3C). IFN-α2b and IFN-α5 effects were almost identical over the broad range of concentrations tested (Supporting Information Fig. 3). The necessary role of IFNAR was revealed by neutralizing anti-human IFNAR2 mAb (Supporting Information Fig. 4). The CD3-redirected cytolytic assay using OKT3 mAb-coated p815 target cells is commonly

used to evaluate the TCR/CD3-triggered cytotoxicity that entails release of perforin and granzymes, and surface relocation of CD107a. Furthermore, Caki-1 cells, sensitive to TRAIL- but not to FasL-induced cell death, can be used as target cells to assess TRAIL-mediated cytotoxicity 15. Figure 3 strikingly shows that IFN-α enhanced CD3-redirected cytotoxicity (Fig. 3D–E) as well as TRAIL-mediated cytolysis (Fig. 3F–G). Neutralizing anti-TRAIL and anti-FasL mAb revealed the exclusive GDC0199 contribution of TRAIL in the lysis of Caki-1 cells (Fig. 3G). Selleck Ganetespib No significant differences were found between the IFN-α2b and IFN-α5 subtypes in any of these assays (Figs. 1–3 and Supporting Information Figs. 1–4). Following CD27- and CD45RA-based phenotypic classifications of CD8+ T

cells 16, negatively selected total CD8+ T cells were sorted into naïve (CD45RAhighCD27high), memory (CD45RA−CD27+) and effector (CD45RA+CD27− and CD45RA−CD27−) cells. For comparative studies, naïve and memory CD8+ T cells were stimulated as above. Regardless of whether cells were naïve or memory, cell division was not noticeable before 72 h of culture and required CD3/CD28-triggering (Supporting Information

Fig. 5A and B). At day 4 of culture, naïve CD8+ T cells from some individuals (3/8) showed a transiently delayed proliferation in the presence of IFN-α (Supporting Information Fig. 5C). However, from day 5, the extent of division was always higher in cells receiving CD3/CD28/IFNAR-derived signals (observed in 8/8 individuals) (Fig. 4A and Supporting Information Fig. 5A and C). By Niclosamide contrast, once division started, CD3/CD28-induced proliferation of memory cells was always delayed by IFN-α (Fig. 4A and Supporting Information Fig. 5B). Interestingly, IFN-α increased the survival of both CD3/CD28-triggered naïve and memory CD8+ T cells (Supporting Information Fig. 5D and E). IFN-α-derived type-3 signals significantly increased the expansion of human naïve CD8+ T cells whereas they reduced the fold expansion of memory CD8+ T cells (Fig. 4B). When the expression of IFN-γ, Granzyme-B and TRAIL was assessed by flow cytometry analysis, we found that IFN-α enhanced the expression of these three effector molecules both in naïve and memory CD8+ T cells (Supporting Information Fig. 6). However, the fold-change increases in protein induction attributable to IFN-α were markedly higher in naïve cells (Supporting Information Fig. 6). Figure 4C shows that regardless of whether the cells were naïve or memory, the amounts of secreted IFN-γ were higher in cells receiving IFN-α as a signal-3.