OC 61.1 - Factor VIII immunization results in expansion of long-lived plasma cells in mice

Background: Factor VIII (FVIII) inhibitors develop in ~30% of patients with severe hemophilia A (HA), limiting hemostatic options for breakthrough bleeding. While emicizumab provides prophylactic hemostasis, FVIII therapy is still desirable for surgery and bleeding control. FVIII immune tolerance induction (ITI) regimens are cumbersome and memory B cell (MBC) depletion has yielded mixed success. A better understanding of antigen-specific plasma cell (PC) subtypes involved in FVIII immune responses may help improve ITI regimens as long-lived PCs (LLPCs) are difficult to eradicate.

Aims: To delineate the contribution of antigen-experienced PCs and MBCs in FVIII immune responses.

Methods: Transgenic mice that express yellow fluorescent protein (YFP) when germinal center B cells are activated by antigen were immunized with FVIII weekly for 6 weeks. Spleen and bone marrow (BM) were harvested and analyzed by flow cytometry for YFP+ MBCs and PCs (CD138+TACI+). Splenic cells were characterized as plasmablasts (CD19+) or PCs (CD19-) and BM cells as short-lived PCs (SLPCs, B220+) or LLPCs (B220-). Neutralizing titer was measured by Bethesda assay from plasma. Data were compared by Mann-Whitney U and Pearson correlation.

Results: Inhibitors developed in 87.5% (16/18) of immunized mice who were compared to 5 inhibitor negative mice. In the spleen, inhibitor positive mice showed enrichment of antigen-experienced PCs (9.4-fold, p< 0.001) and MBCs (3.5-fold, p< 0.01). In the BM, inhibitor positive mice demonstrated enrichment of antigen-experienced LLPCs (2.2-fold, p< 0.05) and MBCs (4.4-fold, p< 0.01). BM LLPCs and splenic PCs correlated linearly with Bethesda titer (r=0.55 and 0.67, respectively, p< 0.05). YFP+ or YFP- plasmablasts or SLPCs were not increased in inhibitor positive mice in the spleen or BM, respectively.

Conclusion(s): These data demonstrate that FVIII-experienced PCs reside in both the spleen and BM. The expansion of BM LLPCs may hinder tolerance attempts with routine ITI and suggest need for immune modulation with higher inhibitor titers.