Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells

Human monocyte-derived dendritic cells (DCs) are capable of expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which allows them to suppress Ag-driven proliferation of T cells in vitro. In DCs that express IDO, the activity of the enzyme is tightly regulated, with the protein being constitutively expressed, but functional activity requiring an additional set of triggering signals supplied during Ag presentation. We now show that triggering of functional IDO obligately requires ligation of B7-1/B7-2 molecules on the DCs by CTLA4/CD28 expressed on T cells. When this interaction was disrupted, IDO remained in the inactive state, and the DCs were unable to inhibit T cell proliferation. Inhibition could be fully restored by direct Ab-mediated cross-linking of B7-1/B7-2. Although both CD4(+) and CD8(+) T cells were susceptible to inhibition once IDO was induced, the ability to trigger functionally active IDO was strictly confined to the CD4(+) subset. Thus, the ability of CD4(+) T cells to induce IDO activity in DCs allowed the CD4(+) population to dominantly inhibit proliferation of the CD8(+) population via the bridge of a conditioned DC. We hypothesize that IDO activation via engagement of B7-1/B7-2 molecules on DCs, specifically, engagement by CTLA4 expressed on regulatory CD4(+) T cells, may function as a physiologic regulator of T cell responses in vivo.     

Enhanced alpha-tocopherol quinone levels and xanthophyll cycle de-epoxidation in rosemary plants exposed to water deficit during a Mediterranean winter

Photosynthesis operates in a constantly shifting balance between efficient capture of solar energy and its rapid dissipation when captured in excess. In an attempt to better understand the role of alpha-tocopherol in plant photoprotection, we examined the changes in alpha-tocopherol quinone (alpha-TQ), in parallel with those of other low-molecular-weight antioxidants, in rosemary plants exposed to water deficit during a Mediterranean winter. Relative leaf water content (RWC) decreased from about 85% to approximately 65% in drought, but plants did not show symptoms of oxidative damage, as indicated by constant Fv/Fm ratios and malondialdehyde (MDA) levels. alpha-TQ was present at concentrations of 20 mmol per 100 mol of chlorophyll, and represented less than 1% of total tocopherol content in non-stressed leaves. Although alpha-tocopherol levels were not significantly altered, alpha-TQ reached up to 36 mmol per 100 mol of chlorophyll under stress (under both high light and after exposure to increasing water deficit at lower light intensities). Furthermore, both alpha-TQ and xanthophyll cycle de-epoxidation were strongly negatively correlated with the relative efficiency of photosystem II photochemistry (phiPSII) at midday. The biological significance of alpha-tocopherol and alpha-TQ in the network of photo- and antioxidative protection mechanisms evolved by plants to withstand stress is discussed.     

Hyponastic leaf growth decreases the photoprotective demand, prevents damage to photosystem II and delays leaf senescence in Salvia broussonetii plants

The influence of leaf angle on the response of plants to high light was studied in Salvia broussonetii, a species endemic of the Canary Islands that shows hyponastic leaf growth. The response of vertical, naturally oriented leaves was compared with that of horizontal, artificially held leaves for 1, 13, 24 and 29 days in terms of photoinhibition [efficiency of photosystem II (PSII)], photoprotection (by the xanthophyll cycle, alpha-tocopherol and beta-carotene) and progression of leaf senescence. Vertical leaves not only showed a decreased photoprotective demand compared with horizontal leaves but also kept the maximum efficiency of PSII (F(v)/F(m) ratio) constant throughout the experiment, thus reflecting the capacity of naturally oriented leaves to avoid photooxidative stress in the field. By contrast, horizontal leaves, which were exposed to higher light intensities, showed a higher photoprotective demand (reflected by a higher de-epoxidation of the xanthophyll cycle, carotenoid losses and increases in alpha-tocopherol), damage to PSII (as indicated by decreases in the F(v)/F(m) ratio) and accelerated leaf senescence, which was associated with cell death after 24 days of high light exposure. It is concluded that hyponastic leaf growth prevents photoinhibition and decreases the photoprotective demand of leaves by reducing the incident light, which helps maintaining leaf vigor and delaying the progression of leaf senescence in S. broussonetii plants. Hyponastic leaf growth is therefore one of the first photoprotection mechanisms activated in this species to avoid the negative impact of high-light stress in the field.     

