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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    • "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.
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    • "Limiting dilution assays suggested that pathogen-specific CD8 T cells were exceedingly rare among responding cells. However, technical innovations, such as the development of MHC I tetramers (Altman et al., 1996), revealed that antigen-specific CD8 T cell responses were 10-to 100-fold bigger than initially thought, precipitating a substantial revision in conceptualization of the immune response (Murali-Krishna et al., 1998). Memory CD8 T cells are present within secondary lymphoid organs (SLO), blood, and the rest of the organism (nonlymphoid tissues [NLT], as well as primary lymphoid organs such as thymus and bone marrow). "
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    ABSTRACT: Memory CD8 T cells protect against intracellular pathogens by scanning host cell surfaces; thus, infection detection rates depend on memory cell number and distribution. Population analyses rely on cell isolation from whole organs, and interpretation is predicated on presumptions of near complete cell recovery. Paradigmatically, memory is parsed into central, effector, and resident subsets, ostensibly defined by immunosurveillance patterns but in practice identified by phenotypic markers. Because isolation methods ultimately inform models of memory T cell differentiation, protection, and vaccine translation, we tested their validity via parabiosis and quantitative immunofluorescence microscopy of a mouse memory CD8 T cell population. We report three major findings: lymphocyte isolation fails to recover most cells and biases against certain subsets, residents greatly outnumber recirculating cells within non-lymphoid tissues, and memory subset homing to inflammation does not conform to previously hypothesized migration patterns. These results indicate that most host cells are surveyed for reinfection by segregated residents rather than by recirculating cells that migrate throughout the blood and body. Copyright © 2015 Elsevier Inc. All rights reserved.
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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.
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