Induction of Activated T Follicular Helper Cells Is Critical for Anti-FVIII Inhibitor Development in Hemophilia a Mice

The development of neutralizing anti-FVIII antibodies (inhibitors) is one major complication of FVIII protein replacement therapy in hemophilia A patients. While multiple lines of evidence indicate that the immune response against FVIII is CD4 T cell-dependent, the role of T follicular helper (TFH) cells, a newly identified subset of CD4 T cells, in FVIII inhibitor development is unknown. TFH cells are a critical player in germinal center formation, which provide cognate help to B cells and are fundamentally required for the generation of T cell-dependent B cell responses. Here we explored the activation and induction of TFH cells and their roles in anti-FVIII inhibitor production in FVIII-immunized mice.

We first confirmed that inhibitor development depends on CD4 T cells as depletion of CD4 T cells during FVIII immunization in FVIII^null mice prevents inhibitor production. We then characterized the features of activated TFH cells in the spleen of immunized FVIII^null mice by flow cytometry analysis. Activated TFH cells were defined as CD4⁺Foxp3^-CD44⁺CD62L^-CXCR5⁺PD-1⁺. These cells express high levels of transcription factors BCL6, ICOS, and CD40L. We showed for the first time that intravenous FVIII immunization induced activation and accumulation and/or expansion of TFH cells in the spleen of FVIII^null mice. The percentage of CXCR5^hiPD-1^hi TFH cells within the effector CD4 population and the absolute numbers of activated TFH cells per spleen significantly increased in inhibitor-producing mice compared to saline-treated controls and non-inhibitor-producing mice (19.6 ± 1.6 %, 8.1 ± 1.2%, and 7.6 ± 1.4%, respectively; 14.4 ± 2.0×10⁴, 3.55 ± 0.8×10⁴, and 3.5 ± 0.9×10⁴, respectively). There was no TFH cell expansion observed in other secondary lymphoid organs (mesenteric and inguinal lymph nodes), suggesting that the spleen is the primary site for TFH activation when FVIII is administered intravenously.

Our results showed that FVIII inhibitor-producing mice had increased GL-7⁺germinal center TFH cells together with increased germinal center formation in response to FVIII immunization and the emergence of TFH cells correlated with titers of anti-FVIII inhibitors. This indicates that a functional relationship exists between TFH cell activation and FVIII inhibitor production after FVIII immunization. To determine the extent to which the anti-FVIII inhibitor development is related to CXCR5⁺ TFH cells, we used a mixed BM chimera model consisting of a CD4 T cell-specific CXCR5 deficiency (CD4-CXCR5^-/-). Compared with CD4-CXCR5^+/+ control mice, there was a significant decrease in the inhibitor titer in CD4-CXCR5^-/- mice after FVIII immunization (111.7 ± 46.2 vs 4.43 ± 3.53 BU/ml, respectively). Our results showed that CXCR5^+/+ TFH cell-specific deletion impaired anti-FVIII inhibitor production, confirming the essential role of CXCR5^+/+ TFH cells for the generation of FVIII-neutralizing antibodies.

To examine specificity of TFH cell responses after FVIII immunization, we used an ex vivo T-cell proliferation assay. In vitro FVIII restimulation induced the antigen-specific proliferation of splenic CD4 T cells from FVIII-primed FVIII^null mice and those proliferating cells expressed the TFH hallmark transcription factor BCL6. These results demonstrated that antigen-specific TFH cells were induced after FVIII immunization and those cells could specifically respond to FVIII upon restimulation. To evaluate the FVIII-specific memory TFH cell responses in FVIII^null mice, matched pairs of FVIII-primed mice with similar inhibitor titers were rechallenged with FVIII antigen in vivo 2 months after the last immunization. The absolute numbers of TFH cells per spleen in FVIII rechallenged animals were significantly higher than those obtained from saline-injected primed mice (36.7 ± 11 ×10⁴ vs 14.8 ± 3.8 ×10⁴, respectively). These data suggest that memory FVIII-specific TFH cells are persistent which can induce efficient recall TFH responses after FVIII rechallenge.

Together, our results demonstrate that the induction of activated TFH cells in FVIII^null mice is critical for FVIII inhibitor development, suggesting that inhibition of FVIII-specific TFH cell activation might be a promising strategy to prevent anti-FVIII inhibitor formation in hemophilia A patients.