Since anti-CCR4 mAb is currently being developed for clinical use, we believe that molecular-targeted therapy and the blockade of CCL17/CCR4 and CCL22/CCR4 pathways using anti-CCR4 mAb may be a potential approach for NNKTL therapy. Acknowledgments The authors thank Dr. significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on Eprodisate Sodium the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells Eprodisate Sodium against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in Eprodisate Sodium the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb. 0.05 was considered statistically significant. All analyses and graphics were done using GraphPad Prism 5 Eprodisate Sodium (GraphPad Software). Results NNKTL cell lines produce CCL17 and CCL22 To identify the chemokines preferentially produced by NNKTL, the chemokine expression patterns of EBV-positive NNKTL cell lines (SNK-6, SNT-8, and SNK-1) were compared with that of KHYG-1 (an EBV-negative NK-cell lymphoma) using a chemokine protein array. The levels of CCL17 were at least 100-fold higher in all 3 NNKTL cell lines, and the levels of CCL22 were at least ten-fold higher in 2/3 NNKTL cell lines, as compared to KHYG-1 cells (Fig. 1a). To validate these observations, the levels of these chemokines were measured by ELISA in culture supernatants of the above 3 NNKTL cell lines, 2 NK-cell leukemia cell lines (YT and KHYG-1), 3 T-cell leukemia cell lines (Jurkat, MOLT-4, and PEER), LCL, and one Hodgkins lymphoma cell line (HDLM-2). As shown in Fig. 1b, a time-dependent accumulation of CCL17 was observed in the culture supernatants of the EBV-positive NNKTL cell lines SNK-6, SNT-8, and SNK-1 cells. Furthermore, 2 NNKTL lines, SNK-6 and SNK-1, also produced CCL22 in a time-dependent manner, while SNT-8 produced a smaller amount of this chemokine. As reported previously [31, 32], CCL17 and CCL22 were observed in culture supernatants of LCL and HDLM-2 cell lines. In contrast, even after 72 h in culture, CCL17 and CCL22 were barely detected in the culture supernatants of the other cell lines tested. Previously, we investigated various EBV characteristics, including the expression of latent membrane protein-1 (LMP-1) in the cell lines used in the present study [10C12]. When we examined whether LMP-1 expression correlated with chemokine production, we found that the NNKTL cell lines that were positive for EBV and LMP-1 were CCL17 and CCL22 producers, while the YT (EBV-positive but LMP-1-negative) and KHYG-1 (EBV and LMP-1-negative) cell lines did not secrete these chemokines (Fig. 1b). These results suggest that the expression of CCL17 and CCL22 in NK-cell malignancies may be somehow regulated by LMP-1. Open in a separate window Fig. 1 Expression of CCL17 and CCL22 in NNKTL cell lines. a Chemokine protein array analysis of CCL17 and CCL22 expression in NNKTL cell lines (SNK-6, SNT-8, and SNK-1) and KHYG-1 cells. The shows the NNKTL cell/KHYG-1 expression ratios, which were calculated as spot intensity of NNKTL cells divided by spot intensity of KHYG-1 cells. The production of CCL17 was 183-fold higher in SNK-6, 101-fold higher in SNT-8, and 183-fold higher in SNK-1 compared with KHYG- 1. The production of CCL22 was 13.8-fold higher in SNK-6, 2.11-fold higher in SNT-8, and 12.8-fold higher in SNK-1 compared with KHYG-1. b Supernatants from the indicated cell cultures (2.5 105/mL) in 96-well round-bottomed plates were collected after 24, 48, and 72 h, and CCL17 and CCL22 production was assessed using ELISA. Columns, means of triplicate determinations; indicate mean values. Rabbit Polyclonal to OR2T2/35 Statistical significance was determined using the MannCWhitney test. b There was a significant correlation between the CCL17 levels and the CCL22 levels in the sera from NNKTL patients. Statistical significance was determined using the Spearman rank correlation CCR4 is expressed on NNKTL cell lines and in NNKTL tissues Next, we determined whether NNKTL cell lines expressed CCR4, the receptor for CCL17 and CCL22 [21, 22]. Flow cytometric analysis revealed that CCR4 was expressed on the surface of 3 NNKTL cell lines (Fig. 3a). To confirm the expression of CCR4 Eprodisate Sodium in NNKTL tissues, immunohistochemistry.