The Function of Tocopherols and Tocotrienols in Plants

Referee: Dr. Kozi Asada, Department of Biotechnology, Faculty of Engineering, Fukuyama University, Gakuencho 1, Fukuyama 729-0292, Japan Tocopherols and tocotrienols, which differ only in the degree of saturation of their hydrophobic prenyl side chains, are lipid-soluble molecules that have a number of functions in plants. Synthesized from homogentisic acid and isopentenyl diphosphate in the plastid envelope, tocopherols and tocotrienols are essential to maintain membrane integrity. α-Tocopherol is the major form found in green parts of plants, while tocotrienols are mostly found in seeds. These compounds are antioxidants, thus they protect the plant from oxygen toxicity. Tocopherols and tocotrienols scavenge lipid peroxy radicals, thereby preventing the propagation of lipid peroxidation in membranes, and the ensuing products tocopheroxyl and tocotrienoxyl radicals, respectively, are recycled back to tocopherols and tocotrienols by the concerted action of other antioxidants. Furthermore, tocopherols and tocotrienols protect lipids and other membrane components by physically quenching and reacting chemically with singlet oxygen. The scavenging of singlet oxygen by α-tocopherol in chloroplasts results in the formation of, among other products, α -tocopherol quinone, a known contributor to cyclic electron transport in thylakoid membranes, therefore providing photoprotection for chloroplasts. Moreover, given that α-tocopherol increases membrane rigidity, its concentration, together with that of the other membrane components, might be regulated to afford adequate fluidity for membrane function. Furthermore, α-tocopherol may affect intracellular signaling in plant cells. The effects of this compound in intracellular signaling may be either direct, by interacting with key components of the signaling cascade, or indirect, through the prevention of lipid peroxidation or the scavenging of singlet oxygen. In the latter case, α-tocopherol may regulate the concentration of reactive oxygen species and plant hormones, such as jasmonic acid, within the cell, which control both the growth and development of plants, and also plant response to stress.     

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]     

Engineering DNA nanoparticles as immunomodulatory reagents that activate regulatory T cells

Nanoparticles containing DNA complexed with the cationic polymer polyethylenimine are efficient vehicles to transduce DNA into cells and organisms. DNA/polyethylenimine nanoparticles (DNPs) also elicit rapid and systemic release of proinflammatory cytokines that promote antitumor immunity. In this study, we report that DNPs possess previously unrecognized immunomodulatory attributes due to rapid upregulation of IDO enzyme activity in lymphoid tissues of mice. IDO induction in response to DNP treatment caused dendritic cells and regulatory T cells (Tregs) to acquire potent regulatory phenotypes. As expected, DNP treatment stimulated rapid increase in serum levels of IFN type I (IFN-αβ) and II (IFN-γ), which are both potent IDO inducers. IDO-mediated Treg activation was dependent on IFN type I receptor signaling, whereas IFN-γ receptor signaling was not essential for this response. Moreover, systemic IFN-γ release was caused by TLR9-dependent activation of NK cells, whereas TLR9 signaling was not required for IFN-αβ release. Accordingly, DNPs lacking immunostimulatory TLR9 ligands in DNA stimulated IFN-αβ production, induced IDO, and promoted regulatory outcomes, but did not stimulate potentially toxic, systemic release of IFN-γ. DNP treatment to induce IDO and activate Tregs blocked Ag-specific T cell responses elicited in vivo following immunization and suppressed joint pathology in a model of immune-mediated arthritis. Thus, DNPs lacking TLR9 ligands may be safe and effective reagents to protect healthy tissues from immune-mediated destruction in clinical hyperimmune syndromes.     

Immunoglobulin Fc fragment tagging allows strong activation of endogenous CD4 T cells to reshape the tumor milieu and enhance the antitumor effect of lentivector immunization

A major problem with current cancer vaccines is that the induction of CD8 immune responses is rarely associated with antitumor benefits, mainly owing to multiple immune suppressions in established tumor lesions. In this study, we investigated if and how activation of endogenous CD4 T cells could be achieved to influence the suppressive tumor milieu and antitumor effect. We engineered a lentivector (lv) to express a nominal fusion Ag composed of hepatitis B surface protein and IgG2a Fc fragment (HBS-Fc-lv) to increase the magnitude of CD8 response but, more importantly, to induce effective coactivation of CD4 T cells. We found that, remarkably, immunization with HBS-Fc-lv caused significant regression of established tumors. Immunologic analysis revealed that, compared with HBS-lv without Fc fragment, immunization with HBS-Fc-lv markedly increased the number of functional CD8 and CD4 T cells and the level of Th1/Tc1-like cytokines in the tumor while substantially decreasing the regulatory T cell ratio. The favorable immunologic changes in tumor lesions and the improvement of antitumor effects from HBS-Fc-lv immunization were dependent on the CD4 activation, which was Fc receptor mediated. Adoptive transfer of CD4 T cells from the HBS-Fc-lv-immunized mice could activate endogenous CD8 T cells in an IFN-γ-dependent manner. We conclude that endogenous CD4 T cells can be activated by lv expressing Fc-tagged Ag to provide another layer of help--that is, creating a Th1/Tc1-like proinflammatory milieu within the tumor lesion to boost the effector phase of immune responses in enhancing the antitumor effect.     

