Ag Tetramers Research Articles

MAIT cell-MR1 reactivity is highly conserved across multiple divergent species

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by MHC-I related protein 1 (MR1), via an αβ T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved MHC-I like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology thereby allowing the use of selected species-mismatched MR1–antigen (MR1–Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1–Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1–Ag tetramers and utilized these alongside previously developed human, mouse and pig-tailed macaque MR1–Ag tetramers to characterize cross-species tetramer reactivities. MR1–Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1–Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1–Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1–Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1–Ag tetramers. Collectively, our results validate the use and limitations of species-mismatched MR1–Ag tetramers in comparative immunology studies.

Validation of Ligand Tetramers for the Detection of Antigen-Specific Lymphocytes.

The study of Ag-specific lymphocytes has been a key advancement in immunology over the past few decades. The development of multimerized probes containing Ags, peptide:MHC complexes, or other ligands was one innovation allowing the direct study of Ag-specific lymphocytes by flow cytometry. Although these types of study are now common and performed by thousands of laboratories, quality control and assessment of probe quality are often minimal. In fact, many of these types of probe are made in-house, and protocols vary between laboratories. Although peptide:MHC multimers can often be obtained from commercial sources or core facilities, few such services exist for Ag multimers. To ensure high quality and consistency with ligand probes, we have developed an easy and robust multiplexed approach using commercially available beads able to bind Abs specific for the ligand of interest. Using this assay, we have sensitively assessed the performance of peptide:MHC and Ag tetramers and have found considerable batch-to-batch variability in performance and stability over time more easily than using murine or human cell-based assays. This bead-based assay can also reveal common production errors such as miscalculation of Ag concentration. This work could set the stage for the development of standardized assays for all commonly used ligand probes to limit laboratory-to-laboratory technical variation and experimental failure caused by probe underperformance.

Study on mode shifts of localized surface plasmon cavity in Ag nanowire tetramer

The interaction between noble metal nanowires can induce the local surface plasmonic resonance effect, thereby enhancing the distribution of electric field in the nanostructures, which is of very important significance in improving the fluorescence characteristics and enhancing the sensitivity of sensors. In this study, we design several types of tetramers based on precious metals Ag nanostructures, including cylindrical and prismatic Ag tetramers, and by changing the arrangement and the rotation angle of prism nanowires, we simulate the rotation-angle dependent electric field distribution and electric field intensity of <i>X</i> component , and also discuss the physical mechanism of the relationship between the resonant peak position of absorption spectrum and the change of mode volume. The results show that in the Ag nanowires tetramer structure, the electric field in the cylindrical structure is not enhanced obviously, but the electric field in the prismatic structure is greatly enhanced, and an electric dipole resonance mode is produced in the gap between tetramers. The polarization of plasma resonant cavity revels that the morphology plays a decisive role in generating the hot spots, After changing both the combination mode of tetramer nanowires and the rotation angle of the four-prism, the local surface exciton resonance of the unrotated asymmetric tetramer structure is most ideal and has resonance intensity higher than the that of symmetrical four-prism structure. Therefore, our results provide a structural model and theoretical parameters for the enhancement of electric field intensity by local surface plasmon resonance effect.

Theoretical study of O2 interaction with subnanometer-sized Ag clusters supported on defective SiO2 surface

The effect of the nature of paramagnetic adsorption centers of the dehydroxylated silica surface - nonbridging oxygens (NBO) or silicon atoms with dangling bond (E′) - on the oxidation of adsorbed subnanometer-sized silver clusters Agn (n = 3, 4, 7) has been clarified by density functional theory using embedded cluster models. The interaction with NBO centers results in the formation of positively charged Ag moieties, while at E′ centers metal species remain rather neutral. At both surface defects, the electronic and structural properties of Ag species resemble the features of free Agn+ clusters. As a result, O2 molecular adsorption on the supported Ag clusters follows the trends established for cationic species. O2 is weakly adsorbed in a terminal mode on the Ag trimers and heptamers and is strongly bound to the Ag tetramers in a bridge mode. The stability of O2 molecular and dissociative adsorbed forms at the supported Ag3 and Ag7 species is similar, whereas on Ag4 cluster O2 dissociation is preferred. The heights of the O2 dissociation barriers are determined by the initial activation of molecularly adsorbed O2 and the deformation of supported metal clusters along the reaction pathway. The high activation energies make O2 dissociation unlikely and manifest that small Ag clusters trapped by silica paramagnetic defects highly resistant to the oxidation.

