Development and validation of cashew patches.
To identify the optimal excipient for patch manufacturing, lyophilized cashew protein extract was solubilized in either PBS 1X, NaCl 0.45% or phosphate buffer 0.1M and deposited on epicutaneous patches. Deposits were dried and then resolubilized using distilled water and analyzed by ELISA or SDS PAGE (Figure S2 ). Cashew-specific IgG1 or IgE generated in orally sensitized mice reacted with all extract deposits, demonstrating that their immunogenicity was preserved (Figure S2A ). Moreover, the electrophoretic profiles of all deposits were comparable to that of the initial protein extract (Figure S2B ). However, the best protein integrity was obtained with phosphate buffer, as demonstrated by the absence of aggregated proteins in the pellet, that was selected as the preferred excipient. Patches loaded with fluorescent cashew proteins were applied to cashew-sensitized mice for 48 hours to evaluate the capacity of cashew patches to deliver allergens to skin dendritic cells (DCs). To control for the degree of sensitization, cashew-specific IgG1, IgG2a and IgE were quantified from plasma collected before patches were applied. All mice presented similar antibody titers that were consistent with previous experiments (data not shown). The number of fluorescent DCs was then measured from BLNs (Figure 3 ). A significant increase of cashew-positive DCs was observed in mice that received cashew patches compared to those receiving excipient patches. Of note, this increase was more pronounced in cDC2 dermal DCs and Langerhans cells than in cDC1 dermal DCs. Overall, these data demonstrate that cashew patches can be produced while maintaining allergen integrity and that they are able to deliver allergens to skin DCs, especially Langerhans cells and cDC2.
EPIT with cashew patches increased cashew-specific IgG2a in sensitized mice .
Mice were sensitized orally to cashew as described above and received cashew patches for up to 16 weeks, at a rate of 1 patch per week for 48 hours per application (EPIT). Plasma was collected every two weeks during treatment to measure the evolution of cashew-specific IgE, IgG1 and IgG2a (Figure 4 ). A transient increase of cashew-specific IgE was observed in EPIT mice compared to the sham group, with a peak level at week 12 post-treatment followed by a significant decrease (Figure 4A ). Similarly, an increase of cashew-specific IgG1 was observed in EPIT mice, with a peak level at week 10 post-treatment followed by a significant decrease (Figure 4B ). Finally, a progressive and continuous increase in cashew specific IgG2a was observed in treated mice compared to the sham group (Figure 4C ). Overall, these data indicate that EPIT to cashew can strongly modulate cashew-specific antibody responses.
EPIT with cashew patches protects sensitized mice against IgE-mediated anaphylaxis .
Mice were sensitized and treated as described above. At the end of the treatment period (i.e. 8, 12 or 16 weeks), mice were challenged orally to cashew (Figure 5 ). A clear protective effect was observed in EPIT mice compared to the sham group and this protective effect was further improved with increasing length of treatment. This protection was characterized by a decrease in both temperature drop (Figures 5A, 5C and 5E ) and clinical symptoms (Figures 5B, 5D and 5F ). To evaluate the capacity of EPIT to protect against mast-cell activation, plasma was collected immediately after oral challenge to measure the level of mMCP-1 and mMCP-7 (Figure 6 ). A sharp and significant decrease of both mMCP-1 and mMCP-7 was observed in EPIT mice compared to the sham group. Of note, the prolongation of the treatment period does not induce further decrease in the plasmatic concentration of these two proteases. Overall, these data indicate that EPIT to cashew can efficiently protect mice against IgE-mediated anaphylaxis induced by oral challenge.