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Chronic lymphocytic leukemia (CLL) is the most prevalent human leukemia and is characterized by the progressive accumulation of long-lived malignant B cells. Here we show that human B-CLL cells selectively express high levels of Toll-like receptor 9 (TLR9) mRNA and proteins. Treating B-CLL cells with TLR9 agonists, type B CpG oligodeoxynucleotides (CpG-B ODNs), induces significant morphologic and phenotypic activation, altered cytokine production, reversal of signal transducer, and activator of transcription 1 (STAT1) phosphorylation state, followed by profound apoptosis of B-CLL cells that is CpG-B ODN treatment time- and dose-dependent. TLR9-CpG ODN ligation-induced apoptosis of B-CLL cells is confirmed by viable cell counts, annexin V/propidium iodide and tetramethyl-rhodamine ethylester staining, Western blots of the activation, and cleaved caspases and poly (ADP-ribose) polymerase. Triggering TLR9 by CpG-B ODN leads to nuclear factor-κB-dependent production of autocrine interleukin-10, which activates JAK/STAT pathway-dependent tyrosine phosphorylation of STAT1 proteins and thereby provokes an apoptosis pathway in B-CLL cells. Treating B-CLL cells in vitro or in vivo with CpG-B ODN reduces the number of leukemia cells that engraft in NOD-scid mice. These findings provide new understanding of CpG ODN-mediated antitumor effects and support for the development of TLR9-targeted therapy for human CLL.

Type 1 interferon–producing cells (IPCs), also known as plasmacytoid dendritic cell (DC) precursors, represent the key effectors in antiviral innate immunity and triggers for adaptive immune responses. IPCs play important roles in the pathogenesis of systemic lupus erythematosus (SLE) and in modulating immune responses after hematopoietic stem cell transplantation. Understanding IPC development from hematopoietic progenitor cells (HPCs) may provide critical information in controlling viral infection, autoimmune SLE, and graft-versus-host disease. FLT3-ligand (FLT3-L) represents a key IPC differentiation factor from HPCs. Although hematopoietic cytokines such as interleukin-3 (IL-3), IL-7, stem cell factor (SCF), macrophage–colony-stimulating factor (M-CSF), and granulocyte M-CSF (GM-CSF) promote the expansion of CD34+ HPCs in FLT3-L culture, they strongly inhibit HPC differentiation into IPCs. Here we show that thrombopoietin (TPO) cooperates with FLT3-L, inducing CD34+ HPCs to undergo a 400-fold expansion in cell numbers and to generate more than 6 × 106 IPCs per 106 CD34+ HPCs within 30 days in culture. IPCs derived from HPCs in FLT3-L/TPO cultures display blood IPC phenotype and have the capacity to produce large amounts of interferon-α (IFN-α) and to differentiate into mature DCs. This culture system, combined with the use of adult peripheral blood CD34+ HPCs purified from G-CSF–mobilized donors, permits the generation of more than 109 IPCs from a single blood donor.

