Browsing by Author "Daniell, Henry"
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Item Distinct functions and transcriptional signatures in orally induced regulatory T cell populations(Frontiers Media, 2023-10-26) Biswas, Moanaro; So, Kaman; Bertolini, Thais B.; Krishnan, Preethi; Rana, Jyoti; Muñoz-Melero, Maite; Syed, Farooq; Kumar, Sandeep R. P.; Gao, Hongyu; Xuei, Xiaoling; Terhorst, Cox; Daniell, Henry; Cao, Sha; Herzog, Roland W.; Pediatrics, School of MedicineOral administration of antigen induces regulatory T cells (Treg) that can not only control local immune responses in the small intestine, but also traffic to the central immune system to deliver systemic suppression. Employing murine models of the inherited bleeding disorder hemophilia, we find that oral antigen administration induces three CD4+ Treg subsets, namely FoxP3+LAP-, FoxP3+LAP+, and FoxP3-LAP+. These T cells act in concert to suppress systemic antibody production induced by therapeutic protein administration. Whilst both FoxP3+LAP+ and FoxP3-LAP+ CD4+ T cells express membrane-bound TGF-β (latency associated peptide, LAP), phenotypic, functional, and single cell transcriptomic analyses reveal distinct characteristics in the two subsets. As judged by an increase in IL-2Rα and TCR signaling, elevated expression of co-inhibitory receptor molecules and upregulation of the TGFβ and IL-10 signaling pathways, FoxP3+LAP+ cells are an activated form of FoxP3+LAP- Treg. Whereas FoxP3-LAP+ cells express low levels of genes involved in TCR signaling or co-stimulation, engagement of the AP-1 complex members Jun/Fos and Atf3 is most prominent, consistent with potent IL-10 production. Single cell transcriptomic analysis further reveals that engagement of the Jun/Fos transcription factors is requisite for mediating TGFβ expression. This can occur via an Il2ra dependent or independent process in FoxP3+LAP+ or FoxP3-LAP+ cells respectively. Surprisingly, both FoxP3+LAP+ and FoxP3-LAP+ cells potently suppress and induce FoxP3 expression in CD4+ conventional T cells. In this process, FoxP3-LAP+ cells may themselves convert to FoxP3+ Treg. We conclude that orally induced suppression is dependent on multiple regulatory cell types with complementary and interconnected roles.Item Investigational new drug enabling angiotensin oral-delivery studies to attenuate pulmonary hypertension(Elsevier, 2020-03) Daniell, Henry; Mangu, Venkata; Yakubov, Bakhtiyor; Park, Jiyoung; Habibi, Peyman; Shi, Yao; Gonnella, Patricia A.; Fisher, Amanda; Cook, Todd; Zeng, Lily; Kawut, Steven M.; Lahm, Tim; Cellular and Integrative Physiology, School of MedicinePulmonary arterial hypertension (PAH) is a deadly and uncurable disease characterized by remodeling of the pulmonary vasculature and increased pulmonary artery pressure. Angiotensin Converting Enzyme 2 (ACE2) and its product, angiotensin-(1-7) [ANG-(1-7)] were expressed in lettuce chloroplasts to facilitate affordable oral drug delivery. Lyophilized lettuce cells were stable up to 28 months at ambient temperature with proper folding, assembly of CTB-ACE2/ANG-(1-7) and functionality. When the antibiotic resistance gene was removed, Ang1-7 expression was stable in subsequent generations in marker-free transplastomic lines. Oral gavage of monocrotaline-induced PAH rats resulted in dose-dependent delivery of ANG-(1-7) and ACE2 in plasma/tissues and PAH development was attenuated with decreases in right ventricular (RV) hypertrophy, RV systolic pressure, total pulmonary resistance and pulmonary artery remodeling. Such attenuation correlated well with alterations in the transcription of Ang-(1-7) receptor MAS and angiotensin II receptor AGTRI as well as IL-1β and TGF-β1. Toxicology studies showed that both male and female rats tolerated ~10-fold ACE2/ANG-(1-7) higher than efficacy dose. Plant cell wall degrading enzymes enhanced plasma levels of orally delivered protein drug bioencapsulated within plant cells. Efficient attenuation of PAH with no toxicity augurs well for clinical advancement of the first oral protein therapy to