Antigen‐specific in vitro expansion of factor VIII‐specific regulatory T cells induces tolerance in hemophilia A mice

Abstract

BackgroundFollowing protein replacement therapy, one‐third of severe hemophilia A patients develop antibodies to factor VIII (FVIII), which also hinders the efficacy of gene therapy. Regulatory T cells (Tregs) have a naturally suppressive function that potentially reduces the immune response to FVIII therapy. Furthermore, antigen‐specific Tregs are functionally much more potent than polyclonal cells. Adoptive transfer of antigen‐specific Tregs can effectively suppress anti‐FVIII antibody responses. ObjectiveDevelop a clinically feasible protocol to enrich and expand Tregs specific to FVIII for suppressing anti‐FVIII immune responses. MethodsRegulatory T cells are isolated from FVIII‐sensitized mice, sorted on CD25high markers, and expanded specifically with FVIII, antigen‐presenting cells, and interleukin 2 (IL 2). Subsequently, Tregs are further cultured with anti‐CD3/anti‐CD28 beads, anti‐Crry antibodies, and IL 2 to achieve 10‐fold to 20‐fold expansion. Expanded Tregs are characterized and tested for their suppressive activity in vitro and in vivo. ResultsIn vitro FVIII‐specific suppressive assays indicate that FVIII specifically expanded Tregs are more suppressive than non‐specifically expanded and naive Tregs. Adoptive transfer of expanded Tregs into HemA mice showed that FVIII‐specifically expanded Tregs are significantly more potent in suppressing anti‐FVIII immune responses in FVIII plasmid‐treated HemA mice. Moreover, the FVIII‐specific immune tolerance is maintained after a secondary challenge with FVIII plasmid. ConclusionsOur results demonstrate that the FVIII‐specific sensitization and expansion protocol yields more potent Tregs to suppress anti‐FVIII antibody responses and induce long‐term tolerance to FVIII, increasing the potential for adoptive Treg cell therapy to modulate anti‐FVIII immune responses.