The Tox Gene Encodes Two Proteins with Distinct and Shared Roles in Gene Regulation.

Here we report that the murine Tox gene encodes two proteins from a single mRNA, and we investigate the mechanism of production and function of these proteoforms. The annotated thymocyte selection-associated HMG-box protein (TOX) coding sequence is predicted to produce a 526-aa protein (TOXFL). However, Western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXΔN), whereas the slower-migrating band is TOXFL. The TOXΔN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL/TOXΔN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+CD8+ cells and subsequent differentiation to CD4+CD8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXΔN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion than did TOXΔN, including uniquely regulated cell cycle and other genes.

Changes in Non-Deamidated versus Deamidated Epitope Targeting and Disease Prediction during the Antibody Response to Gliadin and Transglutaminase of Infants at Risk for Celiac Disease.

Celiac disease (CeD) is a conditional autoimmune disorder with T cell-mediated immune response to gluten coupled with antibody production to gliadin and the self-protein tissue transglutaminase (TG2). TG2 contributes to the CeD pathomechanism by deamidating gliadin, thereby generating more immunogenic peptides. Anti-gliadin antibodies may appear before the autoantibody production. The scope of this study was to dissect these early antibody responses by investigating serum samples collected during the PreventCD prospective double-blind study, where infants with high CeD risk were randomized to 200 mg daily gluten intake or placebo from 4 to 6 months of age, followed by frequent blood testing on regular gluten consumption in both groups. After primary gluten intake, children with or without later CeD produced IgA and IgG antibodies which preferentially recognized non-deamidated gliadin peptides. At CeD development with anti-TG2 seroconversion, there was a significant increase in the antibody reaction toward deamidated gliadin peptides (DGP), with maturation in the binding strength for the PEQPFP gamma-gliadin core peptide. The earliest produced autoantibodies targeted TG2's celiac epitope 2. Our results reveal a qualitative change in the gliadin-directed humoral immune response at the time when anti-TG2 antibodies appear, but anti-DGP antibodies in the absence of anti-TG2 antibodies are not disease-predictive.

Usefulness of recombinant γ-gliadin 1 for identifying patients with celiac disease and monitoring adherence to a gluten-free diet.

BACKGROUND: Celiac disease (CD) is an inflammatory disease of the small intestine caused by an immunologic hypersensitivity reaction to dietary wheat gluten. OBJECTIVES: We sought to clone, express, and perform IgA epitope mapping of a CD-specific wheat antigen and to study its usefulness for identifying patients with CD and monitoring adherence to a gluten-free diet. METHODS: A synthetic gene coding for γ-gliadin 1 (GG1) was expressed in Escherichia coli. Recombinant γ-gliadin 1 (rGG1) was purified and characterized biochemically, structurally, and immunologically by using sera from patients with CD and control subjects. Overlapping GG1 peptides were synthesized for IgA and IgG epitope mapping. GG1 and peptide-specific antibodies were raised for tracing GG1 in cereals and dietary wheat products and to study its resistance to digestion. RESULTS: rGG1 was expressed and purified. rGG1-based IgA ELISAs performed in populations of patients with CD and control subjects showed a specificity of 92.9%, which was higher than that of gliadin extract (e). Furthermore, it allowed monitoring of adherence to a gluten-free diet in patients. A 26-amino-acid peptide from the proline-glutamine-rich repetitive N-terminal region was identified as the immunodominant IgA epitope. GG1-related antigens were found in rye, barley, and spelt but not in oat, rice, or maize. GG1 was detected in dietary wheat products after baking, and in particular, the major IgA epitope-containing region was resistant against digestion. CONCLUSIONS: rGG1 and its epitope might be useful for identifying patients with CD, monitoring treatment, and studying the pathomechanisms of CD and development of preventive and therapeutic strategies.

CD8+ T Cell Fate and Function Influenced by Antigen-Specific Virus-Like Nanoparticles Co-Expressing Membrane Tethered IL-2.

