1. Recognition of diverse primate MIC molecules by human Vδ1 γδ T cells specific for MICA and MICB. (A D)C1R cells transfected with the cDNAs for chimpanzee Patr-MIC1, rhesus monkey Mamu-MIC1, African green monkey Ceae-MIC2, or owl monkey Aotr-MIC1 expressed the encoded MIC proteins on the cell surface (filled profiles), as shown by immune fluorescence stainings and flow cytometry.
Kinetics of HLADM and HLA-DR association in wild-type Pala cells. Cells were pulse labeled with methionine for 15 min and chased in the presence of an excess of methionine and cysteine for the indicated periods of time (h). Class II and DM molecules were immunoprecipitated at each time point from CHAPS or Triton X-100 solubilized lysates (as indicated above each gel) with the DR-specific mAb L243 or with a polyclonal rabbit antiserum sp ecific for the DM heterodimer (αDM) and analyzed by SDSPAGE.
HLA-DM associates with DR in a post-Golgi compartment. Pala cells were pulsed labeled wit methionine for 15 min and chased for the indicated periods of time (h). DM-DR complexes were immunoprecipitated at each time point from CHAPS solubilized lysates with anti-DM (αDM). DM-associated class II molecules were eluted with 0.5% DOC and the supernatants re-precipitated with DR β chain-specific mAb HB10A (A) or control mAb W6/32 (B). The precipitates were either treated with Endo H or mock treated and analyzed by SDS-PAGE.
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HLA-DM associates with αβLIP and αβSLIP complexes. (A) Pala cells were pulse labeled for 15 min with methionine and chased for 2 h in the presence of an excess of methionine and cysteine. Class II, DM and I chain molecules were immune precipitated from CHAPS solubilized lysates with anti-DM (αDM) or control anti-C23V (αC23V) or from Triton X-100 solubilized lysates with anti-DM, DR-specific mAb L243, I chain-specific mAb PIN.1.
3. CD4+CD25+ immune regulatory T cells represent a unique lineage of thymic-derived cells that potently suppress both in vitro and in vivo effectors T cell function. We analyzed CD4+CD25+ and CD4+CD25− T cells by DNA microarray, identifying 29 genes differentially expressed in the resting subpopulations, and 77 that were differentially expressed following activation. Most of these genes were elevated in the CD4+CD25+ population, suggesting a previously activated phenotype. Among these were a number of genes that antagonize signaling, including members of the SOCS family, which may contribute to their anergic phenotype. Multiple cell surface receptors also had increased expression in CD4+CD25+ cells, including GITR, a member of the TNF receptor super family. Importantly, antibodies to GITR abrogated suppression, demonstrating a functional role for this receptor in regulating the CD4+CD25+ T cell subset.
4. Immersion of preformed, ordered alkanethiolate SAMs into dilute solutions of the conjugated molecules results in two-dimensional matrix isolation of conjugated adsorbates in the host SAM. The post-immersion host SAM matrix shows retained alkanethiolate ordering with the guest molecules inserted both singly into boundaries between SAM structural domains and in bundles at substrate step edges. Inserted molecules of lengths in the 15 Å range adopt surface orientations similar to those of alkanethiolate molecules at all compositions, including the pure conjugated SAMs. In contrast, the configuration of an inserted, long conjugated molecule (40 Å), varies monotonically with the final SAM composition. When inserted at decreasing fractions into an n-octanethiolate matrix, the average tilt of the long molecular axis decreases and approaches alignment with the host SAM. Combined quantum chemical calculations and IRS data for the short guest−host SAMs support a picture of a dense local environment of the host SAM around the guest molecules, despite their insertion at host defects. These results have important implications for designing electronic devices based on the addressing of individual, fully conjugated molecules self-assembled at gold electrodes.
The structure of a series of alcohol-terminated bi-functional long-tail organosilane films with varying numbers of superimposed monolayer of the cell, prepared on smooth, hydrophilic silicon substrates by the layer-by-layer self-assembly approach, has been investigated with the purpose of elucidating details of the molecular organization and the intra- and interlayer modes of binding in such films. To this end, experimental results obtained by synchrotron X-ray scattering, micro-Raman, and Fourier transform infrared (FTIR) spectroscopic techniques have been combined and compared. A comprehensive analysis of all data demonstrates that the studied multilayer films consist of stacks of uncorrelated discrete monolayer, the inner molecular order of which is preserved with the growing total number of superimposed layers. Similar to self-assembled films of long-tail silages with terminal −COOH groups,1 the intermolecular binding in the present films is characterized by partial intra- and interlayer covalent bond formation. The molecular hydrocarbon tails are perpendicularly oriented on the layer planes, forming a densely packed rotator phase like hexagonal lattice with a molecular surface area of 20 Å2 and a lateral correlation length of the order of 16 molecular diameters.