Leukemia involving rearrangements of the MLL gene is resistant to standard therapies and is often a fatal disease. MLL gene-rearrangements are commonly associated with infant-leukemia and secondary leukemias. New therapeutic strategies are needed to achieve longer survival and improve cure rates in patients with these and other refractory leukemias. Toll-like receptor (TLR) agonists are known as potent immune stimulatory agents that can elicit host anti-tumor effects in murine tumor models. We hypothesized that targeting TLRs expressed on leukemia cells with TLR agonists may have direct antitumor effects against leukemia cells. In this study, we investigated the effects of TLR agonists specific for TLR3, 4, 7, and 9, (i.e., polyinosine-polycytidylic acid (Poly(I:C)), monophosphoryl lipid A (MPL), imiquimod (IMQ), and CpG oligodeoxynucleotides (CpG ODN)), in MLL-AF9 knock-in mice that develop myeloid leukemia akin to human MLL-AF9 disease. In contrast to Poly(I:C), MPL, and CpG ODN, treatment of MLLAF9 cells with TLR7 agonist IMQ significantly increased the surface expression of CD40, CD54, CD80, and CD86 on MLL-AF9 cells in vitro. TLR7 mRNA and protein expression in MLL-AF9 cells were confirmed by real-time RT-PCR and intracellular staining/FACS analysis. Most importantly, TLR7 agonist strongly inhibited the in vitro MLL-AF9 cells in a drug dose- and treatment time-dependent manner. Whereas MLL-AF9 cells proliferated rapidly in culture with more than 40-fold increase of cell number in 5 days, the addition of IMQ at 5 mcg/ml fully inhibited the growth and induced profound apoptosis of MLL-AF9 cells with less than 2% of leukemia cells left at day 5 of culture. IMQ-mediated apoptotic death of MLL-AF9 was confirmed by viable cell counts, TMRE staining, Western blots and intracellular staining of the cleavage of caspases and PARP. Preincubation of MLLAF9 cells with caspases 8 and 10 inhibitors effectively blocked IMQ-induced apoptosis and sustained the proliferation of leukemia cells in cultures. To further determine the intracellular pathways engaged by IMQ, microarray gene expression profiles of 24-hour IMQ-treated vs. untreated MLL-AF9 cells were compared. Gene Set Enrichment Analysis (GSEA) showed that IMQ treatment resulted in up-regulated expression of a set of proapoptotic genes (e.g., p53, Bax, caspase 8, Apaf-1, etc) involved in apoptotic pathways. To determine whether IMQ pre-treatment of MLL-AF9 cells would prolong survival due to an apoptotic effect, cohorts of NOD-scid IL2Rgamma mice were i.p. injected with a lethal dose of MLL-AF9 cells with or without pre-incubation with IMQ. Mice receiving 5×106 untreated MLL-AF9 cells resulted in uniform lethality in 4 weeks. In contrast, mice receiving the same lethal dose of MLL-AF9 cells pretreated with IMQ had a significant prolonged survival, confirming in vitro findings that IMQ-treated MLL-AF9 cells undergo apoptosis. Administration of IMQ (daily i.p. injection at 1 mg/kg for 5 days) strongly inhibited leukemia cells growth and significantly prolonged the survival time of MLLAF9 mice. Flow cytometry results confirmed that residual MLL-AF9 cells recovered from IMQ treated mice were apoptotic and had up-regulated expression of cleaved caspases and PARP. In summary, our results demonstrate that TLR7 targeting of MLL-AF9 cells can directly induce apoptosis of MLL-AF9 cells in vitro and in vivo, providing new insights into the TLR-targeted therapy of refractory or relapsed leukemias.

Acute myeloid leukemia (AML) is a common form of acute 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 improve cure rates in AML patients. Toll-like receptor (TLR) agonists have been shown to elicit anti-leukemia effects in murine AML models. However, TLR expression profile of human AML cells is unknown. We analyzed TLR1-10 mRNA expression in purified AML cells from 41 patients with different AML subtypes (M0, M1, M2, M3, M4, or M5; n > 5 per group) by real-time RT-PCR. The majority of AML samples expressed high level of TLR2, 4, 7, 8, low level of TLR1, 5, 9, 10, and undetectable level of TLR3. Significant higher TLR4 and TLR7 expressions were detected on M4 and M5 subtypes of AML cells. Triggering TLR4 or TLR7 with specific TLR agonists (Monophosphoryl Lipid A or Imiquimod) significantly increased the surface expression of molecules essential for T cell activation (CD54, CD80, CD86) on AML M4/M5 cells and enhanced T-cell mediated proliferative responses against AML cells. Thus, TLR signaling enhances the immunogenicity of AML M4/M5 cells and makes them more suitable targets for T cell mediated attack. Most importantly, TLR7 agonist strongly induced apoptotic death of primary AML M4/M5 cells and inhibited the growth of TLR7-expressing AML cell lines (U937, HL-60, KG-1) in culture in a drug dose dependent manner. The addition of TLR7 agonist at 10 ug/ml fully induced apoptosis of AML cells and inhibited the growth of AML cell lines, as confirmed by viable cell counts and TMRE staining. Intracellular staining demonstrated that TLR7 agonist treatment significantly down-regulated the signal transducer and activator of transcription (STAT)3 and STAT5 protein expression in AML cells. These results suggest that TLR7 agonist-induced apoptosis of AML cells is likely via inhibition of STAT3 and/or STAT5 signaling pathway. To study the in vivo effects of TLR7 agonist against human AML cells, primary AML M4/M5 cells or a monocytic AML cell line (U937) were injected i.p. into NOD-SCID IL2Rgamma mice with or without subsequent TLR7 agonist treatment. Mice receiving TLR7 agonist treatment (1 mg/kg daily i.p. infusion for 5 days) significantly reduced tumor burden with substantially lower numbers of engrafted leukemia cells detected in these xenograft mice. Flow cytometry results confirmed that residual AML cells recovered from mice treated with TLR7 agonist were apoptotic with down-regulated expression of TMRE and STAT3/STAT5, confirming previous in vitro findings that TLR7 agonist-treated AML cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established human AML is being investigated using these xenograft mouse models. In summary, our results provide the first report of TLR expression profile of human AML cells and demonstrate that TLR targeting of AML cells can increase the immunogeneicity of leukemia cells and directly induce AML apoptosis in vitro and in vivo, providing new insights into the biology and therapy of AML.

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.