Therapeutic Toll-like receptor agonists directly influence mouse and human T cell lymphoma cell viability and cytokine secretion.

Mycosis fungoides (MF) and S é zary syndrome (SS) are the most common forms of cutaneous T cell lymphoma (CTCL), and most frequently manifest as CD4 CD45RO malignant T cells with T cell receptor clonality [1]. MF and SS are often refractory to standard chemotherapeutic treatments, which expose patients to toxicity often without substantial benefi t. Because of the limited effi cacy of existing treatments, several novel immune-modulating therapies for MF and SS are under investigation, including imiquimod and oligodeoxynucleotides (ODNs), ligands for Toll-like receptor 7 (TLR7) and TLR9, respectively. Imiquimod is already Food and Drug Administration (FDA) approved for the treatment of basal cell carcinoma, actinic keratoses and condyloma. In a published case report, administration of the synthetic TLR7 ligand imiquimod successfully treated a cancerous skin plaque that was resistant to standard therapy, resulting in disease remission for at least 12 months [2]. Imiquimod treatment resulted in complete clearance of skin plaques in a patient with stage 1A CTCL with a 10-year history of disease [3]. Imiquimod treatment also eliminated skin involvement in a patient with B cell chronic lymphocytic leukemia (B-CLL) [4]. A preliminary study of imiquimod in patients with MF likewise showed a clinical response rate of 50% as measured by the clearance of plaques [5]. Both imiquimod and a synthetic CpG-containing ODN TLR9 agonist have shown the ability to enhance the host immune response in patients with CTCL [6,7]. Kim et al . recently reported the results of a phase I clinical trial of ODN 2006 (CpG 7909) in the treatment of a small cohort of patients with CTCL with refractory disease, showing that treatment was well tolerated and demonstrated anti-tumor activity in patients [8]. Th e ability of imiquimod to alter cell viability has precedent in studies of other skin cancers, where imiquimod has been found to induce apoptosis of malignant skin cells [9]. TLRs are most widely known for their role in pathogen recognition during the innate immune response. Upon detection of TLR agonists, antigen presenting cells (APCs) up-regulate costimulatory molecules and become primed to activate other immune cells. Anti-cancer studies employing TLR agonists are centered on the hypothesis that stimulation through TLR7 or TLR9, respectively, in APCs will enhance APC uptake and presentation of cancer antigens to other cells of the immune system. Th e alternative hypothesis that TLR ligands may directly aff ect the cancerous T cells themselves has not been investigated, and may have important implications for continued investigation of TLR ligands as therapeutic agents in the treatment of T cell malignancies. Several studies have demonstrated the ability of TLR ligands to aff ect malignant B cells with both proand anti-cancer outcomes, providing precedent for such a hypothesis [10]. We tested the hypothesis that TLR agonists directly alter T cell lymphoma cell biology, fi rst using mouse T cell lymphoma cell lines as a model system (data not shown), and ultimately confi rming these fi ndings on cell lines derived from human patients with CTCL. We focused these studies by stimulating HH, a non-MF/non-SS CTCL cell line, and HuT78, a SS cell line, with ODNs and imiquimod. Cells were stimulated with these ligands for 24, 48 or 72 h and assessed for viability by propidium iodide (PI) stain. By 72 h, the cell line HH demonstrated increased culture viability upon incubation with imiquimod, whereas imiquimod increased cell death in HuT78 cell cultures [Figure 1(A)]. A dose titration of imiquimod revealed maximal viability of HH cells at 5 μ g/mL imiquimod, and maximal death induction of HuT78 cells was observed with 10 μ g/mL imiquimod [Figure 1(B)]. In contrast, ODNs only minimally altered cell line viability [Figure 1(A)]. Th ese human data, taken together with mouse T cell line viability data, indicate that the TLR7 agonist imiquimod has the greatest potential to infl uence malignant T cell viability. We next determined whether TLR ligands alter cytokine secretion by human CTCL cell lines. Human CTCL cell lines were stimulated for 72 h with ODNs or imiquimod, and supernatants were subjected to a bead-based cytokine assay to detect the presence of 51 cytokines, with comparison to TLR ligand-stimulated peripheral blood mononuclear cells (PBMCs) from healthy human blood as a control for ligand activity and assay function (Table I). Soluble vascular cell adhesion molecule 1 (sVCAM-1), regulated upon activation, normal T cell expressed and secreted (RANTES), soluble intercellular adhesion molecule 1 (sICAM-1), interferon inducible protein 10 (IP10) and vascular endothelial growth L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y St an fo rd U ni ve rs ity o n 12 /2 6/ 12