Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells.

Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.

TLR9-independent CD8+ T cell responses in hepatic AAV gene transfer through IL-1R1-MyD88 signaling.

Upon viral infection of the liver, CD8+ T cell responses may be triggered despite the immune suppressive properties that manifest in this organ. We sought to identify pathways that activate responses to a neoantigen expressed in hepatocytes, using adeno-associated viral (AAV) gene transfer. It was previously established that cooperation between plasmacytoid dendritic cells (pDCs), which sense AAV genomes by Toll-like receptor 9 (TLR9), and conventional DCs promotes cross-priming of capsid-specific CD8+ T cells. Surprisingly, we find local initiation of a CD8+ T cell response against antigen expressed in ∼20% of murine hepatocytes, independent of TLR9 or type I interferons and instead relying on IL-1 receptor 1-MyD88 signaling. Both IL-1α and IL-1ß contribute to this response, which can be blunted by IL-1 blockade. Upon AAV administration, IL-1-producing pDCs infiltrate the liver and co-cluster with XCR1+ DCs, CD8+ T cells, and Kupffer cells. Analogous events were observed following coagulation factor VIII gene transfer in hemophilia A mice. Therefore, pDCs have alternative means of promoting anti-viral T cell responses and participate in intrahepatic immune cell networks similar to those that form in lymphoid organs. Combined TLR9 and IL-1 blockade may broadly prevent CD8+ T responses against AAV capsid and transgene product.

CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII.

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by "redirecting" polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

Lifestyle Modification Improves Insulin Resistance and Carotid Intima-Media Thickness in a Metabolically Healthy Obese Prepubescent Population.

OBJECTIVES: The aim of this study was to analyze effects of a 12-month lifestyle modification that involved a Mediterranean diet (MedDiet) and physical activity (PA) program in a population of metabolically healthy obese children (MHOCh). METHODS: We included a population of MHOCh with ≤1 of the following criteria: waist circumference and blood pressure ≥90 percentile, triglycerides >150 mg/dL, high-density lipoprotein-cholesterol (HDL-c) <40 mg/dL, or impaired fasting glucose. After 12 months of intensive lifestyle modification, anthropometric measurements, glycemic and lipid profiles, adherence to the MedDiet, energy intake, PA, body composition, and carotid intima-media thickness (cIMT) were analyzed. RESULTS: One hundred thirty-one MHOCh (70 boys and 61 girls; P = 0.65, age: 7.9 ±â€Š1.3 years, body mass index [BMI]: 24.7 ±â€Š3.5 kg/m2) were included. After 12 months of intervention, a significant decrease in standard deviation (SD) units of body weight (-0.5 ±â€Š0.1; P < 0.001) and BMI (-0.5 ±â€Š0.1; P < 0.001) were observed in the total population. A significant improvement in adherence to the MedDiet (+2 points) and a significant reduction in protein, fatty acids, total fat, and cholesterol intake in the entire population were observed. All participants did more moderate-vigorous PA, which led to a significant increase in lean and total mass and decrease in total fat. Significant improvements in the glycemic profile (insulin levels [-6.6 µIU/mL, P < 0.001] and HOMA index [-1.2, P < 0.001]) were observed. Participants with pathological cIMT values reduced this cardiovascular predictor to normal values. CONCLUSIONS: A 12-month lifestyle modification intervention involving weight loss with MedDiet and PA in MHOCh yielded improvements in MedDiet adherence, lipid intake, moderate-vigorous PA, body composition, insulin resistance, and cIMT.

Role of FoxP3+ Regulatory T Cells in Modulating Immune Responses to Adeno-Associated Virus Gene Therapy.

Adeno-associated virus (AAV) gene therapy is making rapid strides owing to its wide range of therapeutic applications. However, development of serious immune responses to the capsid antigen or the therapeutic transgene product hinders its full clinical impact. Immune suppressive (IS) drug treatments have been used in various clinical trials to prevent the deleterious effects of cytotoxic T cells to the viral vector or transgene, although there is no consensus on the best treatment regimen, dosage, or schedule. Regulatory T cells (Tregs) are crucial for maintaining tolerance against self or nonself antigens. Of importance, Tregs also play an important role in dampening immune responses to AAV gene therapy, including tolerance induction to the transgene product. Approaches to harness the tolerogenic effect of Tregs include the use of selective IS drugs that expand existing Tregs, and skew activated conventional T cells into antigen-specific peripherally induced Tregs. In addition, Tregs can be expanded ex vivo and delivered as cellular therapy. Furthermore, receptor engineering can be used to increase the potency and specificity of Tregs allowing for suppression at lower doses and reducing the risk of disrupting protective immunity. Because immune-mediated toxicities to AAV vectors are a concern in the clinic, strategies that can enhance or preserve Treg function should be considered to improve both the safety and efficacy of AAV gene therapy.

Viral Vector Based Immunotherapy for Peanut Allergy.

Food 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.

Redundancy in Innate Immune Pathways That Promote CD8+ T-Cell Responses in AAV1 Muscle Gene Transfer.

While adeno-associated viral (AAV) vectors are successfully used in a variety of in vivo gene therapy applications, they continue to be hampered by the immune system. Here, we sought to identify innate and cytokine signaling pathways that promote CD8+ T-cell responses against the transgene product upon AAV1 vector administration to murine skeletal muscle. Eliminating just one of several pathways (including DNA sensing via TLR9, IL-1 receptor signaling, and possibly endosomal sensing of double-stranded RNA) substantially reduced the CD8+ T-cell response at lower vector doses but was surprisingly ineffective at higher doses. Using genetic, antibody-mediated, and vector engineering approaches, we show that blockade of at least two innate pathways is required to achieve an effect at higher vector doses. Concurrent blockade of IL-1R1 > MyD88 and TLR9 > MyD88 > type I IFN > IFNaR pathways was often but not always synergistic and had limited utility in preventing antibody formation against the transgene product. Further, even low-frequency CD8+ T-cell responses could eliminate transgene expression, even in MyD88- or IL-1R1-deficient animals that received a low vector dose. However, we provide evidence that CpG depletion of vector genomes and including TLR9 inhibitory sequences can synergize. When this construct was combined with the use of a muscle-specific promoter, transgene expression in muscle was sustained with minimal local or systemic CD8+ T-cell response. Thus, innate immune avoidance/blockade strategies by themselves, albeit helpful, may not be sufficient to prevent destructive cellular responses in muscle gene transfer because of the redundancy of immune-activating pathways.

B cell focused transient immune suppression protocol for efficient AAV readministration to the liver.

Adeno-associated virus (AAV) vectors are used for correcting multiple genetic disorders. Although the goal is to achieve lifelong correction with a single vector administration, the ability to redose would enable the extension of therapy in cases in which initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAbs) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell-targeting protocol using a combination of monoclonal Ab therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression before (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig-mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.

Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion.

Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.

Distinct functions and transcriptional signatures in orally induced regulatory T cell populations.

Oral 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-ß (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 TGFß 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 TGFß 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.

Single-dose AAV vector gene immunotherapy to treat food allergy.

Immunotherapies 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.