Elevated immune suppression in hosts with established acute leukemia. (A-B) Mice were challenged with C1498 intravenously, and the immune response in the liver and spleen was analyzed 20 days later. (A) Flow cytometric identification of Tregs. (B) Percentage of Tregs in the spleens and livers of naïve and leukemia-challenged mice. (C) Symptom-free survival of mice challenged with C1498 and vaccinated 7 days later. One group was depleted of Tregs the day after leukemia challenge. Symbols represent treatment groups: unvaccinated (●), therapeutic α-GalCer vaccination (▪), therapeutic α-GalCer vaccination plus PC61 (▲), and irradiated C1498 cells plus PC61 (▼). Statistical analysis compared therapeutic α-GalCer vaccination plus PC61 to therapeutic α-GalCer vaccination. (D-G) Mice were challenged with C1498 intravenously and euthanized 20 days later. (D) Flow cytometric identification of splenic CD11b⁺Ly6G⁺ MDSCs. (E) The proportion of MDSCs within live splenocytes. (F-G) Splenic CD11b⁺ cells were isolated from naïve or C1498-challenged mice and cultured with CFSE-labeled lymphocytes and anti-CD3 and anti-CD28. A representative histogram of CFSE dilution of (F) CD4⁺ cells and (G) CD8⁺ cells incubated with CD11b cells from naïve mice (black line) or C1498-challenged mice (dotted line); unstimulated (shaded) and graph of reduced CFSE. (H-I) CFSE-labeled lymph node cells stimulated with anti-CD3 and anti-CD28 were cultured with naïve splenocytes or C1498 cells for 72 hours. A representative histogram of CFSE dilution of (H) CD4⁺ and (I) CD8⁺ T cells cultured with naïve splenocytes (black line) or C1498 cells (dotted line); unstimulated (shaded). The percent divided of (H) CD4⁺ cells or (I) CD8⁺ cells. This figure represents 3 experiments, each with 5 mice per group. (B,E-H) *P < .05, **P < .01 (Student t test with Mann-Whitney U test). (C) *P < .05 (log-rank Mantel-Cox test).