The key role of the microenvironment for tumour growth and aggressiveness has been described for different B-cell malignancies, including chronic lymphocytic leukaemia (CLL). The dependency of CLL cells on their microenvironment is supported by several studies showing that CLL cells can be rescued from apoptosis in vitro if co-cultured with stromal cells, fibroblasts, dendritic or nurse-like cells. These cells are known to provide a variety of survival stimuli for CLL cells, mediated via soluble factors and extracellular matrix components, as well as direct cell-cell contact. Notably, some soluble factors are also produced by CLL cells themselves, allowing both autocrine activation of CLL cells, and paracrine recruitment of additional microenvironmental accessory cells (reviewed in Burger et al, 2009a; Caligaris-Cappio, 2003). Recently, the two T cell chemokines CC motif chemokine 3 (CCL3) and CC motif chemokine 4 (CCL4) have been described to be over-produced by CLL cells upon their co-culture with nurse-like cells, as well as B cell receptor (BCR) stimulation (Burger et al, 2009b). In parallel studies by our group, we similarly described the production of CCL3 and CCL4 by CLL cells (Zucchetto et al, 2009), especially when expressing the bad prognosticators CD38 and CD49d (Shanafelt et al, 2004; Gattei et al, 2008). In particular, we observed the release of CCL3 and CCL4 by cultured CLL cells upon

CD38 triggering, and found CCL3 protein in CLL cell cytoplasms from bone marrow biopsies (BMB) of CD38+CD49d+ CLL patients (Zucchetto et al, 2009). Regarding the putative function suggested for these CLL-derived chemokines, Burger et al (2009b), although not providing direct data, extensively discussed the possibility that CCL3/CCL4 secretion by CLL cells may account for recruitment of T lymphocytes at sites of CLL infiltration, thus favouring productive CLL-T lymphocytes interactions. On the other hand, in our study, we described the presence of an abnormally high number of infiltrating CD68+ monocyte/macrophages in CLL-involved areas of BMB from CCL3/CCL4-producing CD38+CD49d+ CLL. We also showed how these monocyte/macrophages contributed to upregulate vascular cell adhesion molecule 1 (VCAM-1) expression by the stromal/endothelial component of the bone marrow (BM) microenvironment, thus promoting VCAM-1/CD49d interactions, which deliver pro-survival signals for CD49d-expressing CLL cells (Zucchetto et al, 2009). This study investigated whether, in addition to monocyte/macrophages, other cell types could be recruited by CLL-derived CCL3/CCL4 in CLL-involved BM microenvironment. To address this issue, we first evaluated by flow cytometry the expression of CCR1 and CCR5, the CCL3/CCL4 receptors, in