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Abstract
Anti-drug antibody formation poses tremendous obstacles for optimal treatment of hemophilia A (HA). In this study, we sought to utilize chimeric receptor-modified natural regulatory T cells (Tregs) to target FVIII-specific memory B cells, which are responsible for persistent anti-FVIII neutralizing antibodies (inhibitors) in HA patients. Thus, CD4+CD25hiCD304+ natural Tregs were FACS sorted from naïve C57BL/6 mice and retrovirally transduced to express a chimeric B-cell antibody receptor (BAR) containing the immunodominant A2 domain of FVIII. Plasmablast-depleted (CD138neg) splenocytes from FVIII immunized FVIII-knockout HA mice served as the source for FVIII-specific memory B cells, which were specifically stimulated in vitro with FVIII and enumerated in a B-cell ELISPOT assays. Adding A2-BAR Tregs (1 per 150 splenocytes), but not conventional T cells, to the CD138– splenocytes significantly suppressed the formation of anti-FVIII antibody secreting cells (ASC), compared to the non-relevant OVA-BAR Tregs control group. The observation that A2-BAR Tregs can suppress the response to FVIII suggests that bystander suppression can occur in the local milieu in this system. Transwell experiments confirmed that the suppression was contact-dependent. Moreover, even in the presence of antibodies to FVIII (so-called inhibitors), similarly prepared CD4+CD25hiCD127low A2-BAR human natural Tregs completely suppressed polyclonal anti-FVIII ASC formation. In conclusion, we demonstrated in vitro that FVIII domain-expressing BAR Tregs could efficiently target and suppress FVIII-specific memory B cells.
Highlights
Hemophilia A (HA) is a hereditary bleeding disorder, caused by mutations in the F8 gene encoding pro-coagulant factor VIII (FVIII) [1]
Analogous to chimeric antigen receptor (CAR) technology that has been successfully used in cancer immunotherapy [10], we developed a chimeric receptor comprising a protein domain antigen linked to transmembrane and intracellular signaling domains CD28-CD3ζ
Transduction efficiencies for A2-B-cell antibody receptor (BAR) and OVA-BAR Tregs were estimated to be 30–70% based on the GFP reporter gene expression
Summary
Hemophilia A (HA) is a hereditary bleeding disorder, caused by mutations in the F8 gene encoding pro-coagulant factor VIII (FVIII) [1]. Despite great improvement in the management of the disease, one remaining major issue is the formation of anti-FVIII neutralizing antibodies (inhibitors), which occur in up to 30% of severe HA and about 5% of moderate and mild HA patients [2]. The only clinically proven strategy to eradicate the inhibitors is called immune tolerance induction therapy (ITI). First described 40 years ago [3], ITI features repeated, high dose FVIII infusions. Targeting FVIII-Specific Memory B Cells until the inhibitor becomes undetectable. Clinical evidence suggests that FVIII-specific memory B cells were deleted in HA patients that had successfully completed ITI [4]. FVIII-specific memory B cells were suppressed in the presence of high dose FVIII in vitro and in vivo using murine HA models [5,6,7]. Restoring tolerance to FVIII is an unmet need [2]
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