TLR9-Regulated IL10 Mediates Immune Inhibitory Activity of Chronic Lymphocytic Leukemia B-Cells
Authors: Ringelstein-Harlev Shimrit , Fanadka Mona , Horowitz Netanel A. , Avivi Irit , Katz Tami 
Published: October 2019

Abstract
Introduction: Chronic lymphocytic leukemia (CLL) cells exhibit inhibitory features of regulatory B-cells (Bregs), which are suspected to modulate cancer immune evasion on the one hand, and to promote immunosuppression leading to opportunistic infections on the other. One of the major mechanisms through which Bregs restrain the immune system is production of the anti-inflammatory cytokine interleukin 10 (IL10). Our previous study has demonstrated that CLL cells generate IL10, inhibit autologous CD4+ T-cell proliferation and induce regulatory T-cells (Tregs) from autologous CD4+ T-cells. All these abilities were found to be triggered by activation of toll like receptor 9 (TLR9) with the agonist CpG-ODN (ODN). Of note, Treg induction was also observed following CD40 activation with CD40 ligand (CD40L), while intracellular IL10 levels and proliferation inhibition were unaffected by this stimulant. Given the magnitude of the regulatory effects induced by ODN, the current study aimed to explore the contribution of TLR9 activation to the control of IL10 generation by CLL cells and to assess the role of this inhibitory cytokine in mediating the above Breg abilities of these cells.
Methods: B-cells were isolated from peripheral blood mononuclear cells (PBMCs) of untreated CLL patients (Rai stages 0-IV). B-CLL cells were stimulated with ODN or CD40L and either examined directly, or co-cultured with isolated autologous CD4+ T-cells. A cytokine array was used to detect and estimate the concentration of a variety of cytokines (n = 42) secreted to the medium. FACS analysis was employed to examine intracellular IL10 levels in CLL cells, either untreated or treated with an IRAK1/4 inhibitor, obstructing upstream TLR signaling, at doses of 2µM or 5µM. For co-culture experiments, CLL cells were transfected with IL10 siRNA or scramble RNA (control), using the nucleofector technology by Amaxa. T-cell proliferation was evaluated using carboxyfluorescein succinimidyl ester (CFSE) staining for cell tracking after stimulation with anti-CD3/CD28 antibodies and interleukin 2 (IL2). Percentage of cells retained in G0 (non-proliferating pool) was assessed for each condition.
Results: The cytokine array showed IL10 to be the only cytokine secreted by CLL cells to the medium under baseline conditions. The secretion was significantly enhanced following ODN stimulation [fold induction >1.65 (N=3)], but not after CD40L activation. Since in our studies, ODN emerged as the sole activator of IL10 generation, levels of this cytokine were assessed following IRAK1/4 inhibition. The results showed a dose-dependent attenuation of intracellular IL10 levels, with percentage of IL10 expression being 5.88 (±2.01) at baseline, 5.18 (±2.01) for low-dose and 4.83 (±1.93) for high-dose IRAK1/4 inhibition (N=5, p<0.05 for 2µM and p<0.01 for 5µM). To define the contribution of IL10 to Breg functions of CLL cells, the impact of IL10 RNA interference was examined. CLL cell transfection with IL10 siRNA diminished the inhibitory effect of these cells on proliferation of co-cultured autologous T-cells, demonstrating a 21% reduction in T-cell inhibition. In the control co-culture, 12.4 (±2.3)% of stimulated CD4+ T-cells remained in G0, whereas in the presence of IL10-depleted CLL cells, only 9.8 (±1.87)% of CD4+ T-cells remained in the non-proliferating pool (N=8, p<0.05).
Conclusions: In the current study, IL10 emerges as the only inhibitory cytokine secreted at biologically relevant levels by CLL cells, and the only one whose quantity significantly increases following ODN-activation. The TLR9 signaling pathway directly controls IL10 generation, which in turn contributes to the inhibition of T-cell proliferation. While normal B-cells are known to respond to TLR9 activation by secreting a variety of cytokines, the exclusive generation of IL10 by activated CLL cells, resulting in T-cell inhibition, may represent a unique clonal quality promoting disease progression.

Disclosures
Avivi: Tel Aviv Sourasky Medical center: Consultancy, Other: consultancy to :BMS Roche.