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

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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 interaction. 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 can be found simultaneously in the same functional subsets directed to a single Ag. blood samples using other tetramers but appear to be more http://www.jimmunol.org/ HLA class II tetramers are now widely used in studies of specific to a given individual (12, 33). An interesting excep- pathogen immunity and vaccine development, in evaluation of tion was seen in tetramer analysis with pMHC corresponding antitumor responses, in allergy monitoring and desensitization to the H5N1 influenza strain hemagglutinin, with cross-re- studies, and in autoimmunity (Fig. 1). Examples of each are activity from pre-existing immunity to other influenza strains, summarized below and illustrate particular issues faced in allowing detection of T cells reactive to H5N1, even in the these different applications. absence of direct exposure to this strain (24). Monitoring immunity to infectious pathogens. Class II tetramers With hundreds of potential peptide epitopes to choose

have been used for analysis of a variety of human CD4 T cell from, selecting pMHC tetramers for studying infectious patho- by guest on September 25, 2021 responses to pathogens, including influenza A, Borrelia,EBV, gens or vaccines is challenging. Traditional methods involving CMV, Mycobacterium tuberculosis, human T-lymphotropic functional testing of individual peptides from large libraries virus 1, hepatitis C, anthrax, severe acute respiratory syn- are laborious and, when based on a particular functional param- drome virus, human papillomavirus, and HIV (3, 7, 11–30). eter (e.g., IFN ELISPOT analysis), may omit epitopes that are Ag-specific T cells are detected in peripheral blood, with a very important for other T cell subsets or lineages. To address this broad range of frequency, depending on prior Ag exposure, issue, an unbiased platform has been developed for a tetramer- vaccination history, and timing of immunization. The most binding and flow cytometry screening method that directly significant limitation for these studies is simply the large determines suitable pMHC tetramers. This technique, called number of potential epitopes present in complex organisms; tetramer-guided epitope mapping (TGEM), involves the display of large peptide libraries loaded into HLA class II molecules (Fig. 2). Pools of tetramers are used to probe the T cell population of interest, and positive tetramer binding, assessed by flow cytometry, is deconvoluted in a second flow cytometry step into individual pMHC targets (34, 35). This approach has been applied to a variety of infectious agents, with epitope identification information deposited in the National Institute of Allergy and Infectious Diseases-sponsored Immune Epitope Database and Analysis Resource, IEDB.org. Allergen tetramers: a tool for improved immunotherapy? Transla- tion from a laboratory tool to a clinically useful biomarker is a difficult transition for any technology, but in the area of allergy desensitization therapy, human class II tetramers have their most immediate translational potential. Similar to the study of infectious pathogens, tetramer development for allergens is complicated by the diversity and number of po- FIGURE 1. Molecular design schematic of a class II tetramer molecule, illustrating four pMHC domains tethered to a central streptavidin molecule tential CD4 T cell epitopes. However, for many of the major through flexible linker sequences and leucine zipper motifs to provide allergic disorders, including allergies to foods, pollen, and structural stability. Some of the major areas of tetramer analysis in human dander, there is a large body of knowledge narrowing the disease applications are listed. field of target allergen proteins. The TGEM technique has The Journal of Immunology 2479

FIGURE 2. Epitope identification using a tetramer- guided mapping strategy. Large peptide libraries, sep- arated into sets of pooled sequences, are loaded into the binding groove of recombinant class II molecules and assembled into tetramers. Direct flow cytometry analysis of T cell populations identifies peptides containing actual epitopes representative of the natural immune response. Downloaded from been applied to several of these, resulting in the identification sulin, IA-2, and ZnT8 have all been used to evaluate T cell of epitopes and tetramers suitable for analysis of peripheral frequencies in peripheral blood. Findings from these studies T cell recognition to major allergens (10, 16, 26, 29). In support the concept that the autoreactive repertoire matures allergic individuals, the frequency of tetramer-binding CD4 during disease progression. First, normal individuals, as well as

