Phenotypic Analysis of Antigen-Specific T Lymphocytes

Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5428, USA.
Science (Impact Factor: 33.61). 11/1996; 274(5284):94-6. DOI: 10.1126/science.274.5284.94
Source: PubMed


Identification and characterization of antigen-specific T lymphocytes during the course of an immune response is tedious and
indirect. To address this problem, the peptide-major histocompatability complex (MHC) ligand for a given population of T cells
was multimerized to make soluble peptide-MHC tetramers. Tetramers of human lymphocyte antigen A2 that were complexed with
two different human immunodeficiency virus (HIV)-derived peptides or with a peptide derived from influenza A matrix protein
bound to peptide-specific cytotoxic T cells in vitro and to T cells from the blood of HIV-infected individuals. In general,
tetramer binding correlated well with cytotoxicity assays. This approach should be useful in the analysis of T cells specific
for infectious agents, tumors, and autoantigens.

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Available from: John D Altman, Oct 04, 2015
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    • "Multimerization of soluble pMHC can considerably extend the half-life of this interaction due to the avidity effect, 3 and can thereby produce reagents that stably adhere to the cell surface of T cells bearing a cognate TCR. Peptide-MHC multimers in the form of avidin–biotin-based pMHC tetramers were first used to stain T cells by Altman et al. in 1996 4 and have gone on to transform the analysis of antigen-specific T-cell populations. Peptide- MHC multimers have been used in many thousands of studies and spawned the generation of several commercial companies that sell various forms of these reagents. "
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    ABSTRACT: Analysis of antigen-specific T cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an Ab against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T cell receptor (TCR) is over 20-fold weaker (KD > 1 mM) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anticancer or autoimmune T cell populations, which tend to bear lower affinity TCRs. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Immunology 06/2015; 146(1). DOI:10.1111/imm.12499 · 3.80 Impact Factor
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    • "To investigate whether CLCA1 and TMEM16A associate directly with one another on the cell surface, we adapted an assay commonly used to identify immunological receptor-ligand pairs (Altman et al., 1996). We previously demonstrated that CLCA1 is cut into two fragments by selfcleavage and that the N-terminal fragment is necessary and sufficient to activate CaCCs in HEK293T cells (Yurtsever et al., 2012). "
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    ABSTRACT: Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl- channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.
    eLife Sciences 03/2015; 4(4). DOI:10.7554/eLife.05875 · 9.32 Impact Factor
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    • "Synthetic biotinylated HLA class I monomers were constructed around various peptide sequences (Table 1) as described previously [17] [23]. For HLA class II, constructs were made by deletion of the transmembrane domains of the alpha and beta chains of HLA-DR and HLA-DQ molecules and replacing a 7 amino acid linker followed by leucine zipper ACIDp1 for alpha chains and leucine zipper BASEp1 for beta chains. "
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    ABSTRACT: For the quantification of HLA-specific memory B cells from peripheral blood of sensitized individuals, a limited number of methods are available. However, none of these are capable of detecting memory B cells directed at HLA class II molecules. Since the majority of antibodies that occur after transplantation appear to be specific for HLA class II, our aim was to develop an assay to detect and quantify HLA class II-specific memory B cells from peripheral blood. By using biotinylated soluble HLA class II molecules as detection agent, we were able to develop an HLA class II-specific memory B cell ELISPOT assay. The assay was validated using B cell-derived hybridomas that produce human monoclonal antibodies directed at specific HLA class II molecules. In pregnancy-immunized females, we found memory B cell frequencies ranging from 25 to 756 spots per 10(6) B cells specific for the immunizing paternal HLA class II molecules, whereas in non-immunized males no significant spot formation was detected. Here, we present a novel ELISPOT assay for quantifying HLA class II-specific memory B cells from peripheral blood. This technique provides a unique tool for monitoring the HLA class II-specific memory B cell pool in sensitized transplant recipients. Copyright © 2015. Published by Elsevier Inc.
    Human Immunology 01/2015; 76(2-3). DOI:10.1016/j.humimm.2015.01.014 · 2.14 Impact Factor
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