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Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases. [Cancer Res 2009;69(16):6387–95]

We previously reported that B-cell receptor (BCR) signaling promoted a concomitant decrease of CXCR4 and CD62L membrane expression only in progressive cases of chronic lymphocytic leukemia (CLL). Recently, Quiroga and colleagues raised some challenging issues and pointed out discrepancies between the two reports. In their letter, Quiroga and colleagues questioned our BCR stimulation protocol. Several studies, including ours, reported that soluble anti-immunoglobulin M (IgM) resulted in apoptosis, whereas immobilized anti-IgM or F(ab9)2 fragment both sustained cell survival (1–3). Hence, the increased cell survival following soluble F(ab9)2 exposure observed by Quiroga and colleagues is rather surprising. We showed that downregulation of CXCR4 was associated with a reduced migration toward CXCL12. Our results are in agreement with numerous reports in various cell types including CLL cells (4). Quiroga and colleagues reported a similar CXCR4 decrease but associated to an unexplained increased chemotaxis toward CXCL12. They relied on data from normal B cells isolated from the bone marrow, hypothesizing that several CXCR4 variants confer differential responsiveness to CXCL12. We considered studying BCR stimulation in the context of lymph nodes to be more relevant: (i) BCR stimulation occurs mainly in lymph nodes; (ii) a higher score of BCR-activated targets in lymph nodes versus bone marrow was recently found (5). Therefore, using a lymph node endothelial cell line in our adhesion assays seems a more physiological model than a bone marrow stromal cell line. Quiroga and colleagues reported a decrease of CXCR4 associated with an increase of CD62L upon BCR engagement. In contrast, we found a systematically coordinated downregulation of both CXCR4 and CD62L occurring in the same subset of cells. We thus believe that our experimental data, done on a delineated subset of cells of 30 untreated patients, are stronger than the results expressed as the mean value of a whole cohort as provided by Quiroga and colleagues. Finally, their inhibition experiment of Syk, the major BCR effector, restores CXCR4 and CD62L levels of expression. This result is in line with our data and is in agreement with the clinical observation on patients treated by the Syk inhibitor R788 showing an initial increase in circulating lymphocytes associated to a decrease in lymph node size, indicative of an exit of CLL cells from the lymph nodes. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.