CCR4: TARC Interaction Provides Supplementary Pro-Survival and Proliferative Signals to Chronic Lymphocytic Leukemia Cells.
Authors: Damle Rajendra N , Temburni Sonal , Kaufman Matthew , Kolitz Jonathan E. , Allen Steven L. , Malavasi Fabio , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
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.

Disclosures:
No relevant conflicts of interest to declare.