520. FVIII-Specific In Vitro Expansion of Regulatory T Cells to Mediate Tolerance in Hemophilia A Mice Following Hydrodynamic FVIII Plasmid Challenge
Authors: Bryn M. Smith, Meghan J. Lyle, Carol H. Miao
Journal: Molecular Therapy (2015)

Hemophilia A (HemA), a bleeding disorder, is characterized by a deficiency of clotting Factor VIII (FVIII). The current standard of care is protein replacement therapy, but complications arise in one-third of patients from the development of inhibitory antibodies (inhibitors) to FVIII. The FVIII immune response also hinders the long-term potential for gene therapy treatments in patients with or at high risk of developing inhibitors. In our previous work, we have shown that the adoptive transfer of two-million FVIII-sensitized, naturally suppressive regulatory T (Treg) cells protect recipient HemA mice from developing inhibitors to FVIII after hydrodynamic plasmid injection, thereby allowing a therapeutic level of FVIII to be reached. In order to develop a clinically feasible protocol of adoptive Treg cell transfer therapy, we explored in vitro expansion of cells isolated from FVIII-sensitized and naïve mice, followed by adoptive transfer of these cells into HemA mice later challenged with FVIII plasmid. In vitro FVIII-specific suppressive assays of the expanded cells indicated that FVIII-sensitized Treg cells are 100% suppressive at a ratio of 1:2 responder T cells, compared to 65% suppression with naïve Treg cells. In vivo adoptive transfer results showed expanded Treg cells from both FVIII sensitized and naïve mice are able to regulate anti-FVIII immune responses. To increase the potential in vivo benefit of FVIII-specific Treg cells, we have pursued development of a FVIII-specific expansion protocol by building upon our previous success of non-specifically expanding functionally suppressive Treg cells. After AutoMACS isolation and FACSAria sorting on CD25high marking, Treg cells from FVIII-sensitized mice are incubated for 3 days with APCs, FVIII, and high-dose IL-2 to stimulate preferential survival of FVIII-specific Treg cells. Following FVIII-specific expansion, Treg cells are further expanded with anti-CD3/anti-CD28 beads, anti-Crry, and high-dose IL-2 for 7-8 days to obtain 10 to 20-fold expansion of activated Treg cells. The resulting Treg cells are 80% CD25+Foxp3+ and have 100% FVIII-specific suppressive activity at a ratio of 1:16 responder T cells, compared to <20% activity in non-specifically expanded FVIII-sensitized Treg cells. Adoptive transfer experiments into HemA mice are performed to test in vivo suppressive function of Treg cells expanded with the best FVIII-specific protocol. The prospect of generating a successful method of expanding Treg cells specific to FVIII would increase the translational potential of Treg cell transfer. Fewer cells would be required to achieve tolerance after adoptive transfer, and antigen-specificity could potentially reduce the risk of off-target suppression without compromising long-term tolerance. Our adoptive transfer experiments with expanded cells have yielded encouraging results, proving that Treg cells can be expanded in vitro and effectively suppress anti-FVIII inhibitor response after plasmid challenge. We expect the FVIII-specific expansion protocol to yield even more potent Treg cells to induce long-term tolerance to FVIII in HemA mice.