2.45 CCR4–Ligand Interactions Contribute Pro-Survival and Proliferative Signals to Chronic Lymphocytic Leukemia B Cells

Abstract

B-cell chronic lymphocytic leukemia (B-CLL) exhibits remarkable heterogeneity in the rate of disease progression between patients. In addition to receiving signals via the B-cell antigen receptor (BCR) and Toll-like receptors (TLRs), CLL cells survive by receiving and responding to signals elicited by their interactions with cytokines and chemokines derived from the microenvironment. Here, we report our findings on CC chemokine receptor 4 (CCR4) expression and function in B-CLL. Within the microenvironment, B-CLL cells attract CD4+ CD40 ligand (CD40L)+ T cells by producing the chemokine CCL22. We asked if B-CLL cells themselves could respond to CCL22 by studying the expression of CCR4, a receptor for CCL22 and CCL17. Peripheral blood mononuclear cells from 80 B-CLL patients were screened for expression of CCR4 on CD5+ B cells. CCR4 expression did not differ significantly when cases were grouped on the basis of IGHV gene mutations. However, when segregated by expression of CD38, cases with high numbers of CD38-positive cells (≥ 30%) showed significantly higher percentages of CCR4-expressing cells (52.8 ± 5.2%) than cases with low CD38 positivity (33.7 ± 5.3%; p < 0.01). Surprisingly, among CD38 high-expressers, those with mutated IGHV genes exhibited even more CCR4-expressing cells (78.7 ± 7.8%) than those with unmutated IGHV genes (44.6 ± 6.4%; p< 0.01). Although there was no appreciable increase in the percentage of cells expressing CCR4 when B-CLL cells from 12 cases were cultured with these stimuli for a 3 day period, density of CCR4 expression was increased in response to: (1) crosslinking with monoclonal anti-immunoglobulin M antibodies conjugated to dextran (1.47 ± 0.2-fold); (2) the TLR agonist ODN 2006 (1.63 ± 0.26-fold); (3) crosslinking with anti-CD40 monoclonal antibody plus interleukin-4 (1.9 ± 0.4-fold); and (4) an interaction of CD38 with CD31-expressing fibroblasts (1.27 ± 0.09-fold). Various functional outcomes of CCR4 interaction with its ligands CCL17 and CCL22 were also assayed. Using Phospho-Flow, the phosphorylation of several key cell-signaling intermediates in response to varying concentrations (0.04–25 ng/mL) of CCL17 and CCL22 was assayed and significant increases in phosphorylation of Btk and STAT5 were detected. Furthermore, both CCL17 and CCL22 induced a chemotactic response in CLL cells within a 2 hour exposure when assayed using Transwell cultures. Since the observed migratory responses to the ligands were bimodal in most cases, a comparison of expression of other molecules – CXCR3, CXCR4, CCR4, and CCR7 (molecules involved in homing of cells to sites favoring growth), and CD31, CD38, and CD69 (activation-related molecules) – was performed. The migrated cells consistently showed significantly higher percentages and densities of CD38 expression than the non-migrated cells, suggesting a role for CD38 in the CCR4-mediated downstream pathway. CCL17 and CCL22 also exhibited downstream effects, independently up-regulating anti-apoptotic proteins Mcl-1 and Bcl2 and down-regulating pro-apoptotic molecules Bim, PUMA, and Bid in 25–30% of cases studied. Furthermore they rescued CCR4+ B-CLL cells (25–32%) from apoptosis, as assessed by Annexin V/PI staining after 1 day of exposure. Concomitantly in those cases in which B-CLL cells were rescued from apoptosis at day 1, CCL17 and CCL22 induced proliferation of leukemic cells in a dose-dependent manner (3H-thymidine uptake at day 3). Because CCR4 ligands may exert an accessory role in BCR- and TLR-9-mediated immune responses in B cells, we tested whether CCL17 and CCL22 further modulated proliferative responses initiated by these 2 receptors in a cohort of 31 CLL cases (16 unmutated [U]-CLL and 15 mutated [M]-CLL). CCL17 augmented BCR-mediated B-cell proliferation in 9/16 U-CLL cases (56%), but only in 3/15 M-CLL cases (20%). On the other hand, CCL17 showed an additive effect in promoting TLR-9-mediated cell proliferation in 13/15 M-CLL cases (87%) at 2 ng/nL, a dose that approximates that detected in serum; CCL17 also augmented TLR-9-mediated B-cell proliferation in 6/16 U-CLL cases, but at a 5-fold or higher dose (10–25 ng/mL). Overall, these findings suggest a role for a novel receptor–ligand combination in promoting B-CLL cell survival downstream of BCR and TLR triggering. Therefore, inhibition of CCR4–ligand interactions in vivo may be a novel therapy, preventing migration of CLL cells toward an environment that promotes their survival.