prevent/treat underlying pathology for this disease.Item Plant cell-made protein antigens for induction of Oral tolerance(Elsevier, 2019-11-15) Daniell, Henry; Kulis, Michael; Herzog, Roland W.; Pediatrics, School of MedicineThe gut associated lymphoid tissue has effective mechanisms in place to maintain tolerance to food antigens. These can be exploited to induce antigen-specific tolerance for the prevention and treatment of autoimmune diseases and severe allergies and to prevent serious immune responses in protein replacement therapies for genetic diseases. An oral tolerance approach for the prevention of peanut allergy in infants proved highly efficacious and advances in treatment of peanut allergy have brought forth an oral immunotherapy drug that is currently awaiting FDA approval. Several other protein antigens made in plant cells are in clinical development. Plant cell-made proteins are protected in the stomach from acids and enzymes after their oral delivery because of bioencapsulation within plant cell wall, but are released to the immune system upon digestion by gut microbes. Utilization of fusion protein technologies facilitates their delivery to the immune system, oral tolerance induction at low antigen doses, resulting in efficient induction of FoxP3+ and latency-associated peptide (LAP)+ regulatory T cells that express immune suppressive cytokines such as IL-10. LAP and IL-10 expression represent potential biomarkers for plant-based oral tolerance. Efficacy studies in hemophilia dogs support clinical development of oral delivery of bioencapsulated antigens to prevent anti-drug antibody formation. Production of clinical grade materials in cGMP facilities, stability of antigens in lyophilized plant cells for several years when stored at ambient temperature, efficacy of oral delivery of human doses in large animal models and lack of toxicity augur well for clinical advancement of this novel drug delivery concept.Item Potential Role for Oral Tolerance in Gene Therapy(Elsevier, 2023) Butterfield, John S. S.; Li, Xin; Arisa, Sreevani; Kwon, Kwang-Chul; Daniell, Henry; Herzog, Roland W.; Pediatrics, School of MedicineOral immunotherapies are being developed for various autoimmune diseases and allergies to suppress immune responses in an antigen-specific manner. Previous studies have shown that anti-drug antibody (inhibitor) formation in protein replacement therapy for the inherited bleeding disorder hemophilia can be prevented by repeated oral delivery of coagulation factor antigens bioencapsulated in transplastomic lettuce cells. Here, we find that this approach substantially reduces antibody development against factor VIII in hemophilia A mice treated with adeno-associated viral gene transfer. We propose that the concept of oral tolerance can be applied to prevent immune responses against therapeutic transgene products expressed in gene therapy.Item Preclinical development of plant‐based oral immune modulatory therapy for haemophilia B(Wiley, 2021-10) Srinivasan, Aparajitha; Herzog, Roland W.; Khan, Imran; Sherman, Alexandra; Bertolini, Thais; Wynn, Tung; Daniell, Henry; Pediatrics, School of MedicineAnti‐drug antibody (ADA) formation is a major complication in treatment of the X‐linked bleeding disorder haemophilia B (deficiency in coagulation factor IX, FIX). Current clinical immune tolerance protocols are often not effective due to complications such as anaphylactic reactions against FIX. Plant‐based oral tolerance induction may address this problem, as illustrated by the recent first regulatory approval of orally delivered plant cells to treat peanut allergy. Our previous studies showed that oral delivery of plant cells expressing FIX fused to the transmucosal carrier CTB (cholera toxin subunit B) in chloroplasts suppressed ADA in animals with haemophilia B. We report here creation of the first lettuce transplastomic lines expressing a coagulation factor, in the absence of antibiotic resistance gene. Stable integration of the CTB‐FIX gene and homoplasmy (transformation of ˜10 000 copies in each cell) were