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. Keywords: atopy; cytokine receptors; interleukin-4; intracellular; patients