T cells in peripheral blood, particularly during periods of T1D subjects, have low, but detectable, levels of circulating http://www.jimmunol.org/ allergen exposure, is suitably high, allowing direct detection tetramer-binding CD4 cells. However, these display markers by flow cytometry after tetramer binding. In a recent study of naive lymphocytes in the former group, whereas they are by Wambre et al. (10), multiparameter flow analysis docu- predominantly memory phenotype in the latter, presumably mented progressive shifts in cell surface marker profiles reflect a history of prior activation in vivo. Second, although associated with T cell deviation during s.c. allergen immu- some high-avidity autoreactive T cells are present and can be notherapy. Current clinical desensitization protocols use expanded in culture, the population of tetramer-binding cells allergen extracts, require very long-term administration, and contains both low- and high-avidity TCR for the autoantigen

carry significant risk for adverse allergic response, making it pMHC. By taking advantage of the unique genetics of hu- by guest on September 25, 2021 attractive to consider using tetramer-based immune monitor- man T1D, in which a polymorphism in the proinsulin pro- ing in this context. Also, because one approach to improve moter region controls levels of thymic expression (48, 49), allergy immunotherapy is to use peptides instead of allergen Durinovic-Bello´ et al. (44) demonstrated that the strength of extracts (19, 36), the TGEM technique and tetramer mon- tetramer binding to proinsulin-specific T cells correlated with itoring may help to identify appropriate peptide epitopes for this genotype, directly linking thymic Ag expression with use, particularly if stratification based on HLA genotype is avidity selection for the CD4 repertoire. Third, when T1D needed. patients undergo pancreas transplantation, they essentially Tetramer profiling of low-avidity T cells in autoimmunity. Human receive an immunological challenge with islet autoantigens, in class II pMHC tetramers containing autoantigenic specificities addition to the alloreactivity associated with an organ trans- have been used to study T cell responses in a variety of dis- plant. Tetramer analysis of such subjects, followed for several eases, including type 1 diabetes (T1D), celiac disease, pemphi- years after transplantation, documented both the expansion gus vulgaris, rheumatoid arthritis, multiple sclerosis, and uve- of autoantigen pMHC tetramer-binding CD4 T cells and, itis (37–47). These studies have highlighted a fundamental more remarkably, the persistence of unique clonotypic TCR point: the avidity of the TCR interaction with its pMHC sequences within this population, indicating a recurrence of target is influenced by selection against high-affinity self- memory responses from a pre-existing autoreactive repertoire Ag recognition. Unlike tetramer analyses in the infectious (50, 51). pathogen and allergen studies cited above, in which most of The low frequency and low avidity of T cells specific the responding T cells have a high-avidity binding to ap- for autoantigens present special technical challenges, but sev- propriate pMHC tetramers, tetramer binding to peripheral eral creative approaches have shown promise for improving T cells in the context of autoimmunity displays a much wider tetramer-detection strategies. In a recent study of subjects spectrum of relative strength of interaction. In addition, the with rheumatoid arthritis, class II tetramers were used to test frequency of autoreactive CD4 T cells specific for a particular an intriguing mechanistic hypothesis: posttranslational mod- pMHC–self-Ag complex is quite low in peripheral blood, ification of peptide epitopes creates neoantigenic specific- often less than five per million lymphocytes, requiring large ities targeted in the disease setting (52). James et al. (45) sample volumes and careful handling for reliable detection. also prepared HLA class II tetramers containing citrulline- The most complete autoimmunity studies have been per- modified peptides based on fibrinogen protein sequences formed in the context of T1D, a disease in which several major found in joint tissue, and they documented the presence of autoantigens are well characterized. Class II tetramers specific T cells that bound these tetramers and responded to the for the T1D autoantigens glutamate dehydrogenase, proin- corresponding autoantigen. In celiac disease, where there is 2480 BRIEF REVIEWS: MHC CLASS II TETRAMERS also posttranslational modification of target Ags, class II tet- quence or a too-stringent binding register is a potential con- ramers composed of gliadin pMHC complexes were shown to cern. An important use of noncovalent peptide-loaded bind peripheral CD4 T cells; however, these cells were only pMHC production systems is the ability to assess many dif- detectable when the subjects consumed gluten-containing ferent tetramer-binding specificities at the same time. This has bread for 3 d (53). This finding strongly suggests that mo- been exploited in the TGEM technique described above, in bilization of the Ag-specific cells from tissue to blood was which large mixtures of peptides, most commonly repre- necessary for visualization, and it may provide a generalizable senting the entire protein sequence of potential antigenic strategy, involving Ag challenge, for improving the detection targets, are interrogated to determine which peptides within threshold for rare autoreactive cells. the mixture contain actual T cell epitopes. A related issue, particularly relevant to the analysis of low- Because of the high specificity and sensitivity of TCR rec- avidity T cell responses in autoimmunity, is the definition of ognition for pMHC, tetramer-binding studies can also be a precise peptide-binding register for docking within the class designed to identify unconventional antigenic epitopes. For II molecule. The presence of more than one binding register example, many protein therapeutics that are administered to within long peptides generated during Ag processing can result patients are potentially immunogenic. Analysis of the T cell in presentation of pMHC ligands to distinct TCR, with po- response in these cases, using tetramers containing pMHC tentially different roles in selection and autoreactivity (54, 55). specificities from the therapeutic molecule, can readily identify Tetramer studies of tumor Ags are similar to the autoantigen the specific epitopes that trigger immunogenicity, as illustrated approach, with an opportunity to help inform both disease in a study of Factor VIII administration in hemophilia A (63). Downloaded from mechanism and therapy. Although most tumor Ags are self-Ags Examples of technical variations that are designed to improve or modified self-Ags, intentional immunization with these Ags the efficiency of tetramer production include coexpression is conducted for therapeutic benefit. For example, class II with chaperone proteins (64), biotinylation of the expressed tetramer studies in patients with malignant melanoma after MHC molecule during synthesis (65), modifications of peptide experimental vaccine therapies documented evidence of im- linkers used to dock covalent peptides in the coexpressed MHC munogenicity, measured the effects of adjuvant on T cell groove (66), or the use of cleavable CLIP peptides (7), which http://www.jimmunol.org/ lineage, and have been used to evaluate shifts in T cell sub- take advantage of the natural role of invariant chain residues sets (56–59). Indeed, because the pMHC complex is itself (CLIP) that are a functional intermediate in loading peptides a surrogate for Ag recognition, it is feasible that tetramers into class II molecules. One of the most intriguing innovations might be considered for therapeutic expansion of Ag-specific is the use of entire libraries of class II pMHC molecules plated effector T cells (60). on arrays designed to capture Ag-specific T cells (67), similar to earlier studies using class I tetramers (68). Variations on the tetramer theme

