Monocytes/macrophages assume a critical role in the maintenance and progression of CLL cells. Many studies highlighted that neoplastic B cells shape the phenotypical and functional features of the monocytes/macrophages of the CLL microenvironment. The crosstalk between leukemic B cells and monocytes leads to the polarization of monocytes toward an immunosuppressive M2 phenotype, simultaneously enhancing the survival and expansion of CLL cells. Moreover, a particular population of myeloid cells, namely, nurse-like cells (NLCs), was described in this disease. NLCs are cells of monocytic origin that spontaneously differentiate in vitro in high-density cultures of CLL peripheral blood mononuclear cells (PBMCs)
[14][15]. These cells support leukemic B cell survival, further creating a permissive microenvironment
[16][17]. Importantly, NLCs were also observed in vivo in the lymphoid organs of patients with CLL
[15]. Since their original description
[14], NLCs/CLL cell co-cultures have been used extensively to dissect key cellular and molecular interactions between leukemic B cells and their microenvironment. Researchers reported that NLCs and CLL monocytes display features of the alternative type-2 subset (M2)
[18]. NLCs are characterized by high CD11b, CD163, CD206, HLA- DR and c-MET expression and by the dysregulation of genes involved in immunocompetence
[18][19][20][21]. In addition, researcehrs demonstrated that NLCs and CLL monocytes showed higher expressions of indoleamin 2,3 dioxygenase (IDO) than monocytes from normal controls
[18], and IDO is regarded as a key endogenous immunologic checkpoint with a pivotal impact on tumor-associated immune tolerance
[22]. In line with their immunosuppressive phenotype, CLL monocytes or NLCs significantly inhibited T cell proliferation, and this inhibition was counteracted by the concomitant addition of neutralizing anti-TGFβ or -IL-10 antibodies or IDO inhibitors in cultures. Indeed, healthy monocytes upregulated IDO after their co-culture with CLL cells
[18]. Interestingly, IDO also appeared upregulated in NLCs cultured in hypoxic conditions
[23]. Jitschin R. and collaborators
[24] further reported that untreated CLL patients show a significantly increased frequency of monocytes CD14 + DRlow, which are defined as myeloid-derived suppressor cells (MDSCs) and characterized by high IDO levels expression, that induced the suppression of T cell activation while expanding T regulatory cells (Tregs); the MDSC-mediated modulation of T cells was attributed to their increased IDO activity. The presence in CLL patients of a higher percentage of MDSCs characterized by immunosuppressive features and expressing IDO together with others immunoregulatory molecules/cytokines, such as arginase 1 (ARG1), nitric oxide synthase (NOS2), TGF-β and IL-10, was also described by Zarobkiewicz M and co-author
[25]. It is of further interest to note that the kynurenine–tryptophan ratio, which reflects increased IDO activity, was found to be higher in sera from CLL patients than in normal donors
[26]. Maffei and co-authors
[27] also observed the functional and phenotypic deregulation of monocytes in CLL patients: the gene expression profile analysis of CLL monocytes compared with monocytes from healthy donors, other than suggesting the deregulation of genes involved in phagocytosis and inflammation, evidenced the ability of CLL B cells to “educate” these cells, skewing them toward an immunosuppressive phenotype. Using cytofluorimetric analysis, these authors further evaluated the proportions of the three subtypes of monocytes present in CLL PBMCs: classical (CD14+ CD16−), intermediate (CD14+ CD16+) and non-classical (CD14+/− CD16++). In contrast with normal donors, they observed a significant increase in the intermediate and non-classical populations and a reduced percentage of classical monocytes. Of further note is the finding that healthy monocytes, co-cultured with CLL cells or with their conditioned medium, were induced to upregulate CD16, which is the Fcγ type III low-affinity receptor for IgG (FcγRIIIa). A higher number of monocytes expressing CD16 in CLL patients was also described by Kowalska and co-authors
[28]. Bolzoni M and co-workers
[29] further interestingly reported that sorted bone marrow CD14+ CD16+ cells from myeloma patients were more pro-osteoclastogenic than CD14+ CD16− cells in cultures ex vivo. Additionally, it was demonstrated that the number of bone marrow CD14+ CD16+ cells was higher in patients with active myeloma than in those with monoclonal gammopathy of undetermined significance
[29]. CD16 is a RANK co-receptor, contributing to the amplification of osteoclast differentiation
[30]; this evidence, along with the previous observations, prompted researchers to evaluate, first, the percentage of monocyte subtypes in a cohort of PBMCs from 35 CLL patients, and second, whether the eventual expansion of a particular subset could be related to the enhanced rate of bone erosion previously reported
[31]. Researchers confirmed that the percentages of the two subsets expressing CD16 (intermediate and non-classical) were significantly higher in CLL cases than in PBMCs from normal donors, and researcehrs found a direct correlation between the percentage of intermediate monocytes and the levels of bone erosion. Researchers also demonstrated that the percentage of healthy monocytes expressing CD16, and to a lesser extent RANK and RANKL, was significantly enhanced when they were cultured with CLL-cm alone, IL-10 or TGFβ. It is worth noting that a higher number of monocytes expressing CD16 appeared capable of generating a higher number of large osteoclasts, and, indeed, the addition of an anti-CD16 neutralizing antibody counteracted osteoclast differentiation. Researcehrs further demonstrated that monocytes polarized toward the M2 phenotype, in particular M2c, were more prone to differentiate toward osteoclasts
[31]. In agreement with our observations, Jihyun Yang et al.
[32] observed that M2 monocytes differentiated into osteoclasts more efficiently than M1, especially when pre-activated with IL-10. Gene expression profile analysis of public data showed that the key osteoclastogenic transcription factor NFATC1 was also significantly higher in M2 versus M1 monocytes in CD16+ versus CD16- monocytes and in intermediate and non-classical versus classical monocytes, further supporting the results from our experiments
[31]. Collectively, these findings suggest that the expression of CD16 facilitates the differentiation of monocytes toward osteoclasts and that CD16 could represent a marker of osteoclast precursors in CLL, as was previously indicated for psoriatic arthritis
[33]. It is also worth noting that, in particular contexts, myeloid-derived suppressor cells (MDSCs) may differentiate toward osteoclast precursors, as demonstrated in breast cancer, rheumatoid arthritis or multiple myeloma
[34][35][36][37][38][39]. The observations presented here are schematically summarized in
Figure 2.
The above-reported data may suggest that myeloid cells, expanded under the influence of leukemic B cells and acquiring an immunosuppressive phenotype, may directly or indirectly contribute to bone derangement in CLL patients: together with the production of cytokines, such as TGFβ and IL-10, the upregulation of the FcγRIIIa (CD16) antigen also appeared to contribute to the amplification of osteoclasts differentiation.