maintained in both T1 and T2 generation marker‐free plants. CTB‐FIX expression in lyophilized leaves of T1 and T2 marker‐free plants was 1.0–1.5 mg/g dry weight, confirming that the marker excision did not affect antigen levels. Oral administration of CTB‐FIX to Sprague Dawley rats at 0.25, 1 or 2.5 mg/kg did not produce overt adverse effects or toxicity. The no‐observed‐adverse‐effect level (NOAEL) is at least 2.5 mg/kg for a single oral administration in rats. Oral administration of CTB‐FIX at 0.3 or 1.47 mg/kg either mixed in food or as an oral suspension to Beagle dogs did not produce any observable toxicity. These toxicology studies should facilitate filing of regulatory approval documents and evaluation in haemophilia B patients.Item Role of Orally Induced Regulatory T Cells in Immunotherapy and Tolerance(Elsevier, 2021) Bertolini, Thais B.; Biswas, Moanaro; Terhorst, Cox; Daniell, Henry; Herzog, Roland W.; Piñeros, Annie R.; Pediatrics, School of MedicineOral antigen administration to induce regulatory T cells (Treg) takes advantage of regulatory mechanisms that the gastrointestinal tract utilizes to promote unresponsiveness against food antigens or commensal microorganisms. Recently, antigen-based oral immunotherapies (OITs) have shown efficacy as treatment for food allergy and autoimmune diseases. Similarly, OITs appear to prevent anti-drug antibody responses in replacement therapy for genetic diseases. Intestinal epithelial cells and microbiota possibly condition dendritic cells (DC) toward a tolerogenic phenotype that induces Treg via expression of several mediators, e.g. IL-10, transforming growth factor-β, retinoic acid. Several factors, such as metabolites derived from microbiota or diet, impact the stability and expansion of these induced Treg, which include, but are not limited to, FoxP3+ Treg, LAP+ Treg, and/or Tr1 cells. Here, we review various orally induced Treg, their plasticity and cooperation between the Treg subsets, as well as underlying mechanisms controlling their induction and role in oral tolerance.Item Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia(Frontiers, 2020-05) Kumar, Sandeep R. P.; Wang, Xiaomei; Avuthu, Nagavardhini; Bertolini, Thais B.; Terhorst, Cox; Guda, Chittibabu; Daniell, Henry; Herzog, Roland W.; Pediatrics, School of MedicineFusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells in the lamina propria of the small but not large intestine. By contrast, no changes in frequencies of CD4+CD25+FoxP3+ T cells were observed. Here we demonstrate that, surprisingly, the adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice significantly suppress antibodies directed against FIX. By contrast, equal numbers of splenic CD4+CD25+ T cells do not have an effect on antibody formation. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine and that suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg. The observation that CD4+CD25-LAP+ Treg migrate to the spleen are useful for the design of clinical protocols.Item Single-dose AAV vector gene immunotherapy to treat food allergy(Elsevier, 2022-07-16) Gonzalez-Visiedo, Miguel; Li, Xin; Munoz-Melero, Maite; Kulis, Michael D.; Daniell, Henry; Markusic, David M.; Pediatrics, School of MedicineImmunotherapies for patients with food allergy have shown some success in limiting allergic responses. However, these approaches require lengthy protocols with repeated allergen dosing and patients can relapse following discontinuation of treatment. The purpose of this study was to test if a single dose of an adeno-associated virus (AAV) vector can safely prevent and treat egg allergy in a mouse model. AAV vectors expressing ovalbumin (OVA) under an ubiquitous or liver-specific promoter were injected prior to or after epicutaneous sensitization with OVA. Mice treated with either AAV8-OVA vector were completely protected from allergy sensitization. These animals had a significant reduction in anaphylaxis mediated by a reduction in OVA-specific IgE titers. In mice with established OVA allergy, allergic responses were mitigated