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MHC Class II Tetramers Gerald T MHC Class II Tetramers Gerald T. Nepom J Immunol 2012; 188:2477-2482; ; This information is current as doi: 10.4049/jimmunol.1102398 of September 25, 2021. http://www.jimmunol.org/content/188/6/2477 Downloaded from References This article cites 69 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/188/6/2477.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. MHC Class II Tetramers Gerald T. Nepom MHC class II tetramers have emerged as an important streptavidin molecules, forming ostensibly tetravalent com- tool for characterization of the specificity and phenotype plexes; hence, the common use of the term “tetramers” for this of CD4 T cell immune responses, useful in a large variety detection method. of disease and vaccine studies. Issues of specific T cell Tetramer assays are widely used for single-cell phenotyping frequency, biodistribution, and avidity, coupled with and enumeration and offer an important advantage over other the large genetic diversity of potential class II restriction methods, such as ELISPOT and single-cell PCR, by enabling elements, require targeted experimental design. Transla- the recovery and further study of sorted cells based on fluo- tional opportunities for immune disease monitoring are rescent tetramer binding in flow cytometry. As a cytometry- based application, tetramers also provide the prospect of driving the rapid development of HLA class II tetramer Downloaded from use in clinical applications, together with innovations ease of use and short assay time, analogous to Ab-based flow in tetramer production and epitope discovery. The cytometry studies. The major limitation to their use is the requirement to have some knowledge of the specific pMHC Journal of Immunology, 2012, 188: 2477–2482. components involved in the recognition events being studied, because these are necessary to construct the tetramer reagents. he concept of using soluble labeled ligands to detect cell A corollary to this element of MHC restriction is the con- http://www.jimmunol.org/ surface receptors is a standard approach for under- sideration that humans have very diverse HLA class II mole- T standing molecular specificity and function. However, cules, so that random sampling or clinical monitoring studies only in the last 15 y has this general strategy been successfully may require the use of many different tetramers to include applied to the analysis of Ag-specific T cells through the use a broad representation of the population. of soluble MHC-peptide ligands that engage the abTCR. Straightforward solutions exist for both of these limita- Originally developed for MHC class I recognition in the tions—HLA diversity and epitope identification—by making context of CD8 T cells (1, 2), over the last decade similar the investment necessary to produce a universal set of class II approaches have been successfully used for MHC class II tetramers for most HLA alleles, as well as by using functional recognition in the context of CD4 T cells for a wide variety of selection of relevant epitopes as a criterion for study design. by guest on September 25, 2021 Ags. From this experience, it is now evident that interrogating With these advances, discussed below, there has been a rapid CD4 T cells using soluble tetramers has matured into a robust expansion of studies using tetramers to study CD4 T cell re- technology, notably useful in numerous translational and sponse to vaccines, allergens, and a variety of Ags associated clinical contexts. However, several constraints that limit their with autoimmune diseases. There has also been an improved use are also now understood. In this Brief Review, key issues understanding surrounding the potential use of tetramers, be- governing CD4 T cell identification by MHC class II tet- cause as the technological problems have been largely solved, ramers are discussed, and examples from human immune there is now realization of some important biological barriers monitoring studies illustrate the lessons learned. associated with interrogating the specific TCR–tetramer in- teraction. Adding specificity to the T cell flow cytometry toolkit Peptide-MHC (pMHC) class II multimers display an anti- Rare CD4 T cell detection using class II pMHC tetramers genic peptide in the class II-binding groove, functioning as A major challenge for studies using HLA class II tetramers is a surrogate for recognition events that occur as part of the T the low frequency of Ag-specific CD4 T cells in peripheral cell interaction with APCs. Various methods for displaying blood. Highly boosted responses, such as tetanus-specific or pMHC class II molecules have been successfully used, ranging influenza-specific CD4 T cells after immunization, display from monomeric pMHC bound to solid substrates or soluble transient levels of Ag-specific cells in the 1:1,000–1:20,000 in solution to higher-order multimers complexed with various range; specific memory T cells without recent boosting are scaffold molecules. The most prevalent form of multimer in more often found in the 1:20,000–1:100,000 range; and rare use today consists of biotin-labeled pMHC displayed on responses or autoantigen-specific responder cells are detectable Benaroya Research Institute, Seattle, WA 98101 Abbreviations used in this article: pMHC, peptide-MHC; T1D, type 1 diabetes; TGEM, tetramer-guided epitope mapping. Received for publication December 7, 2011. Accepted for publication January 6, 2012. Work in the author’s laboratory cited in this article was supported by grants from the Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 National Institutes of Health and the Juvenile Diabetes Research Foundation. Address correspondence and reprint requests to Dr. Gerald T. Nepom, Benaroya Re- search Institute, Immune Tolerance Network, 1201 Ninth Avenue, Seattle, WA 98101. E-mail address: [email protected] www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102398 2478 BRIEF REVIEWS: MHC CLASS II TETRAMERS in the 1:100,000–1:1 million range. Naive T cell frequencies not only are there multiple potential protein targets within to particular pMHC are also ∼1:200,000–1:1 million. Be- each pathogen, but a huge number of processed and presented cause of these low cell frequencies, the first generation of peptides are potentially targets for CD4 T cell recognition. tetramer-based immunoassays used in vitro activation and/or For example, within the protective Ag of Bacillus anthracis, expansion to enable T cell detection (3), somewhat analo- multiple epitopes are present; as expected, the dominance of gous to the historically used techniques of limiting dilution- each epitope varies depending on the HLA genotype of the proliferation assays and ELISPOT analysis. Of course, a ma- individual subject (23). Therefore, selection of tetramers for jor problem with such an approach was the potential for studies of the T cell response to anthrax or similarly complex phenotypic changes introduced by in vitro manipulation; agents involves matching the HLA class II genotype of each therefore, these studies were limited primarily to the estimates subject with appropriately tailored pMHC tetramers (31). In of cell frequency and specificity. contrast, occasionally there are immunodominant epitopes Current second-generation tetramer assays avoid the use of from pathogens that bind promiscuously to multiple HLA in vitro expansion and are either based on single-cell capture class II molecules. When this occurs, production and use of technology (4–6) or on direct flow cytometry staining analysis appropriate pMHC tetramers becomes straightforward. For (7–10). In the latter, stringent “dumping” criteria and en- example, the hemagglutinin protein of influenza A H1N1 richment with magnetic bead-trapping procedures are used to contains a peptide epitope (aa 306–319) that binds to at least remove cells that lack tetramer specificity, enriching the Ag- five common human class II molecules and is recognized by specific population as much as 10,000-fold, allowing for de- T cells in each case. Most people, through either natural ex- Downloaded from tection of rare events (e.g., 10 tetramer-binding T cells from posure or periodic flu vaccination, contain peripheral CD4 20 ml of peripheral blood). In addition, multiparameter flow T cells recognizing this epitope (14). A similar strategy has been cytometry analysis using a variety of mAbs simultaneously adopted to search for conserved epitopes in pandemic strain with tetramer assays allows for direct phenotyping of the Ag- H5N1, after vaccination (32). T cells recognizing additional specific cells, a major advance toward discriminating T cell influenza epitopes
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