Blockade of CD40/CD40 Ligand Interactions Prevents Induction of Factor VIII Inhibitors in Hemophilic Mice but Does not Induce Lasting Immune Tolerance

Abstract

SummaryPatients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.

Reduction of the inhibitory antibody response to human factor VIII in hemophilia A mice by mutagenesis of the A2 domain B-cell epitope

“…[31][32][33][34] The E16 knock-out hemophilia A mice used in this study, along with the genetically similar, phenotypically indistinguishable E17 knock-out mice, develop immune responses to human fVIII that require CD4 ϩ T-cell help 31,32,34 and B7/CD28 and CD40/CD40L costimulation pathways. [35][36][37][38] IgG1, IgG2a, and IgG2b anti-fVIII antibodies are produced, indicating a combined T H 1 and T H 2 response. 34,37-39 fVIII-specific interferon-␥ ϩ (IFN-␥ ϩ ), 38 interleukin-4 ϩ (IL-4), 34 and IL-10 ϩ T cells 34,38 have been identified in immunized mice, which also is consistent with a combined T H 1 and T H 2 response.…”

“…[35][36][37][38] IgG1, IgG2a, and IgG2b anti-fVIII antibodies are produced, indicating a combined T H 1 and T H 2 response. 34,37-39 fVIII-specific interferon-␥ ϩ (IFN-␥ ϩ ), 38 interleukin-4 ϩ (IL-4), 34 and IL-10 ϩ T cells 34,38 have been identified in immunized mice, which also is consistent with a combined T H 1 and T H 2 response. In human hemophilia A, anti-fVIII antibodies are a mixture of IgG1, IgG2, and IgG4 antibodies, [40][41][42][43] again indicating a combined T H 1 and T H 2 response.…”

Effect of B-cell depletion using anti-CD20 therapy on inhibitory antibody formation to human FVIII in hemophilia A mice

“…A similar result was also seen when the experiment was independently repeated with the more complete B-cell depletion using IgG2a anti-CD20 ( Figure 2D). It is known that inhibitor formation is T cell-dependent in both humans and mice, 6,[18][19][20] and it has been reported that B cells are required for optimal CD4 T-cell function. 21 Thus, the elimination of the majority of B cells may be responsible for the lack of boosting.…”

Indoleamine 2,3-dioxygenase attenuates inhibitor development in gene-therapy-treated hemophilia A mice

“…[14][15][16][17][18] The protein B7, which is expressed on the surface of antigen-presenting cells, interacts with CD28 expressed on T cells, thereby activating T-cell proliferation and promoting the humoral response to FVIII. 14,17 Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is a second high-affinity T-cell receptor for the B7 ligand, and is a negative regulatory of T-cell activation.…”