Identification of Tetramers in Silver Films Grown on the Si(001) Surface at Room Temperature.

We describe the atomic structure of the silver film grown on Si(001) at room temperature, as studied by low-temperature scanning tunneling microscopy and density functional theory. Experiment and theory agree on a film structure in which Ag tetramers are identified for the first time. Ag tetramers are found to be adsorbed exclusively at the trough between two Si rows, interacting with four adjacent Si dimers via covalent bonding. Consequently, the π bonds of the Si dimers underneath the silver film are eliminated.

Conversion of toluene into benzyl radical on anatase TiO2 (0 0 1) surface

In this study, the initial step of toluene dissociation, that is, the formation of benzyl radical through the reaction with hydroxyl group on anatase (001) surface is investigated using density functional theory calculations. The calculated results show that the toluene is physisorbed on the pristine TiO2 surface, and it is converted into benzyl radical when it interacting with surface hydroxyl groups through methyl group of toluene. It has been found that the interaction between toluene and TiO2 surface through OH group is enhanced by the presence of Ag tetramer. The calculated reaction energy profile shows that the reaction associated with conversion of toluene into benzyl radical on TiO2-OH and TiO2-OH-Ag surfaces is highly exothermic with an activation energy barrier of 0.56 and 0.53eV, respectively. The results obtained from this work reveal that the toluene dissociation on TiO2 surface is possible only through the surface hydroxyl groups and the presence of Ag tetramer augments the dissociative reaction.

General Approach for Tetramer-Based Identification of Autoantigen-Reactive B Cells: Characterization of La- and snRNP-Reactive B Cells in Autoimmune BXD2 Mice.

Autoreactive B cells are associated with the development of several autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. The low frequency of these cells represents a major barrier to their analysis. Ag tetramers prepared from linear epitopes represent a promising strategy for the identification of small subsets of Ag-reactive immune cells. This is challenging given the requirement for identification and validation of linear epitopes and the complexity of autoantibody responses, including the broad spectrum of autoantibody specificities and the contribution of isotype to pathogenicity. Therefore, we tested a two-tiered peptide microarray approach, coupled with epitope mapping of known autoantigens, to identify and characterize autoepitopes using the BXD2 autoimmune mouse model. Microarray results were verified through comparison with established age-associated profiles of autoantigen specificities and autoantibody class switching in BXD2 and control (C57BL/6) mice and high-throughput ELISA and ELISPOT analyses of synthetic peptides. Tetramers were prepared from two linear peptides derived from two RNA-binding proteins (RBPs): lupus La and 70-kDa U1 small nuclear ribonucleoprotein. Flow cytometric analysis of tetramer-reactive B cell subsets revealed a significantly higher frequency and greater numbers of RBP-reactive marginal zone precursor, transitional T3, and PDL-2(+)CD80(+) memory B cells, with significantly elevated CD69 and CD86 observed in RBP(+) marginal zone precursor B cells in the spleens of BXD2 mice compared with C57BL/6 mice, suggesting a regulatory defect. This study establishes a feasible strategy for the characterization of autoantigen-specific B cell subsets in different models of autoimmunity and, potentially, in humans.

Direct visualization of endogenous Salmonella-specific B cells reveals a marked delay in clonal expansion and germinal center development.

CD4(+) T cells and B cells are both essential for acquired immunity to Salmonella infection. It is well established that Salmonella inhibit host CD4(+) T-cell responses, but a corresponding inhibitory effect on B cells is less well defined. Here, we utilize an Ag tetramer and pull-down enrichment strategy to directly visualize OVA-specific B cells in mice, as they respond to infection with Salmonella-OVA. Surprisingly, OVA-specific B-cell expansion and germinal center formation was not detected until bacteria were cleared from the host. Furthermore, Salmonella infection also actively inhibited both B- and T-cell responses to the same coinjected Ag but this did not require the presence of iNOS. The Salmonella Pathogenicity Island 2 (SPI2) locus has been shown to be responsible for inhibition of Salmonella-specific CD4(+) T-cell responses, and an examination of SPI2-deficient bacteria demonstrated a recovery in B-cell expansion in infected mice. Together, these data suggest that Salmonella can simultaneously inhibit host B- and T-cell responses using SPI2-dependent mechanisms.

