In human desensitizations High Content Screening the level of IgE sensitization varies and is unknown for each patient and the target dose used for desensitization is empirical, which impacts its safety 4, 5, 8. The mechanism of desensitization is not fully understood and we have observed that low antigen doses induce small amounts of extracellular calcium

flux, indicating the mobilization of endoplasmic reticulum stores, enabling functional CRAC channels to open 17. The sequential delivery of low antigen doses during desensitization may provide continued low levels of calcium entry with conformational changes of CRAC and other calcium-related channels locking further calcium entry and blocking signal transduction. Because calcium entry is clearly specifically impaired in our model, since a second non-desensitizing antigen allowed restoration of calcium flux, membrane compartmentalization may be required to exclude signal transduction molecules Roxadustat molecular weight around desensitized receptors. We observed

that in desensitized cells, phosphorylation of STAT6 and p38 MAP kinase was impaired and consequently TNF-α and IL-6 production was diminished. Since earlier studies indicated that STAT6-null BMMCs could not be desensitized 16, it is possible that STAT6 activity is required for desensitization, via a pathway different from the one leading to the acute and late activating responses. Our system is limited by the fact that BMMCs are cultured in IL-3, which may affect cytokine production 24. Nonetheless, this may have an important correlate in human desensitizations since our group has not observed delayed reactions in desensitized patients, confirming that the inhibition of mast cell activation Methisazone during desensitization prevented later hypersensitivity reactions 4, 5. Maintenance of hypo-responsiveness in desensitized cells was not sustained by the presence of an excess of soluble antigen since washed cells remained desensitized. It is possible that bound antigen is equilibrated in desensitized

cells. Earlier studies 12, 13 suggested that the hypo-responsiveness induced by desensitization was due to internalization of antigen/IgE/FcεRI complexes and that the lack of available IgE renders the cells refractory to further stimulation. In contrast, we show here that, unlike activation, internalization of IgE and FcεRI is impaired during specific desensitization (Fig. 4A) and that desensitized cells can be triggered by anti-IgE, since unbound IgE remains accessible and is available for crosslinking (Fig. 4B). Saturating doses of IgE in a co-culture system and the use of higher antigen doses 12 may promote internalization while low doses may redistribute antigen-bound receptor at the membrane level. Moreover, others have shown that low doses of antigen induce antigen-crosslinked receptors to remain mobile on the cell surface 25. In addition, microscopy studies gave us the opportunity to directly look into antigen localization after desensitization.