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Dexamethasone promotes durable factor VIII-specific tolerance in hemophilia A mice via thymic mechanisms.

ABSTRACT: The development of inhibitory antibodies to factor VIII is the most serious complication of replacement therapy in hemophilia A. Activation of the innate immune system during exposure to this protein contributes to inhibitor development. However, avoidance of factor VIII exposure during innate immune system activation by external stimuli (e.g., vaccines) has not been consistently shown to prevent inhibitors. We hypothesized that dexamethasone, a drug with potent anti-inflammatory effects, could prevent inhibitors by promoting immunologic tolerance to factor VIII in hemophilia A mice. Transient dexamethasone treatment during ainitial factor VIII exposure reduced the incidence of anti-factor VIII immunoglobulin G in both a conventional hemophilia A mouse model (E16KO, 77% vs 100%, P=0.048) and a hemophilia A mouse model with a humanized major histocompatibility complex type II transgene (E17KO/hMHC, 6% vs 33%, P=0.0048). More importantly, among E17KO/hMHC mice that did not develop anti-factor VIII immunoglobulin G after initial exposure, dexamethasone-treated mice were less likely to develop a response after re-exposure six (7% vs 52%, P=0.005) and 16 weeks later (7% vs 50%, P=0.097). Similar results were obtained even when factor VIII re-exposure occurred in the context of lipopolysaccharide (30% vs 100%, P=0.069). The ability of these mice to develop immunoglobulin G to human von Willebrand factor, a structurally unrelated antigen, remained unaffected by treatment. Transient dexamethasone administration therefore promotes antigen-specific immunologic tolerance to factor VIII. This effect is associated with an increase in the percentage of thymic regulatory T cells (12.06% vs 4.73%, P<0.001) and changes in the thymic messenger ribonucleic acid transcription profile.

SUBMITTER: Georgescu MT

PROVIDER: S-EPMC6068046 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Dexamethasone promotes durable factor VIII-specific tolerance in hemophilia A mice via thymic mechanisms.

Georgescu Maria T MT Moorehead Paul C PC van Velzen Alice S AS Nesbitt Kate K Reipert Birgit M BM Steinitz Katharina N KN Schuster Maria M Hough Christine C Lillicrap David D

Haematologica 20180419 8

The development of inhibitory antibodies to factor VIII is the most serious complication of replacement therapy in hemophilia A. Activation of the innate immune system during exposure to this protein contributes to inhibitor development. However, avoidance of factor VIII exposure during innate immune system activation by external stimuli (e.g., vaccines) has not been consistently shown to prevent inhibitors. We hypothesized that dexamethasone, a drug with potent anti-inflammatory effects, could ...[more]

PMID: 29674503

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Project description:Essentials The immune response is a significant concern in gene therapy. Platelet-targeted gene therapy can restore hemostasis and induce immune tolerance. CD4 T cell compartment is tolerized after platelet gene therapy. Preconditioning regimen affects immune tolerance induction in platelet gene therapy.SummaryBackground Immune responses are a major concern in gene therapy. Our previous studies demonstrated that platelet-targeted factor VIII (FVIII) (2bF8) gene therapy together with in vivo drug selection of transduced cells can rescue the bleeding diathesis and induce immune tolerance in FVIIInull mice. Objective To investigate whether non-selectable 2bF8 lentiviral vector (LV) for the induction of platelet-FVIII expression is sufficient to induce immune tolerance and how immune tolerance is induced after 2bF8LV gene therapy. Methods Platelet-FVIII expression was introduced by 2bF8LV transduction and transplantation. FVIII assays and tail bleeding tests were used to confirm the success of platelet gene therapy. Animals were challenged with rhF8 to explore if immune tolerance was induced after gene therapy. Treg cell analysis, T-cell proliferation assay and memory B-cell-mediated ELISPOT assay were used to investigate the potential mechanisms of immune tolerance. Results We showed that platelet-FVIII expression was sustained and the bleeding diathesis was restored in FVIIInull mice after 2bF8LV gene therapy. None of the transduced recipients developed anti-FVIII inhibitory antibodies in the groups preconditioned with 660 cGy irradiation or busulfan plus ATG treatment even after rhF8 challenge. Treg cells significantly increased in 2bF8LV-transduced recipients and the immune tolerance developed was transferable. CD4+ T cells from treated animals failed to proliferate in response to rhF8 re-stimulation, but memory B cells could differentiate into antibody secreting cells in 2bF8LV-transduced recipients. Conclusion 2bF8LV gene transfer without in vivo selection of manipulated cells can introduce immune tolerance in hemophilia A mice and this immune tolerance is CD4+ T cell mediated.

Project description:Hemophilia A is a genetic disorder that results in the deficiency of functional factor VIII protein, which plays a key role in blood coagulation. Currently, the majority of hemophilia A patients are treated with repeated infusions of factor VIII protein. Approximately 30% of severe hemophilia A patients develop neutralizing antibodies to factor VIII (known as factor VIII inhibitors) due to treatment, rendering factor VIII protein infusions ineffective. Previously, mice receiving murine IL-2 complexed with α-murine IL-2 mAbs (JES6-1A12) showed a lack of factor VIII inhibitor formation after factor VIII treatment, which was associated with the proliferation and the activation of factor VIII-specific regulatory T cells (Tregs). In this paper, we evaluated if an Fc-fused mutated protein analog of mouse IL-2, named Fc.Mut24, engineered to selectively promote the expansion of Tregs in vivo can modulate factor VIII-specific immune responses. The mice received one intraperitoneal injection of Fc.Mut24. When the regulatory T cell population reached its highest frequency and peak activation, the mice received a hydrodynamic injection of factor VIII plasmid (day 4) followed by a second Fc.Mut24 dose (day 7). Peripheral blood was collected weekly. Flow cytometry was used to characterize the peripheral blood cell populations, while ELISA and Bethesda assays were used to assess the inhibitor concentrations and the functional titers in plasma. The activated partial thromboplastin time assay was used to assess the functional activities of factor VIII in blood. The mice receiving Fc.Mut24 showed a dramatic and transient increase in the population of activated Tregs after Fc.Mut24 injection. Factor VIII gene therapy via hydrodynamic injection resulted in high anti-factor VIII inhibitor concentrations in control PBS-injected mice, whereas the mice treated with Fc.Mut24 produced no inhibitors. Most significantly, there were no inhibitors generated after a second hydrodynamic injection of factor VIII plasmid administered at 19 weeks after the first injection in Fc.Mut24-treated mice. The mice receiving Fc.Mut24 maintained high levels of factor VIII activity throughout the experiment, while the control mice had the factor VIII activity dropped to undetectable levels a few weeks after the first factor VIII plasmid injection. Our data show that human therapies analogous to Fc.Mut24 could potentially provide a method to prevent inhibitor formation and induce long-term immune tolerance to factor VIII in hemophilia patients.

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Dexamethasone promotes durable factor VIII-specific tolerance in hemophilia A mice via thymic mechanisms.

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Dexamethasone promotes durable factor VIII-specific tolerance in hemophilia A mice via thymic mechanisms.

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