Plant‐produced Der p 2‐bearing bioparticles activate Th1/Treg‐related activation patterns in dendritic cells irrespective of the allergic background

To the Editor, Allergen immunotherapy (AIT) is the only disease-modifying treatment for allergic diseases with proven sustained efficacy, but long treatment duration, chronic exposure to aluminium salts and allergic side-effects are major drawbacks. AIT aims to redirect the underlying T helper type 2 (Th2)-skewed immune response towards an allergen-specific mixed T helper 1 (Th1)/regulatory T cell (Treg) response.1 Recently, plant-made enveloped eBioparticles (eBPs) were introduced as an allergen expression platform to display recombinant allergens at high densities on their surface.2 Such eBPs harbouring the recombinant house dust mite (HDM) allergen Der p 2 (eBP-rDer p 2) induced very potent Th1-associated IgG responses upon injection in mice.2 Here, we compared the allergenic and immunogenic potency of soluble and particulate allergen presentation. To assess allergenicity, we performed degranulation assays with basophilic leukaemia cells (RBL-2H3) transfected with the human high-affinity IgE receptor (FcεRI, subclone 30/25).3 The quality of RBL cells to induce degranulation upon cross-linking was assessed by using a purified mouse anti-rat FcεRI antibody (BD Pharmingen, clone BC4). Those cells were passively sensitized with human serum derived from HDM-allergic patients and the release of β-hexosaminidase was measured as readout for degranulation. Our data show that compared to soluble rDer p 2, eBP-rDer p 2 was less potent in triggering β-hexosaminidase release as shown by 10-fold higher median concentrations required to trigger half-maximum mediator release (Figure 1A). This suggests that multivalent high-density allergen presentation on eBPs creates hypoallergenicity. Earlier reports based on eBP-rFel d 1/-rAra h 2 show even (far) higher reductions in basophil degranulation.4, 5 It can be hypothesized that IgE binding capacities and related effector cell activation might be linked to the localization of the respective dominant IgE epitopes, which might either be exposed or shielded off due to the one-directional allergen anchorage on the eBP surface.5 Other factors contributing to deviating allergenic potency might include differences in the density of surface expression as well as size and charge of the eBPs. Besides allergenicity, one of the challenging requirements of AIT is to actively counterbalance an existing Th2-dominated inflammatory response by the induction of a Th1/Treg response. Dendritic cell (DC) activation is pivotal to induce downstream adaptive T- and B-cell responses that are crucial for vaccine effectiveness. Therefore, we assessed the efficacy of eBP-rDer p 2 to modulate the activity of monocyte-derived DCs (moDCs) from non-atopic and HDM-allergic donors. We treated the respective moDCs with equivalent concentrations of soluble rDer p 2 and eBP-rDer p 2, lipopolysaccharide (LPS) as a potent immune activator or the tolerance-associated vitamin D3 derivate calcitriol 1,25(OH)2D3 (VitD3) alone or in co-stimulation with LPS and assessed the expression levels of activation markers (CD80, CD83 and CD86) and tolerogenic markers (PD-L1 and ILT-3)6, 7 by flow cytometry. MoDCs from neither HDM-allergic nor non-atopic controls showed reactivity towards soluble rDer p 2 within a concentration range of 1–30 μg/mL (Figure 1B). In contrast, eBP-rDer p 2 induced CD80, CD83 and CD86 in moDCs from both donor groups. Besides, our data show that eBP-rDer p 2 induced similar expression of PD-L1 and low levels of ILT-3 in both healthy and non-atopic controls. Our findings furthermore show that stimulations with VitD3 treatment induced significantly increased levels of ILT-3 in the non-atopic group compared to HDM-allergic individuals. To assess the levels of secreted cytokines, we harvested supernatants from moDC cultures after 48 h of sample exposure and measured IL-6, IL-10 and IL-12 p70 by ELISA. Soluble rDer p 2 did not trigger a detectable cytokine response in patients or non-atopic controls (Figure 1C). However, we observed a dose-dependent upregulation of IL-6, IL-10 and IL-12p70 production by eBP-rDer p 2-treated moDCs of HDM-allergic and non-atopic donors. For most conditions, no significant differences between the groups were observed. However, our data show a trend towards increased IL-10 levels in the non-atopic group. The combination of LPS and VitD3 induced a much stronger IL-10 (and IL-6) production in non-atopic controls than in allergic subjects, indicating a possible suppression of IL-10 responses in the HDM-allergic group. At 10 μg/mL of eBP, IL-12p70 response was significantly lower