A variety of adjuvants fostering humoral immunity are known as of today. However, there is a lack of adjuvants or adjuvant strategies, which directly target T cellular effector functions and memory. We here determined whether systemically toxic cytokines such as IL-2 can be restricted to the site of antigen presentation and used as 'natural adjuvants'. Therefore, we devised antigen-presenting virus-like nanoparticles (VNP) co-expressing IL-2 attached to different membrane-anchors and assessed their potency to modulate CD8+ T cell responses in vitro and in vivo. Efficient targeting of IL-2 to lipid rafts and ultimately VNP was achieved by fusing IL-2 at its C-terminus to a minimal glycosylphosphatidylinositol (GPI)-anchor acceptor sequence. To identify optimal membrane-anchor dimensions we inserted one (1Ig), two (2Ig) or four (4Ig) immunoglobulin(Ig)-like domains of CD16b between IL-2 and the minimal GPI-anchor acceptor sequence of CD16b (GPI). We found that the 2IgGPI version was superior to all other evaluated IL-2 variants (IL-2v) in terms of its i) degree of targeting to lipid rafts and to the VNP surface, ii) biological activity, iii) co-stimulation of cognate T cells in the absence of bystander activation and iv) potency to induce differentiation and acquisition of CD8+ T cell effector functions in vitro and in vivo. In contrast, the GPI version rather favored memory precursor cell formation. These results exemplify novel beneficial features of membrane-bound IL-2, which in addition to its mere T cell stimulatory capacity include the induction of differential effector and memory functions in CD8+ T lymphocytes.

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.

Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the allergen molecules with only minute amounts of serum. In this article we summarize recent progress in the field of allergen microarray technology and introduce the MeDALL allergen-chip which has been developed for the specific and sensitive monitoring of IgE and IgG reactivity profiles towards more than 170 allergen molecules in sera collected in European birth cohorts. MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed.

A combined biochemical, biophysical and immunological approach towards the identification of celiac disease-specific wheat antigens.

Celiac disease (CD) is an inflammatory affliction of the small bowel caused by an immunological hypersensitivity to ingested wheat antigens affecting almost 1 % of the population. The gliadin fraction of wheat has been shown to contain the pathogenic antigens which react with antibodies and T cells. However, there is only limited knowledge regarding the precise nature of the wheat antigens recognized by IgA antibodies from CD patients and diagnostic tests based on the gliadin fraction have been demonstrated to give frequently false positive results. The aim of this study was the characterization of wheat antigens specifically recognized by IgA antibodies of CD patients. We developed a combined biochemical, biophysical, and immunological approach for the identification of celiac disease-specific wheat antigens. It is based on sub-fractionation of the wheat gliadin fraction using two ion exchange chromatography steps, the localization of CD-specific antigens by immunoblotting with IgA antibodies from CD patients, subsequent digestion followed by electro spray ionization-liquid chromatography/mass spectrometry (LC-ESI-MS/MS) and N-terminal sequencing by Edman degradation. Through the sub-fractionation procedure it was possible to separate CD-specific IgA-reactive wheat antigens from other wheat antigens which were also recognized by IgA antibodies of individuals without CD or by CD patients on gluten-free diet. Analysis by LC-ESI-MS/MS and N-terminal sequencing of the sub-fractions and the proteins specifically recognized by CD patients identified certain γ-gliadins with molecular mass of 37,000 and 45,000 as CD-specific wheat antigens. The CD-specific γ-gliadins with the molecular mass of 37,000 and 45,000 should be useful to study pathomechanisms of the disease and to improve the specificity of diagnostic tests for CD.

Role of individual domains and identification of internal gap in human guanylate binding protein-1.

Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded beta-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif ((103)DXEKGD(108)), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [alpha-(32)P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the alpha-helix of the intermediate region and the (103)DXEKGD(108) motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: alpha-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.