The human pharmacopeia includes IFN-I 6. Direct effects on malignant or virus-infected cells have been considered the main mechanism for the efficacy of IFN-I in therapy. However, IFN-I directly regulates many immune system cells such as NK cells, DC and B- and T-lymphocytes 7. In mice, IFN-α/β are important enhancers

of CD8+ T-cell responses 8. One contributing factor is DC stimulation 9. However, direct effects of IFN-I on DC seem to be insufficient for CD8+ T-cell priming 8, 10. IFN-I also exerts direct effects on murine CD8+ T cells 4, 10–13. The most definitive report came from Kolumam et al.12 who showed that IFN-I directly targets anti-viral CD8+ T cells in vivo allowing their clonal expansion and differentiation into memory cells. Elegant experiments in mice by the group of Mescher 11 have shown that, in addition to signals Liproxstatin-1 order via TCR (signal-1) and CD28 (signal-2), naïve CD8+ T cells require a third signal. Signal-3 delivered by IL-12 or IFN-α is Raf inhibitor required for expansion, acquisition of effector functions and memory differentiation. cDNA microarray analyses show that IFN-α as a signal-3 regulates critical genes involved in CTL functions

14, providing evidences that IFN-α promotes activation and differentiation of CD8+ T cells by sustaining the expression of key genes through chromatin remodeling. There is very scanty information about the effects of IFN-I on human CD8+ T cells and how IFN-I may alter the response of different CD8+ T-cell subsets. Since IFN-α is frequently prescribed to patients with a variety of medical conditions, it is of great importance to determine whether mouse and human CD8+ T cells respond in the same way to this bio-therapeutic agent. Using good manufacturing practice (GMP)-grade recombinant IFN-α and Beads coated with anti-human CD3 and CD28 mAb Oxaprozin to mimic type-1 and type-2 signals, we show that IFN-α provides a strong type-3 signal directly to human CD8+ T cells supporting the acquisition of effector functions. Intriguing distinct IFN-α effects on the expansion of human naïve and Ag-experienced CD8+ T cells are described. Magnetically

sorted untouched CD8+CD45RO− cells were stimulated (7 h) with GMP-grade recombinant IFN-α2b or IFN-α5 and their transcriptional profiles were defined by cDNA microarrays (Series GSE17299, deposited in the Gene Expression Omnibus (GEO) database, accession number GSE17302). In total, 195 genes changed at least two-fold by either IFN-α2b or IFN-α5 and 161 genes were regulated in common. Supporting Information Table 1 groups genes by functional pathways. The regulation of several transcripts involved in cell-mediated cytotoxicity [TNFSF10 (also known as TNF-related apoptosis-inducing ligand (TRAIL), FASLG and PRF1], chemotaxis (CXCL10 and CXCL11) and T-cell homeostatic proliferation (IL15RA) were confirmed by quantitative RT-PCR (Table 1A).