Effects of Toll-like receptor 7 and Toll-like receptor 9 signaling stimulators and inhibitors on chronic lymphocytic leukemia cells ex vivo and their interactions with cladribine

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant B lymphocytes in the peripheral blood that are regarded as poorly antigenic to the host immune system. Nonetheless, they are thought to be able to undergo stimulation and become antigen-presenting cells possibly through Toll-like receptors (TLRs). Several studies have examined the effect of TLR7 and TLR9 stimulation on the biology of CLL cells, revealing contradictory results in terms of cell proliferation and apoptosis. On the other hand, suppression of TLRs has not been studied in CLL so far, and the rationale for this may be increasing evidence of the supportive role of TLR signaling in CLL. In our study we assessed the effect of a synthetic oligodeoxynucleotide with immunoregulatory sequences (IRS 954) on peripheral blood cells from patients with untreated CLL, in terms of expression of costimulatory molecules, production of cytokines and cell viability ex vivo. Agonists of TLR7 (imiquimod, IMI) and TLR9 (oligodeoxynucleotide ODN 2006) acted as positive internal controls. ODN 2006 most markedly induced CD86 expression compared to IMI and IRS 954. Both oligodeoxynucleotides - IRS 954 and ODN 2006 - caused 1.5- and 5-fold increases of CD40 on CLL cells, respectively. Immunostimulatory ODN 2006 induced CD95 expression 1.5-fold. Changes in costimulatory molecule expression were accompanied by a moderate response from CD4 + and CD8 + T lymphocytes. TLR7 and TLR9 agonists led to significantly higher production of interleukin 6 (IL-6) and IL-10. IRS 954 and ODN 2006 markedly increased the concentration of tumor necrosis factor alpha (TNF alpha). IL-17A was significantly decreased by 50% after IMI. IRS 954 and IMI induced significant necrosis at all concentrations, and the effect was augmented by the addition of cladribine (2CdA). ODN 2006 presented a dual effect on cell viability, which was related to disease stage and baseline IL-17A concentration. The addition of 2CdA had little effect in a group where ODN 2006 supported cell survival, and further enhanced cytotoxicity of ODN 2006 in the second group. Inhibitory oligodeoxynucleotides seem to exert promising antileukemic effects regardless of sample background, and thus may become a new modality in CLL. The response of leukemic cells to ODN 2006 varies between samples and cannot yet be predicted.