Autologous Immune Cell Assay to Investigate risk of Processed- and Novel Food-Induced type 2 Inflammation in Peanut Allergy

Abstract Peanut allergy represents a major food-allergy burden, raising concerns about food processing and novel dietary products. Current diagnostics assess primarily allergic endpoints rather than immune mechanisms initiating and maintaining type 2 inflammation, particularly DC2-mediated Th2 polarization. Here, an in vitro autologous monocyte-derived dendritic cell (moDC)-T cell and B cell assay has been established to study immunomodulatory effects induced by unprocessed (P-D) and processed (P-DH (heated) or P-DHG (heated and glycated)) peanut proteins and emerging foods (protein concentrates or whole biomass), related to ex vivo DC2-T cell reactions. CD14+ monocytes, CD4+ T cells and CD19+ B cells were isolated from six peanut-allergic patients’ PBMCs. MoDCs generated with IL4/GM-CSF were exposed (48h) to type 2 polarizing cytokine (DC2) mix, or DC2 mix combined with the food samples. Next, DC2s were co-cultured with T cells (5d), followed by B cells incubation with DC2/T cell supernatant and food samples (10d). Supernatants and cells were analyzed for Th1/Th2/Th-regulatory (Treg) cells, IgE and IgG profiles. DC2 induced a strong Th2 phenotype and activity, P-D DC2 further enhanced IL13 secretion and %Tregs. P-DH DC2 favored Th2, whereas P-DHG DC2 increased IFNγ, with neither increasing %Treg. All increased CD40L+CD25+ memory Th2 cells. Wheat, whey and seaweed biomass had little effect, whereas algae DC2 showed distinct immunomodulatory, adjuvant-like activity. In conclusion, this autologous in vitro assay captures peanut-specific and generic Th2 responses and reactivity to food samples, supporting its use as additional tool to assess type 2-driving potential and allergenicity of emerging foods and processing methods in peanut-allergic patients. Clinical trial registration The current in vitro study was conducted in accordance with the Declaration of Helsinki and approved by the Medical Ethics Review Committee (METC) of the Erasmus MC (NL79534.078.21 MEC-2021-0905) and registrated at International Clinical Trials Registry Platform (NL-OMON51765). Graphical abstractImmune cell illustrations were adapted (1, 2) with permission; permission conveyed through Copyright Clearance Center, Inc. Capsule summary The autologous moDC-T cell-B cell in vitro assay may be used to assess whether processing methods or new foods might have intrinsic capacity to affect type 2 inflammation in peanut-allergic patients. Competing Interest Statement J.G. is head of the Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science at Utrecht University and partly employed by Danone Nutricia Research B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Footnotes ORCID ID and email addresses: Robine Janssen: r.janssen4{at}uu.nl, Alinda J Berends: a.j.berends{at}uu.nl, Marit Zuurveld: m.zuurveld{at}uu.nl, Govardus A. H. de Jong: aard.dejong{at}wur.nl, Dianne B.P.M. Somhorst: dianne.somhorst{at}wur.nl, Anouk Boudewijn: anouk.boudewijn{at}wur.nl, Severina Terlouw†, Sharon Veenbergen: s.veenbergen{at}erasmusmc.nl, Harry J Wichers: harry.wichers{at}wur.nl / harry.wichers{at}gmail.com, Johan Garssen: j.garssen{at}uu.nl, Shanna Bastiaan-Net: shanna.bastiaan{at}wur.nl, Rosalinde Masereeuw: r.masereeuw{at}uu.nl, Nicolette W de Jong: n.w.dejong{at}erasmusmc.nl Abbreviations - AGEs - Advanced glycation end-products - ALEX - Allergy Explorer (ALEX) multiplex array - BAT - Basophil activation test - CCR - C-C chemokine receptor - CD - Cluster of differentiation - CXCR - C-X-C chemokine receptor - DCs - Dendritic cells - ELISA - Enzyme-linked immunosorbent assay - HLA-DR - Human leukocyte antigen - DR - IFNγ - Interferon gamma - IgE/IgG - Immunoglobulin E / Immunoglobulin - G IL - Interleukin - LPS - Lipopolysaccharides - MFI - Median fluorescence intensity - FMO - Fluorescence Minus One - METC - Medical Ethics Review Committee - MHC-II - Major histocompatibility complex class II - MoDC - Monocyte-derived dendritic cell - NP - Not performed - OFC - Oral food challenge - OX40L - OX40 ligand - PBMC - Peripheral Blood Mononuclear Cells - P-D - Dialysed peanut protein - P-DH - Dialysed and heated peanut protein - P-DHG - Dialysed, heated and glycated peanut protein - PPR - Pattern recognition receptor - P1 - Sea Lettuce (Seaweed whole biomass) - P3 - Chlorella vulgaris (Algae whole biomass) - P5 - Cow’s milk (Hydrolyzed whey protein concentrate) - P7 - Wheat (Hydrolyzed Wheat protein concentrate) - PD-L1 - Programmed death-ligand 1 - SEM - Standard error of the mean - SPT - Skin prick test - Th1 - T helper type 1 (cell) - Th2 - T helper type 2 (cell) - UPFs - Ultra-processed foods