IntroductionB-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of a monoclonal population of CD5 ϩ neoplastic B cells in secondary lymphoid organs, marrow, and blood. Because most of the circulating leukemia cells are arrested in the G 0 /G 1 phase of the cell cycle, the primary defect may be one of resistance to programmed cell death rather than accelerated cell division. 1,2 However, CLL cells can rapidly undergo spontaneous apoptosis under culture conditions that support the growth of human B-cell lines. This implies that such ex vivo conditions lack factors necessary for leukemia cell survival or that the resistance to apoptosis is not intrinsic to the leukemia B cell.The leukemia cell microenvironment in the marrow or in secondary lymphoid tissues may contribute to the noted resistance of CLL cells to apoptosis in vivo. 3,4 Normal B-cell development depends on complex interactions with accessory cells that define the so-called specialized microenvironments. T cells and a variety of different types of adherent cells, generally defined as stromal cells, are the main elements of the microenvironment. 3 In patients with CLL, the marrow invariably is infiltrated with CLL B cells, and the pattern and extent of marrow involvement correlates with clinical stage and prognosis. 5,6 As such, interactions with stromal cells in the marrow microenvironment appear to play a role in disease progression and resistance to therapy. [7][8][9] In addition, we found that a small proportion of the mononuclear cells from the blood of patients with CLL can differentiate into large, round, adherent cells that attract CLL cells and protect them from undergoing spontaneous or drug-induced cell death. 10,11 Because these cells share features with thymic nurse cells that nurture developing thymocytes, we designated them nurselike cells (NLCs). Although NLCs differentiate from blood mononuclear cells after several days in vitro, fully differentiated NLCs can be found in the spleen and secondary lymphoid tissue of patients with CLL, 11 where they might play a role in protecting CLL cells from apoptosis in vivo. This model implies that CLL cells depend on specific extrinsic factors from NLCs and other stromal elements for their survival. Conceivably, CLL cells recirculate from the blood through secondary lymphoid tissues and back into the systemic circulation in response to certain chemokines.One such chemokine is stromal cell-derived factor-1/pre-B cell growth-stimulating factor (SDF-1/PBSF), which recently has been designated CXCL12. CXCL12 is a member of a family of chemotactic cytokines (chemokines) that initially were characterized as growth-stimulating factors for B-cell precursors. 12 CXCR4 is a primary physiologic receptor for CXCL12 and functions as a coreceptor for entry of T-tropic strains of HIV-1. Mutant mice with targeted gene disruption of CXCL12 or CXCR4 have defects in the We previously demonstrated that stromal cells can attract CLL cells through the production of CXCL12. 13 In addition, NLCs e...

There is growing evidence that the microenvironment confers survival signals to Chronic Lymphocytic Leukemia (CLL) B-cells that may result in disease progression and resistance to therapy. In the marrow or secondary lymphoid tissues, CLL cells are in close contact with non-tumoral accessory cells, such as mesenchymal stromal cells or nurselike cells. We previously characterized SDF-1 (CXCL12) as a central mediator for CLL cell migration and interaction with the protective microenvironment. Constitutive secretion of CXCL12 attracts CLL cells to stroma or NLC through its cognate receptor, CXCR4. These accessory cells protect CLL cells from spontaneous or drug-induced apoptosis, which is contact-dependent and partially mediated by CXCL12. B-cell receptor (BCR) signaling has been considered another important regulator of CLL cell survival. Typically, CLL cell that lack somatic mutations in the immunoglobulin (Ig) variable region (V) genes and display high levels of the tyrosine kinase ZAP-70 strongly responds to anti-IgM stimulation. Because both, CXCL12 stimulation and BCR signaling may represent important mechanism for maintenance of CLL cell within the microenvironment, we examined whether anti-IgM stimulation affects CXCL12 responses in correlation with the ZAP-70 status. BCR signaling was modulated either by crosslinking the BCR with IgM or by blocking the tyrosine kinase Syk. Effective BCR cross-linking with anti-IgM antibodies was demonstrated by phosphorylation of Syk and p44/42 MAP kinase. In ZAP-70 positive cells, BCR crosslinking resulted in a robust activation of Syk, p44/42 MAP kinases, and protein kinase B (Akt). ZAP-70 negative CLL cells displayed a weaker activation of p44/42 upon IgM crosslinking. Pretreatment of CLL cells with anti-IgM resulted in an enhanced calcium mobilization upon CXCL12 stimulation. This was not due to changes in surface expression of CXCR4. Accordingly, Syk inhibition by piceatannol resulted in a loss of calcium response upon CXCL12 stimulation. Furthermore, anti-IgM stimulation significantly increased CLL cell chemotaxis towards CXCL12 1.4 ± 1.2fold (n=9, p=0.027), and Syk inhibition by piceatannol decreased chemotaxis to 0.6 ± 0.2fold of controls (n=8). In these experiments, we could not detect differences between ZAP-70 positive or negative cells. However, there was a strong difference regarding the spontaneous, CXCL12-dependent migration of CLL cells beneath marrow stromal cells (pseudoemperipolesis). BCR crosslinking significantly increased pseudoemperipolesis of ZAP-70 expressing CLL cells 13.4 ± 21.0fold (n=7, p=0.043), whereas there was no significant increase in pseudoemperipolesis of ZAP-70 negative cells (1.4 ± 0.2fold increase, n=8). Syk inhibition by piceatannol significantly decreased the pseudoemperipolesis of ZAP-70 positive as well as ZAP-70 negative CLL cells to 0.4 ± 0.07 of controls (n=5, p=0.043). Interestingly, spontaneous migration of CLL cells beneath follicular dendritic cells (HK cells) was also significantly enhanced by anti-IgM stimulation, in particular in ZAP-70 positive cases. In summary, BCR signaling enhances calcium mobilization, CLL cell migration to CXCL12, and pseudoemperipolesis beneath marrow stroma or follicular dendritic cells. These data suggest that BCR stimulation co-operates with CXCL12 for localization and/or maintenance of CLL cells within distinct tissue microenvironments.