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Introduction: Prophylactic strategies to prevent the development of allergies by establishing tolerance remain an unmet medical need. We previously reported that the transfer of autologous hematopoietic stem cells (HSC) expressing the major timothy grass pollen allergen, Phl p 5, on their cell surface induced allergen-specific tolerance in mice. In this study, we investigated the ability of allergen-expressing immune cells (dendritic cells, CD4 + T cells, CD8 + T cells, and CD19 + B cells) to induce allergen-specific tolerance in naive mice and identified CD19 + B cells as promising candidates for allergen-specific cell therapy., Methods: For this purpose, CD19 + B cells were isolated from Phl p 5-transgenic BALB/c mice and transferred to naive BALB/c mice, pre-treated with a short course of rapamycin and an anti-CD40L antibody. Subsequently, the mice were subcutaneously sensitized three times at 4-week intervals to Phl p 5 and Bet v 1 as an unrelated control allergen. Allergen-expressing cells were followed in the blood to monitor molecular chimerism, and sera were analyzed for Phl p 5- and Bet v 1-specific IgE and IgG 1 levels by RBL assay and ELISA, respectively. In vivo allergen-induced lung inflammation was measured by whole-body plethysmography, and mast cell degranulation was determined by skin testing., Results: The transfer of purified Phl p 5-expressing CD19 + B cells to naive BALB/c mice induced B cell chimerism for up to three months and prevented the development of Phl p 5-specific IgE and IgG 1 antibody responses for a follow-up period of 26 weeks. Since Bet v 1 but not Phl p 5-specific antibodies were detected, the induction of tolerance was specific for Phl p 5. Whole-body plethysmography revealed preserved lung function in CD19 + B cell-treated mice in contrast to sensitized mice, and there was no Phl p 5-induced mast cell degranulation in treated mice., Discussion: Thus, we demonstrated that the transfer of Phl p 5-expressing CD19 + B cells induces allergen-specific tolerance in a mouse model of grass pollen allergy. This approach could be further translated into a prophylactic regimen for the prevention of IgE-mediated allergy in humans., Competing Interests: RV received research grants from Worg Pharmaceuticals, Hangzhou, China; HVD Biotech, Vienna, Austria; and Viravaxx, Vienna, Austria. He serves as a consultant for Viravaxx and Worg. 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Prickler, Baranyi, Mengrelis, Weijler, Kainz, Kratzer, Steiner, Mucha, Rudoph, Pilat, Bohle, Strobl, Pickl, Valenta, Linhart and Wekerle.)

Introduction: Antibody mediated rejection (ABMR) is a major factor limiting outcome after organ transplantation. Anti-HLA donor-specific antibodies (DSA) of the IgG isotype are mainly responsible for ABMR. Recently DSA of the IgE isotype were demonstrated in murine models as well as in a small cohort of sensitized transplant recipients. In the present study, we aimed to determine the frequency of pre-existing and de novo anti-HLA IgE antibodies in a cohort of 105 solid organ transplant recipients., Methods: We prospectively measured anti-HLA IgE antibodies in a cohort of kidney (n=60), liver, heart and lung (n=15 each) transplant recipients before and within one-year after transplantation, employing a single-antigen bead assay for HLA class I and class II antigens. Functional activity of anti-HLA IgE antibodies was assessed by an in vitro mediator release assay. Antibodies of the IgG1-4 subclasses and Th1 and Th2 cytokines were measured in anti-HLA IgE positive patients., Results: Pre-existing anti-HLA IgE antibodies were detected in 10% of renal recipients (including 3.3% IgE-DSA) and in 4.4% of non-renal solid organ transplant recipients (heart, liver and lung cohort). Anti-HLA IgE occurred only in patients that were positive for anti-HLA IgG, and most IgE positive patients had had a previous transplant. Only a small fraction of patients developed de novo anti-HLA IgE antibodies (1.7% of kidney recipients and 4.4% of non-renal recipients), whereas no de novo IgE-DSA was detected. IgG subclass antibodies showed a distinct pattern in patients who were positive for anti-HLA IgE. Moreover, patients with anti-HLA IgE showed elevated Th2 and also Th1 cytokine levels. Serum from IgE positive recipients led to degranulation of basophils in vitro , demonstrating functionality of anti-HLA IgE., Discussion: These data demonstrate that anti-HLA IgE antibodies occur at low frequency in kidney, liver, heart and lung transplant recipients. Anti-HLA IgE development is associated with sensitization at the IgG level, in particular through previous transplants and distinct IgG subclasses. Taken together, HLA specific IgE sensitization is a new phenomenon in solid organ transplant recipients whose potential relevance for allograft injury requires further investigation., Competing Interests: The 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., (Copyright © 2023 Mucha, Cho, Weijler, Muckenhuber, Hofmann, Wahrmann, Heinzel, Linhart, Gattinger, Valenta, Berlakovich, Zuckermann, Jaksch, Oberbauer and Wekerle.)

Humoral immunity is a major barrier limiting long-term outcome after organ transplantation. Especially, the production of antibodies directed against donor HLA/MHC antigens (i.e. donor-specific antibodies (DSA)) leading to antibody-mediated rejection (ABMR) is considered to be a major factor negatively affecting allograft survival. DSAs of the IgG isotype are routinely measured in transplant patients. However, not all patients diagnosed with IgG-DSA develop ABMR events. Therefore, research in better understanding the mechanisms of ABMR is of great importance. We recently demonstrated the production of MHC-specific IgE upon allograft rejection in mice and in transplant patients. IgE is classically connected with allergy and is known to be important for the humoral defense against helminths and worms. However, its role in autoimmune diseases and cancer has been reported recently as well. The concentration of IgE in blood is extremely low compared to other antibody isotypes. Therefore, detection of MHC-specific IgE from serum requires methods of high sensitivity. Since MHC-specific IgG-typically present at much higher serum levels-develops as well, high specificity is also required of IgE detection methods. In the murine model we developed an enzyme linked immunosorbent assay (ELISA) using MHC monomers for measurement of MHC-specific IgE, allowing us to distinguish between specificities of antibodies against different class I and class II antigens. For measurement of functional activity of MHC-specific IgE in vitro , a release assay using a rat basophil cell line (RBL-2H3) was established. For functional analysis of MHC-specific IgE in vivo , a cutaneous hypersensitivity reaction assay was adapted for this purpose using MHC monomers. Humanized RBL-2H3 cells transfected with cDNA coding for the human-high affinity IgE receptor were used for functionality measurement of donor-specific IgE in sensitized transplant patients. For detection of HLA-specific IgE, a bead assay was adapted, using beads expressing single HLA antigens. The aim of this publication is to demonstrate currently established methods for the detection and characterization of MHC-specific IgE in the murine and human setting., Competing Interests: The 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., (Copyright © 2020 Weijler, Mucha, Farkas, Baranyi, Pilat, Cho, Muckenhuber, Hopf, Wahrmann, Linhart, Valenta and Wekerle.)