Misoriented Bi dimers blocking Ag nanowire growth along the Bi nanoline: a first-principlesstudy

Growth of Ag nanostructures on the Bi nanoline template (a double line of Bi dimersself-assembled on the Si(001) surface) is examined at the level of the generalized gradientapproximation. The aim is to find whether Ag adsorption induces the misorientation of Bidimers (i.e. those oriented transversely), because the misoriented dimers would block Agnanowire growth along the line. The calculation finds that the Ag tetramer on misorientedBi dimers is more stable than that on oriented dimers. To grow Ag wires hencerequires suppression of the transformation to the misoriented configuration. Thecalculated energy barriers indicate that it is possible to suppress the transformationwithout inhibiting the Ag migration to the Bi nanoline. Furthermore, the greaterstability of Ag wire nuclei compared to less wetting ones such as the icosahedralAg55 cluster also supports the feasibility of Ag wire growth. On the other hand, allowing themisorientation can be useful in growing uniform Ag nanoclusters; an optimum size(∼50 atoms) is found for the pyramidal islands that are bounded by misoriented Bi dimers.

High efficiency TCR gene transfer into primary human lymphocytes affords avid recognition of melanoma tumor antigen glycoprotein 100 and does not alter the recognition of autologous melanoma antigens.

The alpha- and beta-chains of the TCR from a highly avid anti-gp100 CTL clone were isolated and used to construct retroviral vectors that can mediate high efficiency gene transfer into primary human lymphocytes. Expression of this TCR gene was confirmed by Western blot analysis, immunocytometric analysis, and HLA Ag tetramer staining. Gene transfer efficiencies of >50% into primary lymphocytes were obtained without selection for transduced cells using a method of prebinding retroviral vectors to cell culture vessels before the addition of lymphocytes. The biological activity of transduced cells was confirmed by cytokine production following coculture with stimulator cells pulsed with gp100 peptides, but not with unrelated peptides. The ability of this anti-gp100 TCR gene to transfer high avidity Ag recognition to engineered lymphocytes was confirmed in comparison with highly avid antimelanoma lymphocytes by the high levels of cytokine production (>200,000 pg/ml IFN-gamma), by recognition of low levels of peptide (<200 pM), and by HLA class I-restricted recognition and lysis of melanoma tumor cell lines. CD4(+) T cells engineered with this anti-gp100 TCR gene were Ag reactive, suggesting CD8-independent activity of the expressed TCR. Finally, nonmelanoma-reactive tumor-infiltrating lymphocyte cultures developed antimelanoma activity following anti-gp100 TCR gene transfer. In addition, tumor-infiltrating lymphocytes with reactivity against non-gp100 melanoma Ags acquired gp100 reactivity and did not lose the recognition of autologous melanoma Ags following gp100 TCR gene transfer. These results suggest that lymphocytes genetically engineered to express anti-gp100 TCR may be of value in the adoptive immunotherapy of patients with melanoma.

The H4b minor histocompatibility antigen is caused by a combination of genetically determined and posttranslational modifications.

Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.

Interaction of tetrameric silver with ammonia in AgCs-rho zeolite

Cationic silver clusters formed in γ-irradiated AgCs-rho zeolite have been studied by EPR spectroscopy. Tetrameric silver is formed efficiently in the dehydrated form of zeolite as well as in samples exposed to ammonia. However, the hyperfine splitting of the Ag43+ pentet in AgCs-rho/NH3 zeolite is distinctly smaller due to the shift of spin density from silver to nitrogen nuclei, suggesting the formation of an Ag43+–ammonia complex. The observed 14N and 15N superhyperfine splitting from interacting 14NH3 and 15NH3 molecules unequivocally proved the presence of a multicore Ag43+(NH3)n complex. Based on the simulation of 14N and 15N superhyperfine interactions a structure of the complex was proposed in which two ammonia molecules are located close to the octagonal windows of two neighboring α-cages and four further molecules are placed in next-neighboring α-cages interacting with the Ag tetramer through square windows. When dehydrated AgCs-rho zeolite containing radiation-induced Ag43+ clusters is exposed to ammonia the Ag hyperfine splitting of silver tetramer decreases from 13.9 to 10.7 mT proving that Ag43+ clusters are located in the sites accessible to adsorbates.