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Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice.

ABSTRACT: Factor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ?30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cell-mediated transgenesis (T2F8Tg). Both endothelial expression approaches were associated with a strikingly robust immune response. Following in situ T2F8LV-mediated EC transduction, all FVIIInull mice developed inhibitors but had no detectable plasma FVIII. In the transgenic approach, the T2F8Tg mice had normalized plasma FVIII levels, but showed strong sensitivity to developing an FVIII immune response upon FVIII immunization. A single injection of FVIII with incomplete Freund adjuvant led to high titers of inhibitors and reduction of plasma FVIII to undetectable levels. Because ECs are putative major histocompatibility complex class II (MHCII)-expressing nonhematopoietic, "semiprofessional" antigen-presenting cells (APCs), we asked whether they might directly influence the FVIII immune responses. Imaging and flow cytometric studies confirmed that both murine and human ECs express MHCII and efficiently bind and take up FVIII protein in vitro. Moreover, microvascular ECs preconditioned ex vivo with inflammatory cytokines could functionally present exogenously taken-up FVIII to previously primed CD4+/CXCR5+ T follicular helper (Tfh) cells to drive FVIII-specific proliferation. Our results show an unanticipated immunogenicity of EC-expressed FVIII and suggest a context-dependent role for ECs in the regulation of inhibitors as auxiliary APCs for Tfh cells.

SUBMITTER: Shi Q

PROVIDER: S-EPMC7252558 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice.

Shi Qizhen Q Carman Christopher V CV Chen Yingyu Y Sage Peter T PT Xue Feng F Liang Xin M XM Gilbert Gary E GE

Blood advances 20200501 10

Factor VIII (FVIII) replacement therapy for hemophilia A is complicated by development of inhibitory antibodies (inhibitors) in ∼30% of patients. Because endothelial cells (ECs) are the primary physiologic expression site, we probed the therapeutic potential of genetically restoring FVIII expression selectively in ECs in hemophilia A mice (FVIIInull). Expression of FVIII was driven by the Tie2 promoter in the context of lentivirus (LV)-mediated in situ transduction (T2F8LV) or embryonic stem cel ...[more]

PMID: 32453842

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Project description:Factor VIII (FVIII)-neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 contains an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells from HA subjects. The aim of this study was to identify amino acid substitutions to deimmunize this epitope while retaining procoagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The shortest DRB1*01:01-binding peptide was FVIII2194-2205, and residues important for affinity were identified as F2196, M2199, A2201, and S2204. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation of clones specific for the WT sequence. T-cell clones that were stimulated by recombinant WT-FVIII-C2 (rWT-FVIII-C2) protein did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic peptide includes a naturally processed T-cell epitope. Additional amino acid substitutions at F2196 and M2199 were evaluated by peptide-MHC class II (MHCII)-binding assays, T-cell proliferation assays, epitope prediction algorithms, and sequence homologies. Six B-domain-deleted (BDD)-FVIII proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W, or M2199R were produced. Proliferation of T-cell clones and polyclonal lines in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A was reduced compared with responses to WT-BDD-FVIII. The BDD-FVIII-F2196K sequence modification appears to be the most promising sequence variant tested here, due to its effectiveness at eliminating DRB1*01:01-restricted immunogenicity, low potential immunogenicity in the context of other MHCII alleles, expression level comparable to WT-BDD-FVIII, and retained procoagulant activity. These results provide proof of principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.

Dataset Information

Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice.

Publications

Unexpected enhancement of FVIII immunogenicity by endothelial expression in lentivirus-transduced and transgenic mice.

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