Comprehensive analysis of human IL‐4 receptor subunits shows compartmentalization in steady state and dupilumab treatment

Background Insight into the pathomechanism of atopic diseases demonstrated a pivotal role of the cytokines interleukin‐4 (IL‐4) and IL‐13, which has spurred the development of tailored therapeutics targeting their common IL‐4 receptor (IL‐4R). However, several aspects of the IL‐4R system remain ill‐defined in humans. Methods We used multicolor spectral flow cytometry to characterize IL‐4R subunit expression in 28 human immune cell subsets on protein and mRNA levels and assessed their subcellular distribution by applying a specifically adapted protocol that avoided influence of fixation and permeabilization on fluorochrome and antibody performance. In patients, we investigated possible changes in IL‐4Rα distribution before and during treatment with dupilumab, a monoclonal antibody‐targeting IL‐4Rα. Results Whereas all immune cell subsets investigated expressed IL‐4Rα and common γ chain protein and mRNA, expression of IL‐13Rα1 was restricted to myeloid and B cells. Interestingly, some cells contained considerably more intracellular IL‐4R protein than on their surface. Naive B cells were found to carry the highest levels of IL‐4Rα distributed evenly between surface and intracellular space, whereas IL‐4Rα was found predominantly in intracellular pools in neutrophils. In patients with atopic diseases treated with dupilumab, we observed that engagement of IL‐4Rα by dupilumab resulted in internalization of the antibody and decreased total IL‐4Rα expression. Notably, even after months of treatment not all intracellular IL‐4Rα molecules were occupied by dupilumab, indicating the presence of a “dormant” intracellular IL‐4Rα pool that could be mobilized upon certain extrinsic or intrinsic cues. Conclusion Collectively, our findings suggest that distinct human immune cell subsets contain surface and intracellular IL‐4R pools, which are differently affected by targeted biologic treatment. All human immune cell subsets express interleukin (IL)‐4 receptor (IL‐4R) subunits at mRNA and protein levels, conferring sensitivity to IL‐4 and/or IL‐13. IL‐4R subunits compartmentalize in a cell‐ and IL‐4R subunit‐specific manner between surface and intracellular location. Dupilumab therapy results in downregulation of IL‐4Rα and endocytosis of dupilumab, but a “dormant” pool of intracellular IL‐4Rα molecules remains unoccupied.Abbreviations: γc, common gamma chain; EC50, half‐maximal effective concentration; IL‐4R, interleukin‐4 receptor; IL‐4Rα, α subunit of interleukin‐4 recepto