Differential Effects Of Triggering Via Toll Like Receptors In Stable Or Aggressive Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood, secondary lymphoid tissues and bone marrow of functionally defective clonal B lymphocytes with prolonged survival in vivo. Despite therapeutic achievements have been accomplished in the management of this disease, however CLL remains incurable partially because of the resistance to apoptosis of CLL B-cells and of the altered immune system of CLL patients. CLL is hetereogenous, but its evolution is mostly slow, probably linked to some level of immune control of the leukemic cells. Indeed, clinical observations have been reported of spontaneous remissions associated with the intense immunological activity following a viral infection. Clinical responses have also been observed after treatment by immunomodulating cytokines and long-term survival is described, without disease, after allogeneic stem cells transplantation. All these data suggest that immunotherapy could be useful in the treatment of CLL, possibly as an adjuvant therapy after classical immunochemotherapy schedules.

Toll like receptors (TLR) are proteins of the innate immune system belonging to the family of Pattern Recognition receptors (PRRs). Recognition of their ligands by the TLR present on neutrophils, macrophages, dendritic cells and B-cells are important in the initiation of adaptive immune responses. TLR-7 and 9 are expressed by CLL B-cells (Grandjenette, Hematologica, 2007). Previous studies have shown that stimulation of TLR-9 by CpG ODN (oligodinucleotides) induces an activation of CLL B-cells while triggering TLR-7 increases in vitro apoptosis of these cells. Here we show that these effects of TLR-9 and TLR-7 stimulation differ depending on the clinical form (stable or aggressive) of the disease and on the mutational status of CLL B-cells.

In vitro stimulation with three doses of Imiquimod R837 or ODN CpG M362 was carried out for three days on cells from 40 patients (22 stable, 18 aggressive, mutational status known for 35, 18 IgVH mutated, 17 unmutated). Flow cytometry was used to measure apoptosis, proliferation after carboxyfluorescein succinimidyl ester (CFSE) labeling and modulation of surface differentiation antigens. Signaling pathways after incubation were further studied by antibody arrays and western blot.

Spontaneous apoptosis occurring in vitro was demonstrated to involve the mitochondrial pathway. CLL B-cells were also confirmed to proliferate strongly and produce large amounts of IL-6 and IL-8 upon triggering by phorbol myrsistate (positive control), this compound almost completely aborting in vitro apoptosis.

Cells from patients with a stable disease were significantly more prone to rapid apoptosis after TLR-7 triggering with Imiquimod, compared to cells from patients with an aggressive disease which displayed only spontaneous apoptosis. This rapid apoptosis in stable patients involved the p38 MAP-Kinase pathway. It was concomitant to an important production of IL-8 and IL-6.

Conversely, CpG ODN conferred a protection against apoptosis to CLL B-cells from patients with an aggressive disease. This was accompanied by the activation of numerous anti-apoptotic proteins in the cells. CpG ODN also significantly increased CLL B-cells proliferation, concomitantly to the phosphorylation of Erk and Akt proteins. ODN finally increased the expression of CD20 and CD19 on the cells’surface.

The same differences in reactivity were observed comparing mutated (∼stable) and unmutated (∼aggressive) cases.

These data indicate that CLL B-cells from patients with a stable or aggressive (mutated/unmutated) disease answer differently when triggered through their surface TLRs. This might have an incidence on the behavior of these cells in vivo, in answer to stimulations by microbial compounds naturally binding these structures. These properties could also be used to develop adjuvant immunotherapies by loading CpG ODN-activated CLL B-cells with autologous apoptotic fragments issued from stimulation of part of the same cells with Imiquimod.