OC 37.2 - Follicular regulatory T cells promote anti-FVIII inhibitor development in hemophilia A mice

Background: The development of anti-FVIII inhibitory antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. Our previous study demonstrated that the induction of activated follicular helper T (Tfh) cells in FVIIInull mice is critical for FVIII inhibitor development. Follicular regulatory T (Tfr) cells are a specialized subset of T cells identified in the germinal center (GC) that can modulate Tfh cell activation of B cells and antibody development.

Aims: To investigate the role of Tfr cells in FVIII immune responses.

Methods: Tfr cells in the spleen of immunized FVIIInull mice were characterized by flow cytometry analysis. Tfr deficient Foxp3-Cre Bcl6fl/fl (BCL6FC) mice were used to determine the role of Tfr cells in anti-FVIII inhibitor generation.

Results: FVIII immunization increased the activated Tfr cells in the spleen of mice that produced anti-FVIII inhibitors. The percentage of activated Tfr cells significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (0.88±0.2%, 0.33±0.16%, and 0.36±0.2%, respectively). The Tfh/Tfr ratio also significantly increased in inhibitor-producing mice, suggesting a preferential expansion of Tfh cells relative to Tfr cells in inhibitor-producing mice. Loss of Tfr cells led to significantly decreased neutralizing anti-FVIII inhibitor levels, with titers in BCL6FC mice markedly lower than in wild-type mice upon rhF8 immunization (22±12 and 145±82 BU/ml, respectively). However, the total anti-FVIII IgG titers were similar between the BCL6FC and wild-type groups, suggesting that Tfr cells help generate high affinity anti-FVIII antibodies. FVIII immunization could still efficiently induce Tfh cell activation in BCL6FC mice. However, both GC Tfh cells and GC B cells were reduced in FVIII immunized BCL6FC mice, suggesting that Tfr cells promote FVIII-specific GC responses.

Conclusion(s): we found that FVIII immunization induces Tfr cell activation/expansion and that Tfr cells have an unexpected helper role in promoting anti-FVIII inhibitor generation.