Assessment of Bet v 1-Specific CD4  T Cell Responses in Allergic and Nonallergic Individuals Using MHC Class II Peptide Tetramers 1 Laurence Van Overtvelt, 2 * Erik Wambre, 2 * Bernard Maille `re, † Eric von Hofe,  Anne Louise, ‡ Anne Marie Balazuc, ‡ Barbara Bohle, # Didier Ebo,** Christophe Leboulaire, § Gilles Garcia, ¶ and Philippe Moingeon 3 * In this study, we used HLA-DRB1*0101, DRB1*0401, and DRB1*1501 peptide tetramers combined with cytokine surface capture assays to characterize CD4  T cell responses against the immunodominant T cell epitope (peptide 141–155) from the major birch pollen allergen Bet v 1, in both healthy and allergic individuals. We could detect Bet v 1-specific T cells in the PBMC of 20 birch pollen allergic patients, but also in 9 of 9 healthy individuals tested. Analysis at a single-cell level revealed that allergen-specific CD4  T cells from healthy individuals secrete IFN- and IL-10 in response to the allergen, whereas cells from allergic patients are bona fide Th2 cells (producing mostly IL-5, some IL-10, but no IFN-), as corroborated by patterns of cytokines produced by T cell clones. A fraction of Bet v 1-specific cells isolated from healthy, but not allergic, individuals also expresses CTLA-4, glucocorticoid-induced TNF receptor, and Foxp 3, indicating that they represent regulatory T cells. In this model of seasonal exposure to allergen, we also demonstrate the tremendous dynamics of T cell responses in both allergic and nonallergic individuals during the peak pollen season, with an expansion of Bet v 1-specific precursors from 10 6 to 10 3 among circulating CD4  T lymphocytes. Allergy vaccines should be designed to recapitulate such naturally protective Th1/regulatory T cell responses ob- served in healthy individuals. The Journal of Immunology, 2008, 180: 4514 – 4522. T ype I allergy is caused by an inappropriate Th2 response to allergens from the environment (1–3), leading to anti- gen-specific IgE production as well as recruitment and activation of proinflammatory cells (e.g., eosinophils and mast cells) in mucosal target organs (4 – 6). In contrast, under similar exposure conditions, tolerance to allergens is maintained in non- allergic individuals, possibly as a consequence of various nonex- clusive mechanisms involving either 1) immune ignorance of nonpathogenic antigens, 2) anergy or deletion of allergen-reactive T cells, or 3) the induction of a protective allergen-specific CD4 + T cell response. Specifically, a potential role of IL-10-producing T cells has been suggested in the induction and maintenance of pe- ripheral tolerance to allergens, either in the context of natural re- sponses in healthy individuals, or as a consequence of successful immunotherapy protocols (7–13). Understanding the nature of such CD4 + T cells responses in healthy individuals is critical to improve current allergy vaccines, with the assumption that natural responses are protective against allergic inflammation, and thus that allergen-specific immunotherapy should recapitulate such im- mune responses (14). MHC class II peptide tetramer technology has been recently used succesfully in the field of allergy to char- acterize allergen-specific CD4 + T cell responses (15–17). Soluble multimeric tetramers comprise fluorescently labeled recombinant MHC class II molecules, each bound to a high-affinity T cell epitope (18 –21). Altogether, MHC class II peptide tetramers con- stitute high-avidity reagents allowing the detection of epitope-spe- cific CD4 + T lymphocytes. Birch (Betula verucosa) pollen is causing one of the most com- mon tree pollen allergies in Europe and North America. More than 95% of birch pollen (BP) 4 allergic patients exhibit IgEs against the major allergen Bet v 1, and up to 60% of these patients react solely to Bet v 1 (22, 23). To analyze in detail T cell responses to Bet v 1, we developed MHC class II peptide tetramers as a means to detect CD4 + T lymphocytes specific for the Bet v 1 141–155 im- munodominant epitope following in vitro stimulation with Bet v1 141–155 -Ii (invariant chain)-Key/epitope hybrid peptides (24). Using this approach, we demonstrate that specific T cells can be detected in PBMC of both allergic and nonallergic individuals, albeit with dramatically different patterns of T cell polarization. Materials and Methods Patients and HLA-DR typing Citrated peripheral blood was obtained from BP or house dust mite (HDM) allergic patients recruited at Ho ˆpital Be ´cle `re (Clamart, France), University *Recherche et De ´veloppement, Stallerge `nes SA, Antony; † Service d’Inge ´nierie Mo- le ´culaire des Prote ´ines, Commissariat a ` l’Energie Atomique, Gif sur Yvette; ‡ Institut Pasteur, Paris; § Beckman Coulter, Marseille; ¶ De ´partement de Pneumologie, Ho ˆpital Be ´cle `re, Clamart, France;  Antigen Express, Worcester, MA 01605; # Department of Pathophysiology, Medical University of Vienna, Vienna, Austria; and **Department of Immunology, Allergy and Rheumatology, Antwerp University, Antwerp, Belgium Received for publication September 11, 2007. Accepted for publication January 27, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was founded by the Fonds zur Fo ¨rderung der wissenschaftlichen For- schung (SFB-F1807-B04), Austria. E.W. is a recipient of a Convention Industrielle de Formation par la Recherche fellowship from the French government. 2 L.V.O. and E.W. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Philippe Moingeon, Staller- ge `nes, 6 rue Alexis de Tocqueville, 92183 Antony, France. E-mail address: pmoingeon@stallergenes.fr 4 Abbreviations used in this paper: BP, birch pollen; DC, dendritic cell; ECD, PE- Texas red; GITR, glucocorticoid-induced TNF receptor; HDM, house dust mite; Ii, invariant chain; PC7, phycoerythrin-cyanine 7, PC7; rBet v 1, recombinant Bet v 1; TCC, T cell clone; Treg, regulatory T. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org