ALBERT

Description: Key Points Rapamycin and Flt3L are synergistic in Treg induction when coadministered with antigen, resulting in improved tolerance induction. pDCs are required for efficient Treg induction and selectively expanded with Flt3L/rapamycin because of high mTOR activity.

Description: Inhibitor (neutralizing anti-drug antibody) formation against factor VIII (FVIII) is currently the most serious complication of FVIII replacement therapy for hemophilia A. The role of innate immune signals in this adaptive immune response is unclear. Lipopolysaccharide (LPS, the major component of the outer membrane in Gram-negative bacteria), which activates the innate immune sensor toll-like receptor 4 (TLR4), enhances inhibitor formation in hemophilia A mice. Intriguingly, earlier data suggested that activation of the endosomal DNA sensor TLR9 may actually suppress inhibitor formation. However, we demonstrated that a TLR9 agonist induced inhibitor formation against a factor IX transgene product in gene therapy for hemophilia B, which resulted from activation of monocyte-derived dendritic cells (moDCs) and enhancement of T follicular helper (Tfh) cell responses. Tfh cells drive germinal center (GC) formation and T cell help-dependent antibody formation. The aim of this study was to elucidate the role of TLR9 signaling in FVIII inhibitor formation in hemophilia A mice. Hemophilia A (F8e16-/-) B6/129 mice were co-injected IV (n=4) with FVIII (1.5 IU) and ODN-1826 (a class B CpG oligodeoxynucleotide, 50 µg), which is a TLR9 agonist, or injected on two consecutive days (n=3), first with ODN-1826 and then with FVIII on the next day. Control mice received FVIII only (n=5). Injections were performed once weekly for 4 weeks. Blood samples, spleens and subiliac (superficial inguinal) lymph nodes were collected for ELISA and Bethesda assays, and flow cytometry analysis. In our analysis, Tfh were defined as CD4+CXCR5+PD1+Bcl6- cells, while GC B cells were defined as CD19+GL7+CD95+. Tfh cell response and GC formation in the spleen were robustly enhanced by the TLR9 agonist compared to mice injected with FVIII only (2-fold for Tfh frequencies and 6-fold for GC B cells when co-injected; P〈 0.05). As a result, inhibitor titers increased 〉400-fold (on average from 6.4 to 2746.5 BU/ml). In contrast, anti-FVIII IgG1 levels increased only 2.5-fold. The differences between mice injected on two consecutive days and the FVIII-only group were not statistically significant. A time-course experiment was also carried out to monitor progress of immune response to FVIII (1.5 IU administered once weekly) in the absence of TLR9 agonist over time: 4 (n=4), 6 (n=4) and 8 weeks (n=5) after the initial antigen challenge. Inhibitor titers continued to rise beyond the fourth week of antigen challenges, reaching 46-fold higher values at eighth week (from 3 BU/ml in the fourth week to 138 BU/ml in the eighth week, p

Description: Key Points Crosspriming of AAV capsid-specific CD8+ T cells requires cooperation between distinct subsets of DCs. Innate immune sensing of the viral DNA genome induces cross-presentation of viral capsid in trans.

