Activation of Nucleic Acid-Sensing Toll-Like Receptors Induces Proliferation, Cytokine Production, Immunogenic Phenotype, and Plasma Cell Differentiation of CLL Cells and Immunoglobulin Production.

Weak immune function of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the function of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of B-cell function in innate and adaptive immunity, we asked to what extent stimulating two nucleic acid-sensing TLRs affected the activation and differentiation of CLL B cells. Isolated CLL cells were treated with either the TLR7-activating ligand, gardiquimod, or the TLR9-activating ligand ODN 2006-G5. CLL cells were also treated with the contents of apoptotic leukemic B cells, produced by repeated freezing and thawing. Ability to induce proliferation, as defined by CFSE dilution studies, cytokine production, differentiation, and altered surface molecule expression was measured. Both TLR 7 and TLR 9 ligands individually induced high levels of proliferation and plasma cell differentiation of CLL B cells. A dramatic increase in percentage of dividing cells was seen with simultaneous TLR7 and TLR9 activation, while blocking the TLR 7/9 pathway by pre-treating CLL B cells with chloroquine or bafilomycin reduced the proliferative capacity by 90%. Furthermore, stimulating CLL B cells with TLR7 and 9 ligands significantly up-regulated activation (CD38, CD69 and CD83), adhesion (CD54), and other immunophenotypic (CD40, CD80 and CD86) markers. Enzyme immunoassays of culture supernatants after TLR 7 and TLR 9 stimulation indicated that the levels of soluble IgM increased considerably, although levels of cytoplasmic IgM were unchanged. Production of IL6 and IL10 was also induced. We propose that ligands for TLRs 9 and 7 stimulate CLL B cells to proliferate and differentiate into plasma cells in the absence of T-cell help. In addition these stimuli alter CLL cell surface phenotype to a more immunogenetic profile, providing a rationale for CpG oligonucleotides and gardiquimod as humoral vaccine adjuvants for both normal immune reactions and potentially for anti-leukemic therapy.