These PD 0332991 results encourage us to apply combination therapy of therapeutic mAbs with IL-21. Although the effect of IL-21 on murine NK cells has been extensively elucidated, there is still limited information about the effect of IL-21 on human NK cells (Skak et al, 2008b). IL-21 had only moderate effects on surface levels of inhibitory (CD158a and CD158b) and activating NK-cell receptors (NKG2D) in human NK cells (Skak et al, 2008a). Moreover, IL-21 was able to increase both mRNA and protein levels of the effector molecules perforin and granzyme-B, as well as enhance NK cytotoxicity against K562 (Skak et al, 2008a). In this study, we provided novel findings that IL-21 induced the significant upregulation of CD247 molecules on NK cells, important molecules for Fc receptor (CD16)-dependent NK killing (Whiteside, 2004).

These results suggested that the upregulation of CD247 molecules was one of the mechanisms behind IL-21-enhanced ADCC in patients with ESCC. However, we did not detect the upregulation of perforin and granzyme-B in response to IL-21 treatment in this study. Although the reason for this discrepancy is currently unknown, it may be due to the difference in in vitro culture condition, for example, unfractionated PBMCs vs purified NK cells, or incubation time. In this study, we showed that IL-21 could directly act on NK cells, as one of the mechanisms behind IL-21 enhances ADCC activity, as it was shown that purified NK cells treated with IL-21 could enhance ADCC activity.

In addition, it was previously shown that IL-21 indirectly enhanced NK cell function through cytokine production such as IFN-��, when PBMCs were treated with IL-21 (Roda et al, 2007). Thus, it is likely that IL-21 has pleiotrophic roles in a wide variety of cells, leading to the enhancement of ADCC activity (Roda et al, 2006). In this study, we showed that the highest dose of IL-21 was sometimes less effective for the enhancement of ADCC than Carfilzomib IL-21 at lower levels. It is likely that there might be some plateau effect of IL-21 at the highest dose in some patients, although further experiments are needed to clarify the mechanisms. A phase I study involving IL-21 monotherapy for metastatic melanoma or renal cell carcinoma reported that monotherapy was well tolerated and exhibited anti-tumour activity in some patients (Davis et al, 2007; Thompson et al, 2008), thereby suggesting that IL-21 may have an anti-tumour effect as a monotherapy. However, this study clearly showed that IL-21 could efficiently enhance impaired ADCC activity in ESCC patients, suggesting that combination therapy of Trastuzumab or Cetuximab with IL-21 might result in the enhancement of the anti-tumour effect.