Fig 2: PD-1 stimulation induces apoptosis in human FVIII- and FIX-specific B cells.(A) MFI of PD-1 expression by FVIII- and FIX-specific and nonspecific B cells from 9 healthy donors detected by flow cytometry. *P < 0.05 by 1-way ANOVA with Dunn’s post hoc test. NS, not significant. (B) Example of PD-1 expression by FVIII-specific B cells from 1 representative donor. (C–F) Percentage of apoptotic FVIII-specific (C), FIX-specific (D), non–FVIII-specific (E), and GFP-specific (F) B cells without and after incubation with a PD-L1 chimeric protein that specifically stimulates PD-1 in vitro. (G) The presence of FVIII-specific CD4+ T cells was analyzed in blood samples of an HLA-matched patient sample (n = 3) who had undergone successful ITI. (H) The percentage of CD25+CD127– (Tregs) of HLA-matched tetramer+CD4+ cells is depicted as a representative example. (I) The geometric MFI of PD-L1 was analyzed on FVIII-specific CD4+ HLA-matched Tregs versus nonspecific Tregs from the same patient blood sample. *P < 0.05 by 1-way ANOVA with Dunn’s post hoc test (A) or paired, 2-tailed Student’s t test (C–F and I). NS, not significant.

Fig 3: High-dose FVIII treatment expands antigen-specific Tregs and increases their PD-L1 expression.(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.

Fig 4: PD-L1+ Tregs are necessary and sufficient to suppress FVIII-specific B cells in vivo.(A) Experimental scheme. WT (black, n = 5) and Foxp3-LuciDTR (orange, n = 5) mice were intravenously injected with 2 IU/mouse of rhFVIII at weekly intervals. Foxp3+ Tregs were depleted by injecting 15 ng/g mouse DTX intraperitoneally on day –1 and 0. (B) ELISA-based quantification of the FVIII-specific IgG antibody titer in the serum of WT and Foxp3-LuciDTR mice. (C) Number of FVIII-specific B cells in spleens of WT and Foxp3-LuciDTR mice after treatment with rhFVIII by flow cytometry. (D) Mean fluorescence intensity (MFI) of PD-1 on FVIII-specific B cells of splenocyte suspensions. (E) Early apoptotic cells are presented as percentage of annexin V+ and Hoechst– FVIII-specific B cells after in vitro restimulation with 0.25 µg rhFVIII overnight. (F) Experimental setup. Tregs (1 × 106) isolated either from WT or Pd-l1–/– mice were injected into HemA mice. Starting on the next day, HemA (red, n = 9) and HemA mice that received Tregs from WT (blue, n = 9) or Pd-l1–/– (purple, n = 9) mice were intravenously injected with 2 IU/mouse of rhFVIII at weekly intervals. (G) FVIII-specific IgG antibody titers measured by ELISA in the serum. (H) The percentage of residual active FVIII in the plasma of rhFVIII-treated mice. (I) Number of FVIII-specific B cells in spleens of HemA mice with or without Treg transfer after rhFVIII treatment. (J) Proportion of splenic PD-1+ FVIII-specific B cells and (K) PD-1 MFI on FVIII-specific B cells. (L) Early apoptotic cells presented as annexin V+ and Hoechst– FVIII-specific B cells after in vitro restimulation with 0.25 µg rhFVIII overnight. *P < 0.05; **P < 0.01 by unpaired 2-tailed Student’s t test (B–E) or 1-way ANOVA with Bonferroni’s post hoc test (G–L).