Associations between HLA Class I Alleles and Escape Mutations in the Hepatitis B Virus Core Gene in New Zealand-Resident Tongans

The full repertoire of hepatitis B virus (HBV) peptides that bind to the common HLA class I molecules found in areas with a high prevalence of chronic HBV infection has not been determined. This information may be useful for designing immunotherapies for chronic hepatitis B. We identified amino acid residues under positive selection pressure in the HBV core gene by phylogenetic analysis of cloned DNA sequences obtained from HBV DNA extracted from the sera of Tongan subjects with inactive, HBeAg-negative chronic HBV infections. The repertoires of positively selected sites in groups of subjects who were homozygous for either HLA-B*4001 (n = 10) or HLA-B*5602 (n = 7) were compared. We identified 13 amino acid sites under positive selection pressure. A significant association between an HLA class I allele and the presence of nonsynonymous mutations was found at five of these sites. HLA-B*4001 was associated with mutations at E77 (P = 0.05) and E113 (P = 0.002), and HLA-B*5602 was associated with mutations at S21 (P = 0.02). In addition, amino acid mutations at V13 (P = 0.03) and E14 (P = 0.01) were more common in the seven subjects with an HLA-A*02 allele. In summary, we have developed an assay that can identify associations between HLA class I alleles and HBV core gene amino acids that mutate in response to selection pressure. This is consistent with published evidence that CD8⁺ T cells have a role in suppressing viral replication in inactive, HBeAg-negative chronic HBV infection. This assay may be useful for identifying the clinically significant HBV peptides that bind to common HLA class I molecules.The most potent nucleoside/nucleotide analogue drugs used to treat chronic hepatitis B reduce serum hepatitis B virus (HBV) DNA to undetectable levels in over 90% of subjects (5, 10). It was originally hoped that such a substantial reduction in viral titers would reverse T-cell tolerance for HBV antigens (17, 30) and lead to an immune response that permanently suppressed the virus, thus removing the need for expensive, lifelong drug therapy. However, HBeAg seroconversion rates of under 30% suggest that suppression of HBV replication is not sufficient to reverse the defects (4, 15) in the HBV peptide-specific CD8⁺ T-cell compartment that occur in these patients. A therapeutic vaccine that stimulated a diverse repertoire of functional CD8⁺ T cells could make a valuable contribution to management of chronic hepatitis B.The first step in designing a therapeutic vaccine that will suppress viral replication without exacerbating chronic hepatitis B (15) is to identify the HBV peptides that stimulate functional CD8⁺ T cells by binding to the most common HLA class I alleles. These peptides may contribute to the antigen component of a vaccine and to the design of assays for use as correlates of immunity in trials of antiviral therapies. Although some of the HBV peptides that bind to four HLA-A alleles have been published (3, 19, 25, 28), a much wider repertoire of peptide-HLA interactions needs to be identified. There is no established method for finding them (32). Adding pools of peptides to peripheral blood mononuclear cells in enzyme-linked immunospot assays is the most commonly used technique (4), but it has disadvantages. Pools of peptides contain epitopes that are not produced by in vivo antigen-processing mechanisms (32), and the influence of these epitopes on complex mixtures of T cells with degenerate antigen receptors is unknown. False-positive and false-negative results are possible. In addition, it cannot be assumed that the ability of a T cell to secrete gamma interferon in an enzyme-linked immunospot assay correlates with its ability to place clinically significant selection pressure on the virus in vivo.We are proposing an alternative approach, which should lead to the identification of the most clinically significant wild-type peptide antigens. This is to assess the influence of HLA class I alleles on the repertoire of escape mutations (3, 18) encoded in the HBV DNA extracted from the sera of HBeAg-negative subjects with an inactive chronic HBV infection. A functional CD8⁺ T-cell repertoire (15, 22) develops in these subjects at the same time the virus in their sera accumulates amino acid mutations (2). Phylogenetic analysis can distinguish those amino acid mutations that have arisen as a result of positive selection pressure from those that have arisen as a result of random processes (31). CD8⁺ T cells are likely to have placed selection pressure on any of the nonrandom amino acid mutations that preferentially occur in patients with a specific HLA class I allele. It should be possible to obtain the precise amino acid sequences of the peptides that contain these amino acids using immunological assays.This study was carried out with Tongan subjects who are homozygous for one of two common HLA-B alleles. Since there is significant linkage disequilibrium within the HLA class I locus in Tongan people (1), this has allowed two groups of subjects with distinct HLA class I haplotypes to be studied. In addition, we restricted the study to subjects infected with a genotype C3 HBV.