only in mice treated with an AAV8-OVA vector expressing OVA from an ubiquitous promoter. In conclusion, an AAV vector with a liver-specific promoter was more effective for allergy prevention, but higher OVA levels were necessary for reducing symptoms in preexisting allergy. Overall, our AAV gene immunotherapy resulted in an expansion of OVA-specific FoxP3+ CD4+ T cells, an increase in the regulatory cytokine IL-10, and a reduction in the IgE promoting cytokine IL-13.Item Suppression of anti-drug antibody formation against coagulation factor VIII by oral delivery of anti-CD3 monoclonal antibody in hemophilia A mice(Elsevier, 2023) Bertolini, Thais B.; Herzog, Roland W.; Kumar, Sandeep; Sherman, Alexandra; Rana, Jyoti; Kaczmarek, Radoslaw; Yamada, Kentaro; Arisa, Sreevani; Lillicrap, David; Terhorst, Cox; Daniell, Henry; Biswas, Moanaro; Pediatrics, School of MedicineActive tolerance to ingested dietary antigens forms the basis for oral immunotherapy to food allergens or autoimmune self-antigens. Alternatively, oral administration of anti-CD3 monoclonal antibody can be effective in modulating systemic immune responses without T cell depletion. Here we assessed the efficacy of full length and the F(ab')2 fragment of oral anti-CD3 to prevent anti-drug antibody (ADA) formation to clotting factor VIII (FVIII) protein replacement therapy in hemophilia A mice. A short course of low dose oral anti-CD3 F(ab')2 reduced the production of neutralizing ADAs, and suppression was significantly enhanced when oral anti-CD3 was timed concurrently with FVIII administration. Tolerance was accompanied by the early induction of FoxP3+LAP-, FoxP3+LAP+, and FoxP3-LAP+ populations of CD4+ T cells in the spleen and mesenteric lymph nodes. FoxP3+LAP+ Tregs expressing CD69, CTLA-4, and PD1 persisted in spleens of treated mice, but did not produce IL-10. Finally, we attempted to combine the anti-CD3 approach with oral intake of FVIII antigen (using our previously established method of using lettuce plant cells transgenic for FVIII antigen fused to cholera toxin B (CTB) subunit, which suppresses ADAs in part through induction of IL-10 producing FoxP3-LAP+ Treg). However, combining these two approaches failed to improve suppression of ADAs. We conclude that oral anti-CD3 treatment is a promising approach to prevention of ADA formation in systemic protein replacement therapy, albeit via mechanisms distinct from and not synergistic with oral intake of bioencapsulated antigen.Item Viral Vector Based Immunotherapy for Peanut Allergy(MDPI, 2024-07-13) Gonzalez-Visiedo, Miguel; Herzog, Roland W.; Munoz-Melero, Maite; Blessinger, Sophia A.; Cook-Mills, Joan M.; Daniell, Henry; Markusic, David M.; Pediatrics, School of MedicineFood allergy (FA) is estimated to impact up to 10% of the population and is a growing health concern. FA results from a failure in the mucosal immune system to establish or maintain immunological tolerance to innocuous dietary antigens, IgE production, and the release of histamine and other mediators upon exposure to a food allergen. Of the different FAs, peanut allergy has the highest incidence of severe allergic responses, including systemic anaphylaxis. Despite the recent FDA approval of peanut oral immunotherapy and other investigational immunotherapies, a loss of protection following cessation of therapy can occur, suggesting that these therapies do not address the underlying immune response driving FA. Our lab has shown that liver-directed gene therapy with an adeno-associated virus (AAV) vector induces transgene product-specific regulatory T cells (Tregs), eradicates pre-existing pathogenic antibodies, and protects against anaphylaxis in several models, including ovalbumin induced FA. In an epicutaneous peanut allergy mouse model, the hepatic AAV co-expression of four peanut antigens Ara h1, Ara h2, Ara h3, and Ara h6 together or the single expression of Ara h3 prevented the development of a peanut allergy. Since FA patients show a reduction in Treg numbers and/or function, we believe our approach may address this unmet need.