Biophysical studies and off-rate analysis suggest that multi- by guest on September 25, 2021 valent binding of pMHC to the TCR is essential for successful Conclusions tetramer imaging of Ag-specific T cells. Monomeric and di- After more than a decade of experience, class II tetramer meric forms of pMHC are much less efficient than tetramers analysis has become a regular component of T cell-specificity (61), whereas higher-order multimers, such as octomers, studies across the entire landscape of immunological investi- decamers, or pMHC-coated beads, are all successful reagents, gation. However, a decision to include class II tetramer studies, generally comparable to tetramers. However, a caveat to this as well as how to use them, is not as routine as selecting Abs for interpretation is that streptavidin aggregates during storage, so flow cytometry. There are four key issues that should be that experiments ostensibly using tetrameric forms of pMHC considered and used as the basis for experimental choice. linked to streptavidin may, in fact, be using higher-order First, what is the anticipated frequency of CD4 T cells complexes, at least in part. recognizing immunodominant epitopes in the system? If A variety of methods has been successfully used to manu- there is no prior evidence for immunodominance and no facture pMHC class II tetramers, with expression of HLA information from previous T cell assays regarding prevalence, class II molecules as “empty” (without intentional loading then it is a challenge to select a particular pMHC tetramer that of peptide into the class II Ag-binding groove); “loaded,” will be representative of the T cell response profile. Either in which the expression system is coupled to a facilitated multiple different pMHC can be selected, individually or as peptide-loading process, resulting in production of complete a mixed tetramer pool, or a technique such as TGEM can be pMHC; or “covalent,” in which an MHC expression vector used in pilot studies to directly determine the appropriate is engineered to cotranslationally express the peptide, with pMHC combinations. a flexible and/or removable linker sequence allowing for 1:1 Second, what is the likely biodistribution of CD4 T cells of stoichiometry of peptide/MHC and directed peptide bind- interest? In cases such as celiac disease, as described above, ing into the class II-binding groove. Expression systems have mobilization of the Ag-specific T cells into the bloodstream included mammalian, insect cell, or bacterial sources (62). was accomplished by Ag challenge. This may have general There are advantages to each system, depending on the in- applicability, supported by the observations in T1D that tended type of tetramer assay planned. For example, covalent tetramer-positive cells are much higher after pancreas trans- pMHC are well suited for consistent production systems and plantation or after glutamate dehydrogenase immunization for situations in which a small number of specific pMHC (H. Reijonen, personal communication) compared with un- combinations are used. They are not well suited to screening immunized diabetic subjects. If the Ag-specific cells are at tis- assays involving hundreds of different potential peptide Ags, sue sites of interest, more complex biopsy and in situ histo- and neoepitope exposure due to the presence of linker se- chemistry techniques are needed. The Journal of Immunology 2481

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