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Adenoviruses (Ad) have been extensively studied as gene delivery vectors in gene therapy and as vaccine carriers. The cell-mediated cytotoxicity induced by Ad is of great interest in both applications. However, the mechanism underlying Ad-specific cytotoxic T lymphocyte (CTL) generation and effector function remains unclear. In this study, we used a novel MHC class I tetramer and an in vivo CTL assay to examine the role of CD28, perforin, Fas ligand (FasL), and TNF-α in the generation and function of Ad-specific CTLs in vivo. During the primary response, there was a significant defect in both the generation and in vivo effector function of Ad-specific CTLs in CD28–/– mice, but not in CD4+ T cell-depleted mice or CD4–/– mice. The relative role of CTL effector molecules was assayed by in vivo CTL assay in perforin- or FasL-mutant mice, using donor cells from Fas-deficient or TNFR1/TNFR2-deficient mice. The results indicated that the in vivo CTL activity is mediated mainly by perforin. In the absence of perforin, production of FasL, but not TNF-α, by the CTLs results in lower level Ad-specific killing of target cells. These results provide important implications concerning the development of safe and effective Ad vectors for gene therapy and vaccines.

To determine whether defective T cell apoptosis is associated with the development of a chronic arthritis subsequent to mycoplasma infection, and to determine whether deletion of T cells can prevent the development of this arthritis. B6 wild-type (B6-+/+), B6-lpr/lpr, and B6-gld/gld mice were infected with Mycoplasma pulmonis. The severity of lymphocytic infiltration and joint damage was evaluated, and the degree of recovery of viable mycoplasma from the spleen and joints was determined. Antigen-presenting cells derived from Fas mutant lpr mice (lpr-APC) were transfected ex vivo with an adenovirus (Ad) vector to yield lpr-APC expressing high levels of Fas ligand (lpr-APC-AdFasL), which in turn were transferred intraperitoneally into M pulmonis–infected B6-gld/gld mice. The development of arthritis subsequent to M pulmonis infection and the induction of apoptosis of cells within the synovial tissue and lymph nodes of lpr-APC-AdFasL–treated B6-gld/gld mice were determined. Infection of B6-lpr/lpr and B6-gld/gld mice with M pulmonis resulted in an acute-phase inflammation of the synovium that later developed into a chronic erosive arthritis. Similar infection of B6-+/+ mice resulted only in an acute joint inflammatory response that resolved. Chronic arthritis in B6-gld/gld mice and B6-lpr/lpr was not due to persistent infection, since there were no differences in the rates of clearance of M pulmonis from the joints of B6-gld/gld or B6-lpr/lpr mice compared with B6-+/+ mice. Treatment of infected B6-gld/gld mice with lpr-APC-AdFasL resulted in a significantly decreased incidence of chronic arthritis that was associated with a decrease in lymph node T cells, but not with apoptosis of synovial T cells or fibroblasts. Defective Fas/FasL-mediated apoptosis of T cells is an important factor that rendered arthritis-resistant B6 mice susceptible to the development of a chronic erosive arthritis subsequent to mycoplasma infection. In vivo lpr-APC-AdFasL cell–gene therapy is a safe and effective method for inhibiting the development of this arthritis.