Description: Introduction: Adeno-associated virus (AAV) mediated gene transfer is currently evaluated in multiple Phase I/II and Phase III studies for the treatment of hemophilia. However, immune responses to both the AAV capsid and encoded transgene remain major impediments to clinical translation. Several studies have implicated innate immune sensors such as Toll-like receptors (TLR) and their downstream adaptor molecule MyD88 in sensing viral structures. TLR9-MyD88 signaling has been linked to cross-priming of CD8+ T cell responses to capsid and also to transgene product-specific CD8+ T cell responses. However, little is known about other signaling pathways that may lead to immune activation. Previously, our lab has shown that while liver gene transfer is capable of inducing immunological tolerance to AAV encoded transgene products, vector dose and design play a critical role. For instance, low hepatic gene expression levels may elicit a CD8+ T cell response to the AAV encoded transgene, resulting in loss of the model antigen ovalbumin (OVA) in C57BL/6 mice or of FIX expression in hemophilia B mice. We investigated innate immune sensing pathways that may play a role in initiating transgene specific CD8+ T cell response in the hepatic microenvironment. Further, we determined the contribution of hepatic antigen presenting cells (APC) by selectively depleting/neutralizing APCs and evaluating their effect on presentation of transgene product-derived antigen following AAV8-OVA liver gene delivery. Methods: Wild-type (WT) C57BL6 and specific innate sensing knockout mice on the C57BL6 background were intravenously (IV) injected with a predetermined immunogenic dose (1x109vg) of hepatotropic AAV8-OVA vector (Mol Ther 25:880, 2017). PBMCs were quantified at 4 weeks for OVA-specific CD8+ T cells using a class I MHC tetramer. Hepatic APC types [Kupffer cells, neutrophils, CD103+ dendritic cell (DC), CD11c+ DC, XCR1+ DC] involved in transgene specific CD8+ T cell activation were selectively depleted/inactivated by pre-treatment with gadolinium chloride (GdCl3), Ly6G, CD103 antibody respectively, or by administering diphtheria toxin (DT) to CD11c-DTR and XCR1-DTR mice. This was followed by intravenous administration of AAV8-OVA and CellTrace violet labeled OT-1 cells. Results: Similar to WT mice, TLR9-/-, TLR2-/-, TRIF-/-, IFNaR-/- and MDA5-/- mice developed a CD8+ T cell response indicating that these sensors do not play a role in transgene specific CD8+ T cells response. Interestingly, adaptor protein MyD88-/- mice did not elicit CD8+ T cell response to OVA, implying a MyD88 dependent but TLR9 independent response. Since MyD88 is an essential adaptor protein not only for TLR but also for interleukin-1 (IL-1) signaling pathways, we next analyzed IL-1R-/- mice. Similar to MyD88-/- mice, IL-1R-/- mice did not show OVA specific CD8+ T cells (p=0.006, 0.007 respectively), indicating that transgene-specific adaptive responses are mediated by IL-1R/MyD88 signaling. Kupffer cells and DCs are principal APCs in liver and infiltrating neutrophils could also act as APCs under inflammatory conditions in liver microenvironment. Using proliferation of OT-I cells as readout we tested if any of these cell types are required for presentation to transgene specific CD8+ T cells. In naïve control, GdCl3 treated and a-Ly6G antibody treated mice, OT-I cell proliferation reached 60%, 65% and 48% on average, respectively. Depletion of CD11c DCs substantially reduced the proliferation of OT-I cells to ~6% (p

Description: A serious complication in the treatment X-linked bleeding disorder hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), which compromise traditional replacement therapy. We previously developed an Oral immunotherapy (OTI) based on repeated uptake of a mixture of lettuce plant cells transgenic for heavy chain (HC) or C2 domain of human FVIII fused to cholera toxin B (CTB) subunit [Blood 124:1659; Plant Biotechnol J. 16:1148]. Fusion proteins were transgenically expressed in the chloroplasts. Repeated oral uptake of a mixture of freeze-dried powder of lettuce cells accomplished antigen delivery to the immune system of the small intestine by targeting of the GM1 receptor that is highly expressed on the surface of the gut epithelium, resulting in induction of regulatory T cells (Treg) that suppress inhibitor formation upon subsequent intravenous (iv) FVIII replacement therapy. An alternative to oral antigen delivery is the oral delivery of immune modulatory antibodies. Here, we compared the plant cell-based method with oral delivery of anti-CD3, which has been successful in murine models of autoimmune disease and is currently evaluated in clinical trials. Unlike in iv administration, oral anti-CD3 does not systemically deplete T cells. Hemophilia A BALB/c mice (F8 e16 gene deletion) received oral gavage of a mixture of CTB-FVIII-HC/-C2 (1.5 µg/antigen) expressing lettuce leaf cells 2x/week for 9 weeks. Starting at 4 weeks into the experiment, 1 IU/mouse of BDD-FVIII (Xyntha) was given iv, once per week for 5 weeks. Alternatively, following a published protocol that was successful in other models, 5 µg of hamster anti-murine CD3 was given by oral gavage daily for 5 straight days, followed by 5 weekly iv injections of BDD-FVIII. Control animals (no OTI) developed inhibitors with an average titer of 18 ± 3 BU/ml (n=16). Of these, 88% were high-titer (i.e 〉5 BU/ml, up to 43 BU/ml). Inhibitor formation was significantly reduced in plant cell-treated mice (10 ± 2.5 BU/ml, n=17), with 47% showing no or low-titer inhibitors (