in non-atopic controls compared to allergic donors. Overall, we conclude that soluble rDer p 2 is virtually unable to trigger DC activation, while eBP-rDer p 2 are potent inducers of DC maturation and cytokine production. The triggered responses were mostly unaffected by the atopic status and might, based on the plant-based production platform, likely be linked to glycan-sensing receptors, such as C-type lectin receptors. Besides, we have shown that the eBPs induced the tolerance-associated markers PD-L1 and to a minor extent ILT-3 on moDCs, as well as the production of the regulatory cytokine IL-10 and Th1 cytokine IL-12, which can possibly counteract the allergy-associated Th2 response.8, 9 As we saw a similar induction of PD-L1 and ILT-3 for LPS-treated conditions, it remains unclear if those responses ultimately result in effective allergen-specific Treg induction or rather act as a countermeasure to prevent excessive tissue damage, which is why further studies focusing on tolerance-inducing properties of eBPs are needed. Besides, it should be considered that the presence of IL-4 in the process of moDC generation might interfere with properties related to the allergic status of the donor. This limitation of our study could be circumvented by the usage of a primary DC model. Additionally, we investigated potential cytotoxic effects that eBP-rDer p 2 exposure might have on primary cells, for which we measured the cellular viability of stimulated moDCs. Our data show that eBP treatment in concentrations up to 30 μg/mL did not lead to significant reductions in viability compared to untreated moDCs (Figure 1D), hypothesizing that plant-derived eBPs are well tolerated. Taken together, eBP-rDer p 2 are hypoallergenic and immunomodulatory structures that may favour Th1/Treg immunity. This not only improves therapy safety but might furthermore allow for shortened treatment protocols due to increased immunogenicity. S. Busold, L. Aglas, C. Menage, M. Stigler and S.A. Versteeg contributed to this study by designing the experiments and acquiring, interpreting and analysing the data. L. Auger, R. Desgagnés, L. Faye, A.-C. Fitchette, C. Martel, B. Morel, V. Catala-Stordeur, G. Tropper, L.-P. Vézina and V. Gomord contributed to the concept design, development or manufacturing of the plant-derived bioparticles platform. S. Busold, L. Aglas, V. Gomord, T. B. H. Geijtenbeek and R. van Ree wrote, revised and edited the manuscript. J. A. Layhadi, M. Shamji, E. C. de Jong, T. B. H. Geijtenbeek and R. van Ree supervised the study. All authors critically reviewed the manuscript. We are grateful for the support obtained from the DC4Balance consortium, EAACI and the NVVI. Stefanie Busold received contract research funding and study material from Angany Inc., DC4Balance consortium and the EAACI and NVVI research fellowships. Lorenz Aglas and Maria Stigler received contract research funding and study material by Angany Inc. Louis Vézina and Véronique Gomord are co-founders, board members and respectively CEO and CSO of Angany Inc. Lydia Auger, Réjean Desgagnés, Loïc Faye, Anne-Catherine Fitchette, Caroline Martel, Virginie Stordeur, Guy Tropper, are employees of Angany Inc. or Angany Innovation. Esther C. de Jong and Teunis B. H. Geijtenbeek received funding from the DC4Balance consortium. J. A. Layhadi reports grants via Biomedical Research Funding (Imperial College BRC), all outside the submitted work. M. H. Shamji reports research grants from Immune Tolerance Network, Medical Research Council, Allergy Therapeutics, LETI Laboratorios, Revolo Biotherapeutics and lecture fees from Allergy Therapeutics and LETI Laboratorios, all outside the submitted work. Serge A. Versteeg and Charlotte Menage have no conflict of interest. Ronald van Ree received contract research funding and research material from Angany Inc. and the DC4Balance consortium and besides consulting fees and/or speaker fees from Angany Inc., HAL Allergy BV, Citeq BV, ThermoFisher Scientific, ALK Abello, Reacta Healthcare Ltd., Mission MightyMe and The Protein Brewery and has stock options from Angany Inc. This project was embedded in the DC4Balance consortium supported by Health Holland. The work has further been supported by Angany Inc. (Quebec, Canada) and ANGANY Innovation (Val de Reuil, France), the Austrian Science Funds (Projects P32189) and by the University of Salzburg priority program ‘Allergy-Cancer-BioNano Research Centre’. S. Busold received an EAACI Short-Term Research Fellowship and a travel grant from the Dutch Association of Immunology (NVVI). The data that support the findings of this study are available from the corresponding author upon request.

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