interaction with pruritogens, including SP and compound 48/80, we evaluated LL-37-induced itch using the mouse cheek model. As opposed to pruritogens, which induce scratching in this model, LL-37 does not induce scratching (see Fig E8 in this article's Online Repository at www.jacionline.org).The data presented here demonstrate that SP and compound 48/ 80 activate native Mrgprs but fail to activate Mrgprs in which a single amino acid residue was mutated. In contrast, LL-37 activates both native receptor and mutant receptors. This finding indicates that the site(s) of Mrgpr activation for the pruritogens SP and compound 48/80 differs from the activation site(s) for LL-37. Furthermore, an Mrgpr antagonist, QWF, 2 inhibits the interaction of SP and compound 48/80 with MRGPRX2 but not that between LL-37 and the receptor.These findings have several implications. First, they reveal that the Glu and Asn residues at the end of the fourth TMD and beginning of the fourth extracellular loop of Mrgprs are necessary for the interaction of SP and compound 48/80 with members of this receptor family. Second, not all compounds that activate Mrgprs or induce mast-cell degranulation result in itch. It is possible that LL-37 may interact with other cells such as keratinocytes and additional receptors to induce the release of molecules with antipruritic properties such as semaphorin 3A. 11 Another possibility is that activation of MRGPRX2 on mast cells by SP versus LL-37 may result in differential release of granules and their associated mediators from mast cells. Third, at least some Mrgprs appear to have more than 1 site for signaling followed by distinct behavioral effects. Fourth, although MrgprB2 has been proposed as the mouse ortholog of human MRGPRX2, modeling demonstrates that mouse MrgprA1 and human MRGPRX2 also exhibit topological similarities (see Fig E9 in this article's Online Repository at www.jacionline.org). Taken together, these data provide further support for the possibility that antagonists of MRGPRX2 may be useful for the treatment of itch and inflammation while providing guidance with respect to the development of receptor antagonists.
Dendritic cells (DCs) are the most important antigen presenting cells to activate naïve T cells, which results in the case of Type 1 allergies in a Type 2 helper T cell (Th2)-driven specific immune response towards allergens. So far, a number of different subsets of specialized DCs in different organs have been identified. In the recent past methods to study the interaction of DCs with allergenic proteins, their different uptake and processing mechanisms followed by the presentation to T cells were developed. The following review aims to summarize the most important characteristics of DC subsets in the context of allergic diseases, and highlights the recent findings. These detailed studies can contribute to a better understanding of the pathomechanisms of allergic diseases and contribute to the identification of key factors to be addressed for therapeutic interventions.
Invariant natural killer T (iNKT) cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses. In contrast to conventional T lymphocytes they recognize lipid antigens. The aim of the project is to establish a novel model system, to study iNKT-TCR – ligand interaction. An iNKT reporter cell line (JE6-1
REP-iNKT
) was engineered by introducing the human iNKT-TCR into a human leukemic T cell line carrying an NF-κB-driven fluorescent transcriptional reporter construct. Antigen presenting BW
STIM
cells expressing human CD1d and CD80 were generated. Reporter induction in JE6-1
REP-iNKT
cells was assessed by flow cytometry. CRISPR/Cas9 was used for β2M knock out in JE6-1
REP-iNKT
cells to abrogate CD1d expression and thus excluding antigen self-presentation. Reporter cells were shown to specifically react with iNKT antigens presented via CD1d. Their sensitivity towards α-GalCer was comparable to a murine iNKT hybridoma cell line. In conclusion, we created a novel iNKT reporter platform which, compared to traditional iNKT cell assays, is characterized by a shorter turnaround time and lower costs. It thus facilitates the identification of antigenic structures that drive the activation of iNKT cells in health and disease.
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