The research was funded by the Austrian Science Fund (FWF) DK W 1248-B13 and SFBF4609. ability of K562 cells to present antigenic peptides derived from whole proteins either taken up or endogenously expressed as LAMP-1 fusion protein. In both cases the ability of these cells to process and present peptides derived from whole proteins critically depended on the expression of HLA-DM. We have identified strategies to achieve efficient presentation of allergenic peptides on engineered APC and demonstrate their use to stimulate T cells from allergic individuals. Accessory signals provided by antigen presenting cells (APC) govern the responses of T cells towards cognate peptide-major histocompatibility complex (MHC) molecules. Plxdc1 Attempts to manipulate T cells as well as the generation of T cells to be used for adoptive transfer critically depends on our knowledge of signals that enhance or efficiently inhibit T cell responses. In this context much can be learned from studies on the interaction of natural APCs such as dendritic cells (DC) with T cells but these cells also harbor certain constraints. Due to the plethora of activating and inhibitory ligands provided by professional APC it is difficult to study the role of individual costimulatory or coinhibitory ligands using such cells. In addition, the limited availability of MHC-matched donors and variability in their T cell stimulatory capacity are of concern when using primary APC to study T cell activation processes. The use of engineered antigen presenting cells (eAPC) – often also designated artificial APCs – is an attractive option to stimulate antigen-specific T cells since it allows to provide T cells with accessory signals of choice. The human erythroleukemia cell line K562 is an ideal platform for antigen presentation to human T cells as it can Bendroflumethiazide be furnished with MHC molecules of choice but is devoid of endogenously expressed MHC class I as well as class II (MHCII) molecules, thereby minimizing the stimulation of allo-reactive T cells1. Initial studies have focused on the generation and use of MHC class I expressing K562 cells to stimulate CD8+ T cells specific for antigens derived from pathogens or tumors2,3,4,5. More recently these cells have been shown to be suitable to present MHCII restricted antigens to CD4+ T cells. In this context the focus was also on the stimulation of CD4+ T cells recognizing peptides derived from viruses or tumor antigens6,7. To date such cells have not been used to study CD4+ T cells that contribute to pathological processes. In this context eAPC might be useful to identify signals that efficiently dampen helper T cells that drive aberrant immune responses. Allergen-specific Type 2 helper (Th2) CD4+ T cells play a Bendroflumethiazide central role in initiating and promoting type I allergy8. By inducing class switching of B cells via IL-4 they are responsible for the production of allergen-specific IgE, the major effector molecule in this disease. In addition, they produce IL-13 and IL-5 thereby stimulating airway epithelial cells and eosinophils9,10. Th2 cells also contribute to late phase reactions8. Consequently, allergen-specific Th2 CD4+ T cells are primary targets in attempts to ameliorate IgE-associated allergic disease11 and improved knowledge regarding signals that dampen Th2 responses is desirable. Studies on allergen-specific T cell clones have yielded Bendroflumethiazide invaluable information on immunodominant T cell epitopes of major allergens present in pollen extracts or other allergen sources12,13. Importantly, such clones have been used to isolate cDNAs encoding allergen-specific T cell receptors (TCRs) making it possible to reconstruct the allergen-specific synapse at the molecular level14,15,16. This is a valuable tool for pursuing and testing strategies to counteract Th2 based allergen-specific T cell responses15. They have been used to demonstrate that regulatory T cells and Th1 cells recognizing peptides derived from allergens might reduce symptoms in allergic individuals by directly antagonizing Th2 cells or via other mechanisms15,17. eAPC stably expressing MHCII molecules of choice are valuable for studying mechanisms and strategies for antigen processing Bendroflumethiazide and presentation to CD4+ T cells. Moreover, they might be useful tools to expand and study allergen-specific T cells derived from allergic individuals. Accessory molecules like coinhibitory ligands of choice can be expressed on these cells. Consequently they can be used to identify signals that inhibit allergen-specific T cells or skew them towards a non-Th2 phenotype. Here we report on the use of K562 cells stably expressing MHCII molecules to present immunodominant T cell epitopes from allergens. Jurkat-based T cell reporter cells.