TLR-9 and IL-15 Synergy Promotes the In Vitro Clonal Expansion of Chronic Lymphocytic Leukemia B Cells

Abstract

Clinical progression of B cell chronic lymphocytic leukemia (B-CLL) reflects the clone’s antigen receptor (BCR) and involves stroma-dependent B-CLL growth within lymphoid tissue. Uniformly elevated expression of Toll-like receptor 9 (TLR-9), occasional MYD88 mutations, and BCR specificity for DNA or antigens physically linked to DNA together suggest that TLR-9 signaling is important in driving B-CLL growth in patients. Nevertheless, reports of apoptosis following B-CLL exposure to CpG oligodeoxynucleotide (ODN) raised questions about a central role for TLR-9. Because normal memory B cells proliferate vigorously to ODN + interleukin-15 (IL-15), a cytokine found in stromal cells of bone marrow, lymph nodes, and spleen, we examined whether this was true for B-CLL cells. Through a CFSE-based assay for quantitatively monitoring in-vitro clonal proliferation/survival, we show that IL-15 precludes TLR-9-induced apoptosis and permits significant B-CLL clonal expansion regardless of the clone’s BCR mutation status. A robust response to ODN+IL-15 was positively linked to presence of chromosomal anomalies (trisomy-12 or ataxia telangiectasia mutated (ATM) anomaly + del13q14), and negatively linked to a very high proportion of CD38+ cells within the blood-derived B-CLL population. Furthermore, a clone’s intrinsic potential for in-vitro growth correlated directly with doubling time in blood, in the case of B-CLL with IGHV-unmutated BCR and <30% CD38+ cells in blood. Finally, in-vitro high-proliferator status was statistically linked to diminished patient survival. The above findings, together with immunohistochemical evidence of apoptotic cells and IL-15-producing cells proximal to B-CLL pseudofollicles in patient spleens, suggest that collaborative ODN and IL-15 signaling may promote in-vivo B-CLL growth.

Microenvironmental agonists generate de novo phenotypic resistance to combined ibrutinib plus venetoclax in CLL and MCL

“…We have not observed a dichotomy in spontaneous apoptosis, cellular response, or resistance to IBR+VEN in our CLL or MCL samples pre-incubated with agonist mix, perhaps because our studies were performed in a mixture of agonists designed to emulate some of the complexities of in vivo environments. Our results are in agreement with reports that cytokines can overcome CpG-ODN induced apoptosis in IGVH mutated CLL cells 34,43 .…”

Selective BTK inhibition improves bendamustine therapy response and normalizes immune effector functions in chronic lymphocytic leukemia

“…To evaluate CC‐292 capability to inhibit CLL cell proliferation, CFSE‐labeled primary CLL cells were induced to proliferate by incubating them with a medium containing CpG oligodeoxynucleotide, which triggers growth and cell division in the proliferative centers of CLL patients, and the inflammation‐linked cytokine IL‐15, which is constitutively produced by stromal cells . This medium significantly increased mean percentage of CFSE low viable B cells from 2.6% to 41.8% after 6 days of incubation, and from 3.0% to 50.9% after 9 days (both p < 0.0001; Supporting Information Table S4).…”

“…CLL primary cells (10 7 cells) were labeled with 0.5 μM carboxyfluoresceinsuccinimidyl ester (CFSE) (Thermo Fisher Scientific, Waltham, MA), seeded in 96‐well plates (Falcon, Corning, NY) at 10 5 cells/200 μL, and cultured for 6 and 9 days in an enriched RPMI‐1640 medium used for long‐term cultures, supplemented with 15 ng/mL recombinant human IL‐15 (R&D systems, Minneapolis, MN) to sustain survival. To induce cell proliferation, 0.2 μM CpG DNA TLR‐9 ligand (ODN‐2006; Invivogen, San Diego, CA) was added, together with CC‐292 1 μM in indicated conditions.…”

“…CLL primary cells (10 7 cells) were labeled with 0.5 μM carboxyfluoresceinsuccinimidyl ester (CFSE) (Thermo Fisher Scientific, Waltham, MA), seeded in 96-well plates (Falcon, Corning, NY) at 10 5 cells/200 μL, and cultured for 6 and 9 days in an enriched RPMI-1640 medium used for long-term cultures, 23…”

Network-Based Drug Discovery Identifies Statins as a Repurposing Agent to Improve Activity of Current Therapies in Chronic Lymphocytic Leukemia.

“…In vitro CLL proliferation assay CLL primary cells (10 7 cells) were labeled with 0.5 µM carboxy uoresceinsuccinimidyl ester (CFSE; Life Technologies) as reported previously [13,14]. Brie y, 10 5 cells/200 μL were cultured for 6 days in an enriched RPMI-1640 supplemented with 15 ng/mL recombinant human IL-15 (R&D systems, Minneapolis, MN) to sustain survival and with 0.2 μM CpG DNA TLR-9 ligand (ODN-2006; Invivogen, San Diego, CA) to induce cell proliferation [15]. The percentage of divided cells was determined as the percentage of CD19+ (PE)/Annexin-V-(Paci c Blue) cells showing a decrease in CFSE staining on ow cytometry.…”