CD14+ blood mononuclear cells co-cultured with chronic lymphocytic leukemia (CLL) B cells differentiate into nurselike cells (NLCs) that in turn can support CLL-cell survival in vitro and possibly in vivo. These cells appear similar to lymphoma-associated macrophages, which were identified in secondary lymphoid tissue of patients with follicular lymphoma and appear more prevalent in patients with therapy-resistant disease. To investigate the relationship between NLC and macrophages, we performed studies on macrophages and NLCs that were induced to differentiate from CD14+ blood mononuclear cells in vitro. Consistent with prior studies, we found that NLCs express significantly higher levels of CD68 than macrophages, as assessed via cytoplasmic flow cytometry. However, Affymatrix U113A microarray analysis of gene expression by NLC, macrophages, and monocytes-derived dendritic cells (DCs) from 3 donors revealed major differences in gene expression between DCs versus macrophages or NLCs, but no major differences in gene expression profiles between NLCs and macrophages. Flow cytometric analyses of NLCs and macrophages revealed that these two cell types also shared similar expression levels of CD16, CD32, CD35, CD86, CD58, MHC-II, CD40, and CD54. However, using flow cytometry we found that NLCs (n=9) expressed significantly higher levels than macrophages of the B-cell activating factor belonging to the tumor necrosis factor family (BAFF). Deconvolution microscopy confirmed the differences in BAFF expression and also revealed that NLCs express higher levels of a proliferation-inducing ligand (APRIL) than macrophages. These are two key factors involved in promoting leukemia/lymphoma B cell survival. Moreover, NLCs maintained high-level expression of BAFF even when cultured apart from CLL cells in fresh medium. We investigated whether co-culture of differentiated macrophages with CLL cells could induce the macrophages to express high-levels of BAFF. Although such co-culture induced progressive increase in macrophages-expression of BAFF, the levels of BAFF induced after even 7 days of co-culture were lower than those noted for NLCs. We cultured CD14+ blood monocytes and CLL cells separated across a transwell membrane to determine whether a soluble factor(s) was responsible for the induction of high BAFF levels noted on NLCs. Following several days in culture, the cultured monocytes acquired expression levels of BAFF similar to those detected for NLCs. These studies indicate that monocytes can respond to a soluble factor(s) elaborated by CLL cells to assume properties similar to those of NLCs. Moreover they suggest that NLCs may be a peculiar type of terminally differentiated macrophages-like cells induced by the leukemia-cell population to have properties that promote CLL cell survival. Agents that can block the maturation of monocytes into NLCs or that inhibit the capacity of NLCs to promote leukemia-cell survival may be effective in the treatment of CLL and related lymphoid malignancies.

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