HLA class II peptide tetramers vs allergen-induced proliferation for identification of allergen-specific CD4 T cells.

Background: Fluorescence-labeled MHC class II/peptide tetramer complexes are considered as optimal tools to characterize allergen-specific CD4(+) T cells, but this technique is restricted to frequently expressed HLA class II molecules and knowledge of immunodominant epitopes. In contrast, allergen-stimulated proliferation assessed by CFSE dilution is less sophisticated and widely applicable. The major mugwort allergen, Art v 1, contains only one single, immunodominant, HLA-DR1-restricted epitope (Art v 1(25-36)). Thus, essentially all Art v 1-reactive cells should be identified by a HLA-DRB1*01:01/Art v 1(19-36) tetramer. Methods: We compared specificity and sensitivity of tetramer(+) and allergen-induced proliferating (CFSElo) CD4(+) T cells by flow cytometry. Results: The frequency of tetramer(+) CD4(+) T cells determined ex vivo in PBMC of mugwort-allergic individuals ranged from 0 to 0.029%. After 2-3 weeks of in vitro expansion, sufficient tetramer(+) T cells for phenotyping were detected in 83% of Art v 1(25-36)-reactive T-cell lines (TCL) from mugwort-allergic individuals, but not in TCL from healthy individuals. The tetramers defined bona fide Th2 cells. Notably, Art v 1(25-36)-reactive TCL depleted of tetramer(+) T cells still reacted to the peptide, and only 44% of Art v 1(25-36)-specific T-cell clones were detected by the tetramer. CFSElo CD4(+) T cells contained only 0.3-10.7% of tetramer(+) T cells and very low proportions of Th2 cells. Conclusion: Allergen-specific T cells can be identified by HLA class II tetramers with high specificity, but unexpected low sensitivity. In contrast, allergen-stimulated CFSElo CD4(+) T cells contain extremely high fractions of bystander cells. Therefore, for T-cell monitoring, either method should be interpreted with caution.