Toll-Like Receptor 7-Targeting of Human B-Lineage Acute Lymphocytic Leukemia Induces Immunogeneicity and Apoptosis of Leukemia Cells.

Abstract Acute lymphocytic leukemia (ALL) is the most common childhood leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improved cure rates in both pediatric and adult ALL patients. In this study, we show human B-lineage acute lymphocytic leukemia (B-ALL) cell lines (6/6 tested) and CD19+CD10+ primary B-ALL cells from patients (8/8 tested) express TLR7 mRNA by real-time RT-PCR and TLR7 proteins by Western blot. Triggering TLR7 on B-ALL cells with a TLR7 agonist (imiquimod) significantly increases the cell surface expression of molecules essential for T cell activation (CD40, CD54, CD80, CD86, and HLA-DR), the ligands for NKG2D and ligands for natural cytotoxicity receptors (NKp30, NKp44, and NKp46) which regulate NK-mediate killing. Thus, TLR7 signaling enhances the immunogenicity of B-ALL cells and makes them more suitable targets for T cell and NK cell mediated attack. Most importantly, TLR7 agonists strongly suppress in vitro growth of B-ALL cell lines (RS4;11, BLIN-1) and induces profound apoptosis of primary B-ALL cells from patients in culture in a TLR7 agonist dose-dependent manner. Both t(4;11)-positive RS4;11 cells and t(4;11)-negative BLIN-1 cells proliferate rapidly in culture with a 30–40 fold increases of leukemia cell number in 7 days. The addition of TLR7 agonist at 10 ug/ml fully inhibit the growth of RS4;11 and BLIN-1 cells in culture. Furthermore, TLR7 agonist treatment dramatically induces apoptosis of primary B-ALL cells isolated from the patients (2/2 with t(9;22), 6/6 without t(9;22)) with 0.4%–13.3% leukemia cells left at day 5 of culture. The TLR7 agonist-mediated apoptotic death of B-ALL cells was conformed by viable cell counts, TMRE staining, and, Western blots of the activation and cleavage of caspases. To study the in vivo therapeutic effects of TLR7 agonist against human B-ALL, RS4;11 and BLIN-1 cells were luciferase labeled and injected into NOD/SCID mice. Both RS4;11 and BLIN-1 leukemia cells engrafted in multiple organs (BM, spleen, liver, lymph nodes, kidney) resulting in uniform lethality of RS4;11 mice in 8 weeks and BLIN-1 mice in 12 weeks, respectively. Flow cytometry and tissue staining results confirmed that these organs were massively infiltrated with human CD45+19+ leukemia cells. To determine whether TLR7 preincubation of RS4;11 or BLIN-1 cells would prolong survival due to an apoptotic effect, cohorts of mice were injected with a lethal dose of RS4;11 or BLIN-1 cells with or without pre-incubation with TLR7 agonist. Mice receiving TLR7 agonist pre-pretreated B-ALL cells had a significant increase in long-term survival rate and significant reduction in tumor burden at the time points evaluated. These in vivo results confirm previous in vitro findings and suggest that TLR agonist-treated B-ALL cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established B-ALL is being investigated using these xenograft mouse models. These results form the basis for a clinical trial of systemic TLR7 agonist administration for treating patients with B-ALL. In summary, we have shown that TLR7 targeting increases B-ALL immunogenicity and directly induces B-ALL apoptosis, providing new insights into the biology and therapy of B-ALL.