MicroRNA in Chronic Lymphocytic Leukemia Figure 3. CLL cells express elevated levels of B cell activation markers. Unstimulated donor B cells, anti-IgM-activated donor B cells, or CLL cells were stained for CD69, CD80 or CD86 levels by FACS analysis. Grey filled curve shows the isotype control, open curve shows the specific antibody staining. One representative unactivated donor B cell sample (B-), one anti-IgM-activated B cell sample (B+) and two CLL samples (L30 and L41) are shown. An expanded analysis of donor and patient samples is shown in Table S3. doi:10.1371/journal.pone.0016956.g003 This variability in expression of these miRNAs may be related to the heterogeneous clinical course. Notably, nine of the 12 miRNAs changed in the same direction in CLL cells compared to their changes when B cells are activated (Figure 1). Several factors can affect the direction of miRNA expression changes in CLL cells and activated B cells including the timing of altered miRNA expression, type of stimulation, or that certain changes may be important to the pathobiology of CLL. Interestingly, the expression of the two prognostic miRNAs (miR-29C and miR- 223) varies in activated B cells depending upon the type of stimulation (Table 1). Interrogating the causes and consequences of altered miRNA expression in CLL cells in common with and distinct from altered miRNA expression in activated B cells will improve our understanding of this disease. Another striking finding is that certain CLL signature miRNAs demonstrate similar trends in activated B cells regardless of the mechanism of activation (Table 1). Mir-23a, miR-23b, miR-24, miR- 27b, miR-155, miR-181a, miR-181b, and miR-223 are all altered consistently with activated B cells. However, certain CLL signature miRNAs are altered consistent with specific B cell activators. For example miR-29b and miR-29c are downregulated with IgM and CD40L activation but are upregulated with TLR activation. Two recent reports examine differential miRNA expression in na¨ıve, germinal center, memory and plasma B cell populations [14,34]. Interestingly, miR-29c is reduced in germinal center B cells [14] and in centroblasts [34] but is increased in memory B cells [14]. The pattern of miR-29 family member expression may be affected by the duration of B cell activation and/or to multiple B cell activators present in germinal centers proceed through T cell-dependent (e.g. CD40L) and T cell-independent (e.g. anti-IgM) pathways acting simultaneously. Still, certain alterations in miRNA expression are common with published observations such as downregulated miR- 181b and miR-223 [34] regardless of the mechanism of activation suggesting that B cell activation may converge on common miRNA- regulated pathways. Importantly, these common miRNAs are also present in the CLL signature reported here. Recent reports indicate that certain miRNAs identified in this CLL signature have prognostic value in CLL [30,31,32] Additionally, CLL patients expressing CD38 tend to have higher expression of activated cellular markers, including CD27, CD62L, CD69 and KI-67, and this activation status is correlated with poor clinical outcomes [35,36]. Our study integrates these two PLoS ONE | www.plosone.org 6 March 2011 | Volume 6 | Issue 3 | e16956