Erin Burnett

Anti-CD3 prevents factor VIII inhibitor development in hemophilia A mice by a regulatory CD4+CD25+-dependent mechanism and by shifting cytokine production to favor a Th1 response

Non-Fc-receptor binding anti-CD3 Ab therapy, in the setting of several different autoimmune disorders, can induce antigen-specific and long-lasting immunologic tolerance. Because factor VIII (FVIII) inhibitor formation is the most serious treatment-related complication for hemophilia A patients, we tested the efficacy of anti-CD3 to prevent FVIII inhibitor formation in hemophilia A BALB/c and C57BL/6 mice. A short course of low-dose anti-CD3 significantly increased expression of CD25 and the proportion of CD4+CD25+ regulatory T cells in the spleen and potently prevented the production of inhibitory and non-neutralizing anti-FVIII antibodies in both strains of mouse. Depleting the CD4+CD25+ cells during anti-CD3 therapy completely ablated tolerance to FVIII. Further phenotypic characterization of regulatory cells in tolerant mice showed a consistently higher number of CD4+GITR+ and CD4+FoxP3+ cells in both strains of mice. In addition, in tolerant C57BL/6 mice we observed an increase in CD4+CD25+ CTLA-4+ and CD4+CD25+mTGF-beta1+ cells. Finally, in vitro cytokine profiling demonstrated that splenocytes from tolerant BALB/c and C57BL/6 were polarized toward a Th1-immune response. Taken together, these findings indicate that anti-CD3 induces tolerance to FVIII and that the mechanism(s) regulating this response almost certainly occurs through the generation of several distinct regulatory T-cell lineages and by influencing cytokine production and profile.

A MicroRNA-regulated and GP64-pseudotyped Lentiviral Vector Mediates Stable Expression of FVIII in a Murine Model of Hemophilia A

The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system.

Recombinant and plasma-derived factor VIII products induce distinct splenic cytokine microenvironments in hemophilia A mice

The use of plasma-derived factor VIII (pdFVIII) concentrates in hemophilia A has been reported to result in reduced anti-FVIII antibody formation. In this study, we have investigated whether the cytokine microenvironment induced by pdFVIII has an influence on reducing anti-FVIII antibody titers in hemophilic mice. Microarray and confirmatory quantitative reverse transcription polymerase chain reaction (RT-PCR) experiments show that pdFVIII infusion causes a different transcriptional profile in dendritic cells than recombinant FVIII (rFVIII). Both treatments caused up-regulation of proinflammatory gene expression, but rFVIII and pdFVIII treatments promote expression of genes that induce Th1 and Th2 responses, respectively. Moreover, administration of rFVIII or pdFVIII concentrates resulted in distinct T-cell splenic cytokine microenvironments. rFVIII induced the release of Th1 cytokines and IL-10, whereas pdFVIII induced the release of Th2 cytokines and transforming growth factor-beta. We have also observed high titers of anti-human von Willebrand factor (VWF) antibodies in the pdFVIII-treated mice and propose that this results from antigenic competition. We further investigated the role of this phenomenon using infusions of FVIII and increasing concentrations of recombinant human factor IX (FIX). These studies show an inverse relationship between increasing concentrations of FIX and the production of anti-FVIII antibodies. In summary, these studies report new mechanisms that contribute to reduced anti-FVIII antibody development in hemophilia A after pdFVIII infusions.

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models

Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H. VWF knockout mice received hydrodynamic injections of mouse Vwf cDNA. Lower VWF antigen levels were demonstrated in both homozygous and heterozygous forms for both type 1 mutations from days 14-42. Recombinant protein infusions and hydrodynamic-expressed VWF propeptide to antigen ratios demonstrate that R1205H mouse VWF has an increased clearance rate, while Y1584C is normal. Recombinant ADAMTS13 digestions of Y1584C demonstrated enhanced cleavage of both human and mouse VWF115 substrates. Hydrodynamic-expressed VWF shows a loss of high molecular weight multimers for Y1584C compared with wild-type and R1205H. At normal physiologic levels of VWF, Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild-type VWF. This study has elucidated several novel mechanisms for these mutations and highlights that the type 1 VWD phenotype can be recapitulated in the VWF knockout hydrodynamic injection model.

Immunoglobulin isotypes and functional anti-FVIII antibodies in response to FVIII treatment in Balb/c and C57BL/6 haemophilia A mice

Summary.  Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti‐factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16‐disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T‐ and B‐cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti‐FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII‐treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII‐treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro‐environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain‐dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.