br and B In the animal tumor model the DNT
and B). In the animal tumor model, the DNT cell group and gem-citabine group had more apoptotic tissues than did the blank control group (Fig. 5C). Western blotting revealed that the expression of cleaved caspase-3 in the cancer cell þ DNT group was higher than that in the other two groups (Fig. 5D). Moreover, the DNT cell group and gemcitabine group had higher cleaved caspase-3 expression than did the blank control group (Fig. 5E).
Regulatory T cell populations play a critical role in the regulation of the tumor immunological response. Notably, recent studies have suggested that specific subsets of T Imipenem (referred to as CD3þCD4 CD8 T cells) also play a role in this regulatory process. The majority of T cell receptor (TCR)þ lymphocytes in the periph-eral blood and lymphoid organs of humans express CD4 or CD8 cell markers, whereas only 1e5% of T lymphocytes express TCR but not CD4 or CD8; these T lymphocytes are defined as DNT cells [17,18]. CD8þ T cells, CD3þCD56 þ T cells and NKT cells are antitumor cells with extensively investigated antitumor effects [19e21]. Unlike the aforementioned lymphocytes, DNT cells have not been widely re-ported in studies of cancer immunity, especially in solid cancers, because of their low purity and a lack of effective methods for their expansion. Merims et al.  developed a novel protocol by which DNT cells could be expanded ex vivo in 2 weeks. These ex vivo DNT cells effectively killed CD34 þ leukemic blasts, thus displaying antileukemic activity. By using the abovementioned procedures, Lee et al.  successfully amplified allogeneic human DNT cells that could be used as immunotherapy for acute myeloid leukemia. Here, we obtained enriched DNT cells from only 10 ml peripheral blood collected from volunteers using this novel protocol. In the present study, the growth of Panc-1 and SW1990 cells was inhibi-ted when these cells were cultured together with DNT cells in vitro. In the nude mouse tumor model, DNT cells had an inhibitory effect on the growth of transplanted tumors in vivo. Moreover, apoptosis assays suggested that DNT cells could significantly promote cancer cell and tissue apoptosis. In addition, the antitumor effect of DNT cells was similar to that of the clinically used chemotherapy drug gemcitabine, which may provide further evidence for the clinical use of DNT cell immunotherapy for pancreatic cancer.
Recently, DNT cells specifically killing tumor cells has received increased attention from scholars [13,14], but the specific mecha-nism involved remains unclear. According to several studies, the Fas/FasL pathway is an important signal transduction pathway for apoptosis in cells and tissues [22e25]. When Fas is combined with FasL, Fas recruits the adapter protein Fas-associated death domain (FADD) and caspase-8, transforms into a death-inducing signaling complex (DISC), activates caspase-3 and finally induces cell apoptosis. Caspases can be cleaved into an activated form [26e28]. The abovementioned proteins all belong to the Fas-mediated pathway effector proteins. Based on several studies, Fas, caspase-8, cleaved caspase-8 and cleaved caspase-3 expression is increased when the Fas pathway is activated [29e32]. In a previous study by Young et al. , DNT cells inhibited the growth of lym-phoma via the Fas pathway. In this study, the changes in pathway effector protein expression and the DNT cell antitumor effect were the most likely explanation for the involvement of the Fas signaling pathway in DNT cell antipancreatic effects in vitro. Here, the expression of the pathway effector proteins Fas, caspase-8, cleaved caspase-8 and cleaved caspase-3 in cancer cells cocultured with DNT cells was higher than that in untreated cancer cells. Blocking agent DcR3 competitively binds to FasL, preventing its binding to Fas receptors and reducing death signal transmission . When DNT cells were pretreated with blocking agent DcR3 before cocul-ture with cancer cells, the expression of pathway effector proteins in cancer cells was decreased, and the antitumor effect of DNT cells was significantly reduced. Therefore, the Fas pathway plays a pos-itive role in the antitumor effect of DNT cells on pancreatic cancer cells in vitro.
In the nude mouse model experiment, western blotting and qPCR analyses showed that the expression of pathway effector proteins Fas, caspase-8, cleaved caspase-8 and cleaved caspase-3 in mouse tumor tissues was higher in the DNT cell group than in the
blank control group. This result was similar to that of our in vitro experiment. This finding suggested that DNT cell cytotoxicity to pancreatic cancer in vivo may also be mediated via the Fas/FasL pathway. However, additional in vivo studies are needed for veri-fication and to make our conclusions more convincing. Moreover, the result showed an interesting phenomenon that gemcitabine had similar effects on the activation of Fas pathway compared to DNT cells. But the studies on the inhibitory effects of gemcitabine on the growth of pancreatic cancer via the Fas signaling pathway are lacking. It is still unclear that the mechanism for both DNT cells and gemcitabine through Fas signaling pathway are different or the same. Gemcitabine is a commonly used chemotherapeutic drug. It may have the ability to directly activate the Fas pathway compared to DNT cells which rely on their own FasL. Therefore, further studies are needed to verify our hypothesis.