Interleukin 15 (IL-15) promotes the survival of natural killer (NK) cells by preventing apoptosis through mechanisms unknown at present. Here we identify Bim, Noxa and Mcl-1 as key regulators of IL-15-dependent survival of NK cells. IL-15 suppressed apoptosis by limiting Bim expression through the kinases Erk1 and Erk2 and mechanisms dependent on the transcription factor Foxo3a, while promoting expression of Mcl-1, which was necessary and sufficient for the survival of NK cells. Withdrawal of IL-15 led to upregulation of Bim and, accordingly, both Bim-deficient and Foxo3a-/- NK cells were resistant to cytokine deprivation. Finally, IL-15-mediated inactivation of Foxo3a and cell survival were dependent on phosphotidylinositol-3-OH kinase. Thus, IL-15 regulates the survival of NK cells at multiple steps, with Bim and Noxa being key antagonists of Mcl-1, the critical survivor factor in this process.

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence.

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The purpose of this study was to evaluate recombinant adeno-associated virus (AAV) as an in vivo gene transfer vector for the retina and to explore the possibility of monitoring the expression of green fluorescent protein (GFP) using a noninvasive method. Rats were injected subretinally with rAAV-gfp or rAAV-lacZ. Strong expression of the reporter gene in a circular area surrounding the injection site was observed in retinal whole mounts and tissue sections. Higher magnification revealed that cells demonstrating high levels of green fluorescence were hexagonal in shape, indicating they were retinal pigment epithelium (RPE) cells. Histological observation of retinal sections demonstrated that recombinant AAV specifically transduced RPE cells. Ten animals were injected with rAAV-gfp for longitudinal studies and the fluorescence was monitored by retinal fluorescence photography. The GFP signal was detected in 100% of the animals as early as 2 weeks postinjection and remained present throughout the experimental period of 4 months. After 2 weeks, a gradual increase in the number of transduced cells occurred before reaching maximal levels of GFP expression at 8 weeks. This was followed by a small decrease over 4 weeks before reaching stable expression at 16 weeks. Our results demonstrated that rAAV efficiently transduces rat RPE cells and that retinal fluorescence photography is suitable for monitoring GFP expression. By using this noninvasive technique, we demonstrated that repetitive measurements of GFP expression in vivo in the rAAV-gfp-transduced retina are possible. This study demonstrated that retinal fluorescence photography is a potent tool for studying AAV-mediated gene delivery in the retina.

A degree of conservation of the genes located between class II and class I [central major histocompatibility complex (MHC) genes] is apparent among mammalian species including primates and the mouse. Few others have been analyzed. The caprine MHC is of particular interest, since it has recently been observed that susceptibility to a lentivirus-induced polyarthritis (caprine arthritis) segregates with serologically defined MHC class I antigens. This arthritis resembles, in a number of respects, rheumatoid arthritis in man. Human cDNA probes were used to examine the caprine central MHC and class I and II genes by restriction fragment length polymorphism (RFLP) and by pulsed field gel electrophoresis (PFGE) in order to define the polymorphism and linkage of central MHC genes to class I and class II genes. An outbred population of dairy goats (Saanen, British Alpine, Anglo Nubian, and Toggenberg) was examined for class I and class II RFLPs. Both regions were found to be highly polymorphic. The number of fragments hybridizing to an HLA-B7 probe after Eco RI, Bam HI, Bgl II, or Hind III digestion suggests there may be 10-13 class I genes. The degree of polymorphism was comparable to that reported in the mouse. Limited polymorphism was found in the central MHC genes. The caprine C4 and CYP21 genes were duplicated and demonstrated RFLP with Bam HI, Hind III, Eco RV, and Taq I. An infrequent Taq I C2 polymorphism was found. PFGE revealed substantial conservation of both the order and linkage of the central MHC genes when compared with mouse and man. C4, C2, CYP21, HSP70, and tumor necrosis factor (TNF) genes are all located within 800 kilobase (kb) of the class I loci. Distant from the class I region, the C4, C2, and CYP21 genes are linked on a short genomic segment (180 kb Not I and 190 kb Pvu I fragments). HSP70 cohybridizes with the complement genes on a 380 kb Mlu I fragment. Linkage of HSP70, TNF, and class I genes was found on a single Not I fragment (610 kb). TNF and class I cohybridize on Pvu I (730 kb) and Not I (610 kb) fragments. Conservation of a similar central MHC genomic structure across species argues for functional interaction between the central MHC genes. We postulate selection for these central MHC genes through their role as non antigen-specific regulators of immune response.

These results confirm the importance of this direct visualization system to study vector transgene expression in vivo and support the use of AAV for diseases treatable by targeting RPE cells.

The genetic predisposition to inclusion body myositis (IBM) is probably multifactorial. The deposition of the beta-amyloid protein is a characteristic histological feature of both IBM and Alzheimer's disease (AD). The epsilon 4 allele of apolipoprotein E (APO E) has been strongly associated with familial and late-onset AD. We therefore compared the APO E allele frequencies in a group of 14 patients with IBM with those in a group of patients with other inflammatory muscle diseases and in the general population. The frequency of the epsilon 4 allele in IBM was increased (0.29) compared with that in patients with other inflammatory muscle diseases (0.15) and the general population (0.13) (p < 0.05). These data suggest that APO E genotype may be one of the factors involved in determining the predisposition to the development of IBM.

Polymyositis is regarded as an autoimmune inflammatory muscle disease. A major subgroup of patients have autoantibodies to cellular histidyl-transfer RNA synthetase (HRS). We have analyzed the role of the autoantigen HRS in the induction of murine myositis in a comparative study of inoculation of BALB/c mice with recombinant HRS protein versus naked DNA coding for HRS. Adult BALB/c mice produced antibodies to human HRS following inoculation with HRS protein and adjuvant, but myositis was not observed. Alternatively, expression plasmid DNA constructs encoding full-length and truncated human HRS were inoculated intramuscularly in gene transfer studies. DNA-inoculated mice produced relatively low anti-HRS antibody titers. However, in contrast to recombinant HRS protein-inoculated mice, HRS gene transfer induced pathology with evidence of cellular infiltration of perivascular and endomysial regions of the inoculated muscle. Multiple inoculations of a plasmid construct encoding a hybrid molecule consisting of HRS and the transferrin receptor cytoplasmic tail induced the highest levels of antibodies and persisting cellular infiltration. Unlike HRS, expression of influenza virus hemagglutinin (HA) following inoculation of an HA plasmid did not induce myositis. Transfer of naked DNA constructs expressing HRS is likely to provide valuable information on the autoimmune response to this protein and its role in the development of myositis.