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Immune tolerance against infused FVIII in hemophilia A is mediated by PD-L1+ Tregs
Janine Becker-Gotot, … , Johannes Oldenburg, Christian Kurts
Janine Becker-Gotot, … , Johannes Oldenburg, Christian Kurts
Published September 15, 2022
Citation Information: J Clin Invest. 2022;132(22):e159925. https://doi.org/10.1172/JCI159925.
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Research Article Hematology Immunology

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

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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 4

High-dose FVIII treatment expands antigen-specific Tregs and increases their PD-L1 expression.

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High-dose FVIII treatment expands antigen-specific Tregs and increases t...
(A) Experimental setup for B–K: 2 IU/mouse of rhFVIII was intravenously injected into HemA mice (red, n = 4) or WT mice (black, n = 5) at weekly intervals for 3 weeks, and twice a week for the high-dose FVIII regimen (blue, n = 5). Experimental setup for high-dose FVIII application regimen, used to induce tolerance (short ITI), and the therapeutic regimen. (B) Representative dot plot of CD4+ T cells analyzed for FVIII specificity via tetramer staining of rhFVIII-treated mice (left panel) and FVIII-specific CD4+ T cell count (right panel). (C) Proportion of FVIII-specific Tregs (gated on CD4+tetramer+CD25+CD127– cells) in splenic suspensions of HemA mice after treatment with rhFVIII once or twice a week. (D) PD-L1 expression by FVIII-specific Tregs (CD4+tetramer+CD25+CD127– cells) in splenic cells ex vivo. (E) Number of FVIII-specific B cells measured by flow cytometry ex vivo after once- or twice-per-week treatment with rhFVIII. (F) Proportion of PD-1–expressing FVIII-specific B cells and (G) PD-1 MFI on PD-1+ FVIII-specific B cells in the spleen on day 22. (H) 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. (I–K) Number of FVIII-specific germinal center (GC; I), marginal zone (MZ; J) and follicular (FO; K) B cells in spleens of HemA mice after rhFVIII treatment. Germinal center B cells were identified as B220+FVIII+GL7+ cells, marginal zone B cells as B220+FVIII+CD93–CD21/35+IgM+IgD– cells, and follicular B cells as B220+FVIII+CD93–CD21/35–IgM–IgD+ cells. *P < 0.05; **P <0.01 by 1-way ANOVA with Bonferroni’s post hoc test. NS, not significant.

Copyright © 2023 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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