CCR4: TARC Interaction Provides Supplementary Pro-Survival and Proliferative Signals to Chronic Lymphocytic Leukemia Cells.

Abstract 2327

Poster Board II-304

A wealth of information is accumulating on a role for antigenic stimulation in the natural history of chronic lymphocytic leukemia (B-CLL). Depending on the specificity of antigen:B-cell receptor (BCR) interactions, mature B cells can recognize and respond to microbial antigens via their BCRs and via Toll-like receptors (TLRs), possibly in a costimulatory manner. Chemokines and their receptors (CXCR3, CXCR4 and CCR7) also play critical roles in leucocyte trafficking as well as cell survival and expansion in B-CLL. Here we report our findings on CC chemokine receptor 4 (CCR4) expression and function in B-CLL. PBMCs from 64 B-CLL cases were screened for the expression of CCR4 on CD5⁺ B cells. CCR4 expression did not differ significantly when cases were grouped on the basis of IgV gene mutations. However when segregated by expression of CD38, cases with high CD38 positivity (>30%) showed significantly higher percentages of CCR4-expressing cells (52.8 ± 5.2; p< 0.01) than cases with low CD38 positivity (33.7 ± 5.3%). Surprisingly, among CD38 high expressers, those with mutated IgV genes exhibited even more CCR4-expressing cells (78.7 ± 7.8 %; p< 0.01) than those with unmutated IgV genes (44.6 ± 6.4%). Various functional outcomes of CCR4 interaction with its ligand, Thymus and Activation Regulated Chemokine, TARC (CCL17) were assayed. Using Phosphoflow, phosphorylation of Akt, Erk, MAPK, Syk, Btk, NF-kB, STAT-3 and STAT-5 in response to varying concentrations of TARC (0.04-25ng/ml) was assessed. Significant increases in phosphorylation of Btk and STAT-5 were detected in response to varying doses of TARC in different cases. However, TARC did not elicit noticeable changes in phospho- Akt, -Erk, -MAPK, -Syk, -NF-kB and -STAT-3. Based on the percentages of CCR4-expressing cells in B-CLL clones, TARC specifically induced decreases in surface CCR4 expression within 15 minutes, whereas it also exhibited indirect effects on reduction of surface expression CXCR3 and CXCR4 expression in ∼60% of the cases studied. Furthermore a chemotactic response (migration) of CLL cells (studied using transwell cultures) was observed within a 2 hour exposure in the same dose range as above. TARC also exhibited significant downstream effects. It independently rescued CCR4⁺ B-CLL cells from apoptosis (25 - 32%) as assessed by Annexin V/PI staining after 1 day exposure. Concomitantly in those cases in which B-CLL cells were rescued from apoptosis at day 1, TARC induced proliferation of the leukemic cells in a dose-dependent manner (³H thymidine uptake at day 3). Although there was no appreciable change in percentage of cells expressing CCR4, increases in the density of CCR4 expression were inducible in response to [1] crosslinking BCR with monoclonal anti-IgM antibodies conjugated to dextran (1.47 ± 0.2 fold), [2] TLR agonist, ODN 2006 (1.63 ± 0.26 fold), [3] crosslinking with anti-CD40 mAb + IL-4 (1.9 ± 0.4 fold), and [4] in response to interaction of CD38 with CD31-expressing fibroblasts (1.27 ± 0.09 fold) when B-CLL cells from 8 IgM⁺ cases were cultured with these stimuli for a 3 day period. These findings suggest a novel role for CCR4:TARC interaction in promoting B-CLL cell survival and promoting cell cycle progression downstream of BCR and TLR triggering in the presence/absence of residual T cell help. This knowledge could specifically aid in understanding the progression of disease in the B-CLL cases with mutated IgV genes and high percentages of CD38⁺ cells.