A granular variant of CD63 is a regulator of repeated human mast cell degranulation.

Mast cells are secretory immune cells whose degranulation can provoke acute allergic reactions. It is presently unclear, however, whether an individual mast cell can repeatedly degranulate or turns dysfunctional after a single antigen stimulus. This work thus aims to better define the mast cell life cycle, with particular focus on new target structures for therapeutic or diagnostic approaches in allergy. Monoclonal antibodies were raised against degranulated cord blood-derived human mast cells. A subset of these antibodies that exclusively recognized degranulated mast cells, but did not cross-react with quiescent mast cells or other hematopoietic cell types, became key reagents in subsequent experiments. We identified a granular variant of tetraspanin CD63 as an exclusive molecular marker of degranulated human mast cells. Mutant analyses indicate that a cysteine cluster around residue C170 and protein glycosylation at residue N172 account for the antibody specificity. Here, we show that mast cells, which underwent an initial FcεRI-mediated degranulation, can be degranulated for at least another cycle in vitro. Repeated degranulation, however, requires an IgE/antigen stimulus that differs from the preceding one. Furthermore, the new variant-specific anti-CD63 antibodies effectively impair repeated cycles of mast cell degranulation. Our findings indicate that mast cells are stable, multiple-use cells, which are capable of surviving and delivering several consecutive hits. Surface expression of the novel CD63 variant is a distinguishing feature of such primed cells. Reagents directed against this molecular hallmark may thus become valuable diagnostic and therapeutic agents.