To determine if tumor necrosis factor α (TNFα)–driven proliferation of rheumatoid arthritis synovial fibroblasts (RASF) is associated with up-regulation of the activity of serine/threonine kinase B/Akt and with survival of RASF. Staining of phosphorylated Akt was done using anti–phosphorylated Thr³⁰⁸ Akt antibody. Levels of phosphorylated Akt were analyzed by Western blot and Akt activity was analyzed using a kinase assay. TUNEL staining was used to analyze the cytotoxicity of TNFα treatment or TNFα combined with either the Akt activity inhibitor wortmannin, an adenovirus expressing dominant-negative mutant (AdAkt-DN), or an adenovirus expressing phosphatase and tensin homolog deleted on chromosome 10 (AdPTEN). The levels of phosphorylated Akt were higher in RASF than in osteoarthritis synovial fibroblasts (OASF), as demonstrated by immunohistochemical staining, immunoblot analysis, and an Akt kinase assay. The levels of phosphorylated Akt and Akt kinase activity were increased by stimulation of primary RASF with TNFα (10 ng/ml). Treatment of RASF with the phosphatidylinositol 3-kinase inhibitor wortmannin (50 nM) plus TNFα resulted in apoptosis of 60 ± 8% (mean ± SEM) of RASF within 24 hours. This pro-apoptosis effect was specific for Akt, since equivalent levels of apoptosis were observed upon TNFα treatment of RASF transfected with AdAkt-DN and with AdPTEN, which opposes the action of Akt. These results indicate that phosphorylated Akt acts as a survival signal in RASF and contributes to the stimulatory effect of TNFα on these cells by inhibiting the apoptosis response. This effect was not observed in OASF and may reflect the pathophysiologic changes associated with the proliferating synovium in rheumatoid arthritis.

Tumor necrosis factor α (TNFα) increases the survival and proliferation of human rheumatoid arthritis (RA) cell lines. These experiments were designed to determine if inhibition of nuclear factor κB (NF-κB) nuclear translocation leads to increased apoptosis of TNFα-treated human RA cell lines. We constructed an inhibitor of nuclear factor κB (IκB) dominant-negative adenovirus (AdCMVIκB-DN) and an X-linked inhibitor of apoptosis (XIAP) antisense adenovirus (AdCMVXIAP-AS). Primary RA synovial fibroblast (RASF) cell lines were transfected in vitro, and SV40-transformed RA synovial cell lines in SCID mice were transfected in vivo. Cells were treated with TNFα and analyzed for apoptosis. There was no apoptosis of primary RASF transfected in vitro with AdCMVIκB-DN alone. In contrast, there was apoptosis of >85% of cells treated with AdCMVIκB-DN plus TNFα. Primary RASF in SCID mice also exhibited high levels of apoptosis after in vivo transfection with AdCMVIκB-DN followed by treatment with TNFα. There was no apoptosis after treatment with AdCMVIκB-DN in the absence of TNFα. XIAP is an inhibitor of apoptosis which was up-regulated by TNFα, and this up-regulation was inhibited by AdCMVIκB-DN plus TNFα. Transfection of an AdCMVXIAP-AS gene therapy resulted in increased TNFα-induced apoptosis. AdCMVIκB-DN gene therapy greatly enhances apoptosis due to inhibition of an NF-κB–mediated antiapoptosis signaling pathway, and XIAP is a TNFα-inducible specific inhibitor of apoptosis in RA synovial cell lines. This and other modulators of TNF receptor or the Fas apoptosis pathway may be therapeutically beneficial in facilitating apoptosis of synovial tissue in patients with RA.

ABSTRACTThe clinical application of adenoviral gene therapy currently is impeded by the potent host immune response to the virus, which limits the duration of its effects. In these studies, we investigated the role of TNF-and of a soluble TNF receptor (TNF-bp) in the inflammatory response and expression of a lacZ-expressing adenovirus (AdCMVlacZ) in the liver and lung of mice. The expression of the recombinant adenovirus was studied in mouse liver and lung by determining the activity of the lacZgene product of the adenovirus. The mononuclear cell inflammatory response was determined histologically at different times after intravenous or intranasal administration of AdCMVlacZ. The cytotoxic T cell and antibody response to the adenovirus was determined. Treatment with TNF-bp reduced circulating levels of TNF-, greatly reduced the inflammatory response, and resulted in prolonged expression of lacZfor up to 30 days in the liver and lung after either intravenous or intranasal administration of adenovirus. Treatment with TNF-bp had no effect on anti-adenovirus antibodies and induction of cytotoxic T cells 30 days after administration of AdCMVlacZ. These results indicate that TNF-is the primary factor driving the early inflammatory response leading to elimination of adenovirus-infected cells in the liver and lung and that TNF-bp is capable of inhibiting these effects.