IL-10 Production By CLL Cells Is Enhanced in the Anergic IGHV Mutated Subset and Associates with Reduced DNA Methylation of the IL-10 Locus

The B-cell receptor (BCR) of chronic lymphocytic leukemia (CLL) cells with unmutated (U-CLL) or mutated (M-CLL) immunoglobulin gene heavy-chain variable (IGHV) regions shows a variable degree of anergy, associated with low surface IgM (sIgM) levels and signaling capacity, more evident in M-CLL. DNA methylation is also different between M-CLL and U-CLL. Patients from both subsets are immunosuppressed from the early stages, possibly influenced by the ability of activated CLL cells to produce IL-10, a property of B10 cells.

The aim of this study was to investigate associations between capacity of circulating CLL cells to differentiate into B10 cells and features of anergy. In parallel, the methylation status of the IL-10 gene locus was probed in order to investigate associations between IL-10 gene methylation and production. CLL cells were isolated and cultured +/- TLR9 activation with CpG (ODN-2006). Cytokine secretion was measured in supernatants (Luminex) and IL10 transcript was measured (RT-qPCR) from cell pellets. IL-10 production by CLL cells was measured by intracellular flow cytometry following stimulation with CpG for 24 hours. Epigenetic profiling was performed using MassARRAY and the 450K Array.

We found that activation of CLL cells by CpG consistently induced production and secretion of IL-10 protein, while no or low amounts of pro-inflammatory cytokines were detected. Production of IL-10 was significantly higher in M-CLL than in U-CLL. Levels also correlated with lower sIgM levels and signaling capacity, both features of anergy. A linear correlation was present between secretion, intracellular production and transcript levels of IL-10, suggesting no aberrant post-transcriptional controls.

In the absence of CpG activation, IL-10 transcript levels were also detected at low levels in M-CLL, while they were even lower in U-CLL. To identify a potential basis for the differential expression, DNA methylation analysis of IL-10 gene locus was performed. While the promoter region displayed similarly low levels of methylation in both U-CLL and M-CLL, methylation differences were detected immediately downstream of the promoter within the first intron (approximately +200 to +500 bp, differentially methylated region 1, DMR1) and in the gene body (approximately +1300 to +1800 bp, DMR2). Both DMR1 and DMR2 were markedly more hypomethylated in M-CLL than U-CLL, with the greatest difference detected at cg17067005 within DMR1. Each of these regions display histone H3 lysine 27 acetylation (H3K27ac) in B cells (GM12878 cells), thus are likely to represent functional DNA elements. Analysis of IL-10 transcript levels with methylation demonstrated a mutually exclusive pattern between expression and methylation of both DMR1 and DMR2.

These data document a strong link between capacity to differentiate into B10-like cells and anergy, and suggest an epigenetic component in the regulation of IL-10 production in CLL cells. This capacity may contribute to immunosuppression. While U-CLL appears less able to produce IL-10 on a per cell basis, higher tumor load may compensate, accounting for clinical immunosuppression in both subsets.