Immune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs
Janine Becker-Gotot, Mirjam Meissner, Vadim Kotov, Blanca Jurado-Mestre, Andrea Maione, Andreas Pannek, Thilo Albert, Chrystel Flores, Frank A. Schildberg, Paul A. Gleeson, Birgit M. Reipert, Johannes Oldenburg, Christian Kurts
Janine Becker-Gotot, Mirjam Meissner, Vadim Kotov, Blanca Jurado-Mestre, Andrea Maione, Andreas Pannek, Thilo Albert, Chrystel Flores, Frank A. Schildberg, Paul A. Gleeson, Birgit M. Reipert, Johannes Oldenburg, Christian Kurts
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Research Article Hematology Immunology

Immune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs

Abstract

A major complication of hemophilia A therapy is the development of alloantibodies (inhibitors) that neutralize intravenously administered coagulation factor VIII (FVIII). Immune tolerance induction therapy (ITI) by repetitive FVIII injection can eradicate inhibitors, and thereby reduce morbidity and treatment costs. However, ITI success is difficult to predict and the underlying immunological mechanisms are unknown. Here, we demonstrated that immune tolerance against FVIII under nonhemophilic conditions was maintained by programmed death (PD) ligand 1–expressing (PD-L1–expressing) regulatory T cells (Tregs) that ligated PD-1 on FVIII-specific B cells, causing them to undergo apoptosis. FVIII-deficient mice injected with FVIII lacked such Tregs and developed inhibitors. Using an ITI mouse model, we found that repetitive FVIII injection induced FVIII-specific PD-L1+ Tregs and reengaged removal of inhibitor-forming B cells. We also demonstrated the existence of FVIII-specific Tregs in humans and showed that such Tregs upregulated PD-L1 in patients with hemophilia after successful ITI. Simultaneously, FVIII-specific B cells upregulated PD-1 and became killable by Tregs. In summary, we showed that PD-1–mediated B cell tolerance against FVIII operated in healthy individuals and in patients with hemophilia A without inhibitors, and that ITI reengaged this mechanism. These findings may impact monitoring of ITI success and treatment of patients with hemophilia A.

Authors

Janine Becker-Gotot, Mirjam Meissner, Vadim Kotov, Blanca Jurado-Mestre, Andrea Maione, Andreas Pannek, Thilo Albert, Chrystel Flores, Frank A. Schildberg, Paul A. Gleeson, Birgit M. Reipert, Johannes Oldenburg, Christian Kurts

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Figure 1

PD-1 suppresses the formation of FVIII-inhibiting antibodies in vivo.

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PD-1 suppresses the formation of FVIII-inhibiting antibodies in vivo. (A...
(A) Experimental scheme for B–K. (B and C) ELISA quantification of FVIII-specific IgG antibody titers (B) and Bethesda units (BU) (C) in the serum of HemA (red triangles) and WT mice (white circles) 22 days after weekly injections of 2 IU recombinant human FVIII (rhFVIII). (D) Percentage of active FVIII in the plasma of HemA or WT mice. (E) Gating strategy for splenic FVIII-specific B cells of mice treated once a week with rhFVIII. (F) Quantification FVIII-specific B cell numbers in spleens of naive HemA (n = 5) and WT mice (n = 5) or after rhFVIII treatment by flow cytometry. (G) Left: Representative ELISpot analysis, after coating with rhFVIII and 4-hour incubation with splenocytes. Right: Number of antibody-forming cells (AFCs) per 107 splenocytes. (H) Representative histograms of PD-1 expression on FVIII+ B cells. (I) Proportion of PD-1–expressing FVIII+ B cells; the blue dashed line represents the PD-1 expression of naive B cells. (J) PD-1 expression by FVIII+ B cells. (K) Early apoptotic cells presented as percentage of annexin V+Hoechst– FVIII-specific B cells after in vitro restimulation with 0.25 μg rhFVIII overnight. (L) Experimental setting for M–P. (M and N) ELISA-based quantification of FVIII-specific IgG antibody titers in the serum (M) and numbers of FVIII-specific B cells in spleens (N) of WT mice weekly injected with 2 IU/mouse rhFVIII and treated with an anti–PD-1 antibody (aPD-1, purple) or not (black). (O) Early apoptotic cells presented as percentage of annexin V+ and Hoechst– FVIII-specific B cells after in vitro restimulation with 0.25 μg rhFVIII overnight. (P) Number of CD4+Foxp3+ Tregs in the spleen of treated mice. *P < 0.05; **P < 0.01; ***P < 0.001 by unpaired 2-tailed Student’s t test (B–P) or 1-way ANOVA with Bonferroni’s post hoc test (F). NS, not significant.