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Background: Major histocompatibility complex (MHC) class II tetramers (tetramers) allow to detect allergen-specific CD4(+) T cells at a single-cell level. Limits to this technology include HLA restriction and the need to identify immunodominant T cell epitopes., Objective: Assessing the expression of various activation markers following allergen stimulation to replace tetramer staining., Methods: Peripheral blood mononuclear cells (PBMCs) from 25 birch pollen, grass pollen or house dust mite allergic individuals were stimulated with peptide mixes encompassing immunodominant epitopes from corresponding major allergens. After 2 weeks of in vitro amplification, cells were stained with both the appropriate tetramer and antibodies directed to CD25, CD30, CD39, CD69, CD137, CD154, GITR, HLA-DR and ICOS, before FACS analysis., Results: Following allergen stimulation, percentages of tetramer(+) cells among CD4(+) CD154(+) cells range from 5% to 87%, depending upon donors. As for CD154, a large inter-individual variability is observed in terms of surface expression for all activation markers tested in allergen-stimulated PBMCs. T cells reactive with either tetramers (0.4-10.4% CD4(+) T cells) or anti-marker antibodies (2.2-32.7% CD4(+) T cells), but not both, are observed, reflecting the presence of anergic as well as non-specifically activated cells. Tetramer(+) /marker(+) , tetramer(+) /marker(-) and tetramer(-) /marker(+) cells were compared for their capacity to express cytokines, demonstrating that only the former represent bona fide allergen-specific activated CD4(+) T cells, based upon a higher expression of cytokines or corresponding genes in presence of the allergen., Conclusion and Clinical Relevance: No strict correlation exists between tetramer staining and the expression of multiple activation markers in stimulated CD4(+) T cells. Dual staining allows to discriminate functional tetramer(+) /marker(+) vs. anergic (tetramer(+) /marker(-) ) allergen-specific T cells or non-specifically activated (tetramer(-) /marker(+) ) T cells. Combining tetramer staining with the detection of activation markers helps understanding patient heterogeneity regarding specific CD4(+) T cell responses. This approach has immediate relevance for monitoring immune changes induced during specific immunotherapy., (© 2011 Blackwell Publishing Ltd.)

Background: A better understanding of allergen-specific CD4(+) T cell responses is needed to help improving immunological therapies. Objective To compare CD4(+) T cell responses against seasonal (Bet v 1) and perennial (Der p 1, Der p 2) allergens., Methods: Major histocompatibility complex class II peptide tetramers were engineered to monitor allergen-specific T cell responses. After in vitro expansion, tetramer(+) cells were tested for surface markers using cytofluorometry. Cytokine gene expression and production were assessed using quantitative PCR and cytokine surface capture assays, respectively., Results: Tetramer(+) cells were detected in 19 patients allergic to house dust mites (HDM), seven allergic to birch pollen, 13 allergic to both and nine non-allergics with either an HLA-DRB1(*) 0101, (*) 0301, (*) 1501 or an HLA-DPB1(*) 0401 background. High-avidity T cells are elicited against the immunodominant Bet v 1(141-155) epitope, whereas broader low-avidity T cell responses are induced against Der p 1(16-30) ,(110-124) ,(171-185) and Der p 2(26-40,107-121) epitopes. Responses against Bet v 1 involve effector (CDL62 low, CCR7 low) or central (CD62L(+) , CCR7(+) ) memory cells in allergic and non-allergic individuals, respectively, whereas central memory cells are mostly detected against mite allergens. In non-allergics, both mite and Bet v 1-specific T cells produce IFN-γ and IL-10. In contrast to Bet v 1-driven Th2 responses, mite allergens induce highly polymorphic responses in allergics, including Th1, Th2/Th17 or mixed Th1/Th2 profiles. Mite-specific T cell frequencies in the blood remain in the range of 1-6 × 10(-4) CD4(+) T cells throughout the year., Conclusion: Different memory CD4(+) T cell responses are elicited in the context of chronic vs. seasonal stimulation with the allergen(s). The heterogeneity in the patterns of CD4(+) T cell responses observed in patients allergic to HDMs should be taken into account for specific immunotherapy., (© 2010 Blackwell Publishing Ltd.)

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-gamma 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-gamma), 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 observed in healthy individuals.

Background: We report herein critical methodological principles for assessing, at a single cell level, allergen-specific T cell responses using MHC class II peptide tetramers., Methods: We developed MHC class II peptide tetramers to monitor T cell responses against the immunodominant Bet v 1(141-155) peptide in individuals with either an HLA-DRB1*0101, DRB1*0401 or DRB1*1501 background. In vitro stimulation was performed with serially truncated versions of the Bet v 1(141-155) epitope chemically conjugated to the Ii-Key peptide., Results: Identification of Bet v 1(141-155) as a high-affinity epitope for multiple HLA-DRB1 allotypes led to the development of corresponding tetramers detecting Bet v 1(141-155)-specific T cells with a high specificity and sensitivity. Stimulation with Bet v 1(141-155) Ii-Key conjugate peptides is the most efficient procedure to expand Bet v 1(141-155)-specific CD4+ T cells, allowing to detect such cells in both allergic and healthy individuals. MHC class II Bet v 1(141-155) tetramer-positive T cells produce IFN-gamma and IL-10 in healthy individuals, and IL-5 in allergic patients. Frequencies of Bet v 1-specific CD4+ T cells circulating in the blood of allergic or nonallergic individuals range from approximately 10(-5) to 10(-3) CD4+ T cells, outside or within the pollen season, respectively., Conclusions: MHC class II peptide tetramers are valuable tools to assess allergen-specific T cell responses, both qualitatively and quantitatively. Selection of a high-affinity T cell epitope, as well as optimization of in vitro stimulation conditions to expand rare T cell progenitors are critical success factors in those analyses., ((c) 2008 S. Karger AG, Basel.)