In Vivo Expansion of Treg Cells Prevents Anti-FVIII Antibody Production Following FVIII Protein Replacement Therapy in Hemophilia A Mice

Abstract 23

Inhibitory antibodies formation is a major complication following protein replacement therapy for hemophilia A. Interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb, JES6-1) can induce selective expansion of regulatory T (Treg) cells. In order to address the question whether in vivo expansion of Treg cells can modulate FVIII-specific immune responses following FVIII protein replacement therapy, we treated hemophilia A mice with IL-2/IL-2 mAb complexes (6 μg/mouse) three times per week for 4 weeks, and concurrently with BDD-FVIII protein (0.3U/mouse) three times per week for 4 weeks, followed by BDD-FVIII (1U/mouse) once per week consecutively for another 14 weeks. Compared to control mice (n=3) which produced high-titer anti-FVIII antibodies after treatment with BDD-FVIII protein only for 4 weeks, mice treated with IL-2/IL-2 mAb complexes + BDD-FVIII protein produced no inhibitory antibody titers against FVIII over time. PC61 is an anti-murine CD25 antibody which depletes CD4⁺ CD25⁺ Treg cells. Mice (n=4) treated with IL-2 complexes + BDD-FVIII protein + PC61 for 4 weeks produced high-titer inhibitory antibodies. These data indicate that immunomodulation by 4-week treatment of IL-2/IL-2 mAb complexes successfully suppressed anti-FVIII immune responses by in vivo expanded Treg cells, and induced long-term tolerance to FVIII. A marked 5–7-fold increase in percentages and total numbers of CD4⁺ Foxp3⁺ Treg cells and Foxp3⁺ Helios⁺ T cells were observed in the peripheral blood, spleen and lymph nodes on the peak days during IL-2/IL-2 mAb complexes treatment indicating that most of the expanded Treg cells are thymically derived natural Treg (nTreg) cells instead of peripherally induced adaptive Treg (aTreg) cells. Treg cells maintained at high levels for 4 weeks, and these levels gradually returned to normal within the next 7–14 days. Also, the expanded Treg cells showed a considerable increase in the expression of molecules crucial to their suppressive function, including CD25, glucocorticoid induced TNFR (GITR) and cytotoxic T lymphocyte antigen 4 (CTLA-4) relative to Treg cells from the control mice. The expansion of CD4⁺ Foxp3⁺ populations and their suppressive functions were examined and supported by suppressive, proliferative and cytokines assays using splenic cells isolated from IL-2/IL-2 mAb + FVIII treated mice. In a separate experiment, mice treated with IL-2/IL-2 mAb complexes + full-length FVIII protein for consecutive 4 weeks generated neither inhibitory nor non-inhibitory antibodies against FVIII up to day 60 post treatment. However, these mice produced anti-FVIII antibodies following a second challenge of 4 week infusions of full-length FVIII protein at a lower titer than mouse groups treated with IgG2a isotype control antibody + FVIII and FVIII only. Our results demonstrate the important role of Treg cells in suppressing anti-FVIII immune responses. Treatment of IL-2/IL-2 mAb complexes represents a highly promising strategy for prevention of anti-FVIII antibody formation following either the BDD-FVIII or full-length FVIII protein replacement therapy in hemophilia A mice.