A defect of the entire JNK function resulted in significantly lower levels of AICD when compared with either intact JNK or a single deficiency of each JNK isoform. Open in a separate window Figure 3 Activation-induced cell death (AICD) of CD8+ T cells Mitochonic acid 5 in c-Jun N-terminal kinase (JNK) -deficient mice after acute ectromelia virus (ECTV) infection. to an inhibition of effector T-cell expansion, as both JNK1 and JNK2 had limited effect on the activation-induced cell death of CD8+ T cells, and only JNK2-deficient mice exhibited a significant change in CD8+ T-cell proliferation after acute ectromelia virus infection. However, optimal activation of CD8+ T cells and their effector functions require signals from both JNK1 and JNK2. Our results suggest that the JNK pathway acts as a critical intermediate in antiviral immunity through regulation of the activation and effector function of CD8+ T cells rather than by altering their expansion. stimulation (reviewed in ref. 18), while JNK signalling mechanisms in CTL responses have only been investigated in a limited number of studies.19C21 Ectromelia virus (ECTV) is an orthopoxvirus and a natural mouse pathogen that causes an infection termed mousepox; it is the classical animal model for the study of biologically relevant CD8 T-cell responses (ref. 22C26, and reviewed in ref. 27). TIL4 C57BL/6 (B6) mice are resistant to acute ECTV infection and generate potent cell-mediated responses and a robust T helper type 1 (Th1) response.24,26 Activation of JNK has been shown in recent infection studies using the orthopox virus vaccinia.28,29 Earlier findings indicated that in addition to the T helper response, CTL responses may also be modulated by JNK signalling (reviewed in ref. 18). Considering the very limited information concerning the role of JNK in biologically relevant CTL responses during viral infections,20 we studied in detail whether the JNK pathway within CD8+ T cells is activated proliferation assay to allow stronger and more efficient stimulation of the donor cells. Animals were monitored twice daily, and at different time-points post infection (p.i.), tissue was processed as previously described.26 For virus titration, BS-C-1 cells were cultured under standard tissue culture conditions in minimum essential medium (Gibco Invitrogen, Carlsbad, CA) with 2 mm l-glutamine and 10% heat-inactivated fetal calf serum (Trace Biosciences, Castle Hill, NSW, Australia), Mitochonic acid 5 and the plaque assay was performed as previously described.26 Flow cytometryAll antibodies used for FACS were purchased from BD Pharmingen (San Jose, CA). Annexin V was purchased from eBioscience (San Diego, CA) and B8R20-27 tetramer was synthesized at the Biomolecular Resources Facility of the Australian National University as described elsewhere.26 Surface and intracellular staining was performed using a standard protocol. For Western blotting, the cell lysates with 30 g of protein were subjected to 10% SDSCPAGE and transferred onto 02-m PVDF transfer membrane (Millipore, Billerica, MA). After blocking with 5% non-fat milk for 2 hr, blots were incubated overnight at 4 with anti-JNK (1 : 1000) or anti-phospho-JNK (1 : 1000) antibodies followed by horseradish peroxidase-conjugated secondary antibodies (all purchased from Cell Signaling Technology, Danvers, MA). Signals were developed by using the enhanced chemiluminescence method according to the manufacturer’s protocol (Pierce, Rockford, IL) and visualized by autoradiography. Cytotoxic T lymphocytes assayAntiviral CTL responses were measured using lymphocytes from the spleens and PLN of individual animals at different time-points p.i. A Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI) was used according to the manufacturer’s instructions. ECTV-infected and uninfected MC57G cells (ATCC CRL-2295) were used as targets to detect the MHC class I-restricted killing. CD8+ cell enrichment, adoptive transfer and proliferation assayCD8+ T cells were Mitochonic acid 5 isolated by negative selection using cell sorting from the spleens of uninfected B6.OT-1, JNK1?/?.OT-1 or JNK2?/?.OT-1 mice as previously described.26 Purified naive CD8+ T cells were then labelled with 5 mm CFSE (Molecular Probes, Eugene, OR), and 1 106 cells were transferred into the lateral tail vein of each of the uninfected recipient wild-type (WT), JNK1?/? or JNK2?/? mice. One day after the cell transfer, each.