The T follicular helper/T follicular helper regulatory pathway in FVIII immune responses in mice.

Developing anti-factor VIII (FVIII) inhibitory antibodies (inhibitors) are a significant complication of FVIII protein replacement therapy in hemophilia A. Our previous study demonstrated that follicular helper T (TFH) cells play a critical role in FVIII inhibitor development. Follicular regulatory T (TFR) cells are a subset of forkhead box protein P3 positive (Foxp3+) T cells identified in the germinal center that can modulate TFH cell activation of B cells and antibody development. Here, we report that FVIII immunization significantly increases the TFR cells in the spleens of FVIII inhibitor-producing FVIIInull mice compared with saline-treated controls and non-inhibitor-producing animals. The TFH/TFR ratio significantly increased in FVIII inhibitor-producing mice. The emergence of TFR cells correlated with titers of FVIII inhibitors in FVIII-immunized mice. Using TFR-deficient Foxp3Cre+Bcl6fl/fl (Bcl6FC) mice, we found that the loss of TFR cells led to significantly decreased FVIII inhibitors compared with wild-type (WT) mice on FVIII immunization (24 ± 16 and 131 ± 114 Bethesda unit (BU)/mL, respectively) but not total anti-FVIII IgG levels and that TFR cells regulated IgG subclass switching and FVIII-specific B-cell responses. Interestingly, on FVIII immunization, mice with phosphatase and tensin (Pten) deficiency in Foxp3+ cells (Foxp3Cre+Ptenfl/fl), a model with augmented TFR cells, developed markedly lower FVIII inhibitor titers (8.1 ± 8.6 BU/mL) than WT controls. When CD4Cre+Bcl6fl/fl mice, a TFH- and TFR-deficient model, were immunized with FVIII, none of the animals developed FVIII inhibitors. In conclusion, FVIII immunization induces TFR cell activation and expansion. TFR cells have a dual function in regulating the development of FVIII inhibitors, and the TFH/TFR pathway is pivotal in FVIII inhibitor development in mice.