Foreign antigens are presented by antigen-presenting cells in the presence of abundant endogenous peptides that are nonstimulatory to the T cell. In mouse T cells, endogenous, nonstimulatory peptides have been shown to enhance responses to specific peptide antigens, a phenomenon termed coagonism. However, whether coagonism also occurs in human T cells is unclear, and the molecular mechanism of coagonism is still under debate since CD4 and CD8 coagonism requires different interactions. Here we show that the nonstimulatory, HIV-derived peptide GAG enhances a specific human cytotoxic T lymphocyte response to HBV-derived epitopes presented by HLA-A*02:01. Coagonism in human T cells requires the CD8 coreceptor, but not T-cell receptor (TCR) binding to the nonstimulatory peptide–MHC. Coagonists enhance the phosphorylation and recruitment of several molecules involved in the TCR-proximal signaling pathway, suggesting that coagonists promote T-cell responses to antigenic pMHC by amplifying TCR-proximal signaling.
Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope−epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.
SignificanceThymocyte-expressed molecule involved in selection (Themis) regulates T cell selection. Absence of Themis leads to severely reduced numbers of CD4 and CD8 T cells, indicating a defect in T cell selection. The molecular mechanism of Themis involvement is not clear. Themis was shown to bind to Src-homology domain containing phosphatase-1 (Shp1), which is a known negative regulator of T cell receptor signaling. Here, using a very sensitive technique to measure phosphatase activity from immunoprecipitated proteins, we find that Themis positively regulates Shp1 phosphatase activity in thymocytes. Shp1 activity is reduced in the absence of Themis, thus providing an explanation for why Themis-deficient thymocytes respond more strongly to positive-selecting ligands, resulting in fewer thymocytes reaching maturity.
Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific T cell responses.
We designed conditional ligands restricted to HLA-B*08:01, 2B*35:01, and 2B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context. V C 2015 International Society for Advancement of Cytometry Key terms CD8 T cells; MHC multimer; HLA-B molecules; flow cytometry; conditional ligands THE tracking of specific T cells by use of fluorochrome-conjugated major histocompatibility complex (MHC) multimers for flow cytometry has increased our understanding in all areas of T-cell immunology (1-3). Staining of cells for binding to MHC multimers, combined with a set of antibodies to identify the CD8 T-cell population, represents an attractive way to quantitatively and phenotypically measure the CD8 T cells with certain specificities in a complex cell sample. It is thus possible to directly investigate the frequency and phenotype of these peptide-MHC (pMHC) multimer-specific T cells. The design and use of conditional ligands for the generation of MHC monomers was introduced in 2006 and revolutionized our ability to generate MHC reagents in a high-throughput fashion (4). These are HLA-binding peptides containing an UV-labile bond next to a 2-nitrophenylglycine or 3-amino-3-(2-nitrophenyl)-propionic acid residue (denoted "J") in their amino acid sequence, and can be integrated into the standard refolding procedure. Upon short-term UV light (366 nm) exposure, the amide bond next to the "J" residue is cleaved, and the fragmented peptide can be substituted with another ligand. The rescue of the MHC Class I complex after UV light exposure is dependent on the binding affinity between the ligand and the MHC molecule. Thus, this technique allows high-throughput affinity estimation using an MHC ELISA (5,6) and the generation of large libraries of pMHC complexes for the determinations of antigen-specific T cells using MHC Class I multimers in flow (7,8) and mass cytometry (9). The UV exchange technology has been extended to several MHC Class I variants (2,6,10), but conditional ligands has previously not been defined for some of the most frequently expressed HLA-B alleles.
Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific T cell responses.
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