Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A

Abstract

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

“…44 In brief, 4-5 × 10 5 irradiated (3,000 rad) CD90 depleted splenocytes were cocultured in wells of 96-well plates with 1 × 10 5 splenic CD4 + T cells from pools of three to five mice in 200 µl/well RPMI 1640 complete media 45 supplemented with increasing concentrations of rhFVIII or OVA. After 72 hours of coculture, 1 µCi 3 H-thymidine was added to the media and 18 hours later cells were harvested and incorporation of the radiolabel was determined on a β-scintillation counter (PerkinElmer, Shelton, CT).…”

Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice

“…In most preclinical experiments using immunocompetent hemophilia A murine and canine models, strong immune responses against FVIII after gene therapy have completely inhibited circulating FVIII activity and thus subverted the effect of gene therapy. [2][3][4][5]8,9,[14][15][16] Recent gene transfer studies 1,5,9,[17][18][19][20] indicate that the risk of transgene-specific immune responses depends on multiple factors, including the type and dose of the vector, the expression cassette and tissue specificity of the promoter, the type and level of transgene expression, route of administration, transduced cell type, and the age and the underlying mutation of the gene therapy model. Some of these factors have been extensively reviewed.…”

Muscle creatine kinase/SV40 hybrid promoter for muscle-targeted long-term transgene expression

“…This suggests that the MCK/ SV40 (L) promoter is unlikely to improve transgene expression for a non-immunogenic gene. However, studies of gene transfer of allogeneic factor VIII (20,43,44), IX (45,46), and erythropoietin (19,47) into animals indicate the risk of eliciting immune responses against a transgene, even if it originates from the same species. Thus, we believe that the MCK/SV40 (L) promoter might confer considerable benefit in gene therapy for congenital protein deficiencies in which the therapeutic gene is recognized as foreign by the immune system.…”

“…Immunemediated elimination of cells bearing a transgene product (14,17) and neutralizing antibodies to transgene products (18)(19)(20) might account for the transient expression observed with conventional CMV and CAG promoters. Thus, the above results suggest that the MCK/SV40 (L) promoter has weak immunogenicity, which allows long-term transgene expression.…”

“…In most cases, expression from the CMV promoter peaks 2 to 4 days after delivery of the transgene and declines to the background level within 4 weeks (14)(15)(16). This may be due to immunological elimination of cells bearing a foreign gene product (14,17), since administration of neutralizing antibodies to transgene products (18)(19)(20) also results in such transgene silencing. Thus, the success of long-term gene therapy in part depends upon the ability of the promoter to avoid induction of immune responses to the transgene product.…”