7, 14, 21, and 28 days post-AML injection (10 mice/group). Gal-9 KO mice had a slower tumor growth compared with WT mice 14 and 21 days post-AML injection. Tumor burden was equal at a later phase of disease (day 28). (B) gal-9 KO mice had a significant prolonged survival from AML. (C-E) Naive or AML-bearing gal-9 KO mice (3-4 mice/group) were killed 25 days post-AML injection. Liver leukocytes were isolated for flow cytometry. (C) The percentage of Foxp3⁺ Tregs in CD4⁺ T cells from liver of AML-bearing gal-9 KO mice was similar to naive WT or gal-9 KO mice, while AML induced an increase in percentage of Foxp3⁺ Tregs in WT mice. (D) An in vitro Treg suppression assay was performed with WT and gal-9 KO Tregs. Gal-9 KO Tregs could inhibit CD8⁺ T cells proliferation at a comparable level to WT Tregs. Data were pooled from 2 individual experiments (7 mice total). (E) The percentage of PD-1⁺Tim-3⁺ CD8⁺T cells was increased in the liver of AML-bearing gal-9 KO mice but to a lesser extent than in AML-bearing WT mice. Data were pooled from 2 individual experiments (7 mice total). (F) Impaired IFN-γ, TNF-α, and IL-2 production was found in PD-1⁺Tim-3⁺ CD8⁺ T cells in AML-bearing gal-9 KO mice. Data were pooled from 2 individual experiments (6 mice total).