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5. The intracytoplasmic forms of class II (or Ia) major histocompatibility complex heterodimers are associated with a third glycoprotein, termed the invariant chain (Ii). This specific interaction has led to the view that Ii plays a necessary role in the assembly, intracellular transport, and/or membrane insertion of Ia molecules. To test this hypothesis directly, we have transfected complementary DNA clones that encode murine class II alpha and beta chains into cells that do not express any endogenous Ii messenger RNA (mRNA).
cDNA encoding the human interferon-γ receptor was isolated from a λgt11 expression library using a polyclonal antireceptor antiserum. The gene for this receptor was identified in a cosmid library and transfected into mouse cells. The human interferon-γ receptor expressed in mouse cells displayed the same binding properties as in human cells. However, transfected cells were not sensitive to human IFN-γ, suggesting the need for species-specific cofactors in receptor function. As inferred from the cDNA sequence, the human interferon-γ receptor shows no similarities to known proteins and represents a novel transmembrane receptor. It is most likely the product of a single mRNA and a gene located on chromosome 6.
6. gene targeting techniques to produce mice lacking the invariant chain associated with major histocompatibility complex (MHC) class II molecules. Cells from these mice show a dramatic reduction in surface class II, resulting from both defective association of class II alpha and beta chains and markedly decreased post-Golgi transport. The few class II alpha/beta heterodimers reaching the cell surface behave as if empty or occupied by an easily displaced peptide, and display a distinct structure. Mutant spleen cells are defective in their ability to present intact protein antigens, but stimulate enhanced responses in the presence of peptides. These mutant mice have greatly reduced numbers of thymic and peripheral CD4+ T cells. Overall, this striking phenotype establishes that the invariant chain plays a critical role in regulating MHC class II expression and function in the intact animals.
8. We have analyzed the ability of major histocompatibility (MHC) class II molecules to capture proteins in the biosynthetic pathway and whether this may be associated with MHC class II-dependent antigen processing. When co-expressed with HLA-DR 4 molecules in HeLa cells, influenza hemagglutinin was inhibited from folding and trimerization in the biosynthetic pathway, targeted to endosomal compartments, and rapidly degraded. Due to the interaction with MHC class II molecules, therefore, unfolded forms of hemagglutinin were bypassing the quality control mechanism of the secretary pathway. More important, however, the transport, endocytosis, and rapid degradation of unfolded hemagglutinin in the presence of MHC class II molecules suggest that proteins captured in the endoplasmic reticulum by class II molecules may become substrates for antigen processing and presentation to CD4-positive T cells. In insect cells we show that this phenomenon is not restricted to a few proteins such as hemagglutinin. A highly heterogeneous mixture of proteins from the endoplasmic reticulum including co-expressed hemagglutinin can form stable complexes with soluble HLA-DR α and β chains that were transported into the supernatant. This mechanism may gain biological significance in abnormal situations associated with accumulation of unfolded or malfolded proteins in the endoplasmic reticulum, for example during viral infections.
9. Most protein antigens cannot elicit a T-cell response unless they are processed to peptides, which are then presented to T lymphocytes by surface MHC class II molecules. Recent evidence supports an essential role of the invariant chain associated with class II MHC polypeptides in antigen processing. Mounting evidence indicates that -GalCer responsive T cells are heterogeneous in their reactivities to cellular antigens, suggesting that an individual semi-invariant TCR may be capable of recognizing more than one ligand. Recent crystal structures of CD1b molecules with three different bound lipids indicate that the antigenic features of lipids may be localized over a smaller area than those of peptides, and that the positioning of the polar head group can vary substantially. A model that explains how CD1d-restricted T cells could possess both conserved and heterogeneous antigen specificities, is that different lipid antigens may interact with distinct areas of a TCR due to differences in the positioning of the polar head group. Hence, canonical CD1d-restricted TCR could recognize conserved antigens via the invariant TCR chain, and have diverse antigen specificities that are conferred by their individual TCR chains.
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