CD18 (ITGB2) expression in chronic lymphocytic leukaemia is regulated by DNA methylation-dependent and -independent mechanisms

The integrin lymphocyte function-associated antigen 1 (LFA-1; CD11A/CD18; ITGAL/ITGB2), is a key regulator of lymphocyte trafficking, activation and prolonged lymphocyte residence in lymph nodes (LNs) (Reichardt et al, 2013); yet, little is known about the regulation of ITGAL (CD11A) and ITGB2 (CD18) transcription. Chronic lymphocytic leukaemia (CLL) cells strongly depend on the lymphoid microenvironment, where they transiently localize to receive supportive signals by accessory cells (Burger & Gribben, 2014). Previously, we found that the majority of CLL cells expressed low surface LFA-1 levels as the result of reduced ITGB2 transcription, which was accompanied by impaired LN homing (Hartmann et al, 2009). Dissecting LFA-1 expression in CLL subgroups of different cytogenetics, we observed comparable and correlating CD11A and CD18 surface expression, allowing cytometrical measurements of each subunit as a surrogate for the other (Fig 1A). We found a prominent increase of CD11A and CD18 expression in Trisomy 12 (tri12) harbouring CLL (Fig 1B,C), in accordance to a recent observation (Riches et al, 2014). The data confirmed our previous observation of associated expression of LFA-1 with CD38 and CD49D (ITGA4) (Hartmann et al, 2009), while we found no differences in CLL subgroups defined by the IGHV mutational status or ZAP70 expression (Figure S1A,B). Curiously, we observed lower CD11A levels in subgroups harbouring the unfavourable 17p deletion or the favourable 13q deletion but no association of 11q deletion with CD11A expression (Figure S1C). Tri12 defines a CLL subgroup with high cell proliferation and an increased frequency of Richter transformation, which manifests with lymphadenopathy and LN infiltration (Tsimberidou & Keating, 2005). Recently, we reported that tri12 CLL cells are also characterized by high CD49D expression due to ITGA4 (CD49D) promoter hypomethylation (Zucchetto et al, 2013). Hypothesizing a similar mechanism responsible for regulation of LFA-1 expression, we studied promoter methylation of the rate-limiting CD18 subunit in CLL. The ITGB2 promoter contains 23 potentially methylated CpG motifs (CpG1-CpG23) (Agura et al, 1992). We analysed these sites by bisulfite conversion of genomic DNA from anti-CD19 purified CLL cells followed by nested polymerase chain reaction, cloning and sequencing. Notably, whereas the region closer to the transcription start site (CpG16-CpG23) was unmethylated in CLL, the region from 603 bp to 241 bp upstream of the transcription start site (CpG4-CpG15) was variably methylated (Figure S2). The grade of methylation in these sites inversely correlated with CD18 surface expression (Fig 1D). A high grade of ITGB2 promoter methylation of CpG4-CpG15 was found in CLL samples with low CD18 expression, whereas CD18 high expressing CLL cells, overrepresented in the tri12 CLL group even in presence of a low percentage of tri12+ cells, were mostly unmethylated at the same sites (Figs 1D and S2). Our data indicate regulation of ITGB2 transcription by DNA hypomethylation in quiescent CLL cells of this specific subgroup. Next, we addressed whether CLL cell activation could influence CD18 expression. Therefore, CLL cells were co-cultured in vitro with autologous T cells on a layer of murine fibroblasts and stimulated with IL2/CpG. After a 5-d co-culture, levels of the activation marker CD86 were measured and an up-regulation of CD18 in CD86-positive CLL cells was detected (Figure S3). Particularly, in tri12 CLL, CD86-positive CLL cells in both unstimulated and IL2/CpG-stimulated samples expressed significantly higher CD18 than CD86-negative sub-clones, indicating a higher propensity of tri12 CLL to become activated and up-regulate CD18 expression, even in the absence of strong stimulation (Figure S3). Furthermore, actual cell division occurred on day 5, with significantly higher proliferation rates of tri12 CLL cells (Fig 2A). Within individual samples, CLL cell subpopulations that had either progressed through the cell cycle or remained quiescent showed higher CD18 expression in proliferating CLL cells (Fig 2B). Remarkably, tri12 CLL were capable of undergoing more cell cycles in vitro than non-tri12 (Fig 2C), with continuously increased CD18 levels in each proliferation round (Fig 2D), suggesting a higher intrinsic proliferative capacity of CD18-high-expressing sub-clones or an up-regulation of CD18 expression by signals from the co-culture. In addition to IL2/CpG, other co-culture systems (Asslaber et al, 2013) effectively induced CD18 and CD11A expression in CLL, including activated autologous or allogeneic T cells (in presence of a fibroblast layer) but not CD40LG-overexpressing fibroblasts alone (Figure S4A,B). Thus, secreted cytokines rather than direct CD40/CD40LG interactions seem responsible for this phenomenon. Next, proliferative and quiescent CLL fractions were sorted according to their Cell Trace Violet (CTV) intensity using correspondence