recombinant human full-length fviii (rhffviii, kogenate fs  (Bayer AG)

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells

doi: 10.1016/j.jtha.2022.10.006

Figure Lengend Snippet: Platelet-targeted FVIII (2bF8) transgenic whole bone marrow transplantation does not mimic platelet-FVIII expression loss on busulfan (Bu) conditioning in the inhibitor model. To test if whole bone marrow transplantation mimics platelet-FVIII expression loss on Bu conditioning in hemophilia A mice with pre-existing immunity, whole bone marrow cells from low-expressor (~0.76 mU/108 platelets) 2bF8-ESTg+/− transgenic mice were transplanted into FVIIInull mice preimmunized with recombinant human B-domain deleted FVIII (rhFVIII), and preconditioned with either Bu alone or Bu+ATG. Blood samples were collected for plasma or platelet isolation. Platelet-FVIII expression was determined by a chromogenic assay on platelet lysates. Anti-FVIII inhibitor titers were determined by a modified Bethesda assay. (A) A schematic diagram of the experimental design. (B) Platelet-FVIII expression in recipients under Bu conditioning alone. (C) Platelet-FVIII expression in recipients under Bu+ATG conditioning. (D) Comparison of the platelet-FVIII expression levels between the Bu group and the Bu+ATG group at 6 months after BMT. (E) Anti-FVIII inhibitor titers in the Bu group. (F) Anti-FVIII inhibitor titers in the BU+ATG group. (G) The correlation between platelet-FVIII expression and inhibitor titers at 4 weeks post-BMT. These results demonstrate that 2bF8 transgenic whole bone marrow engraftment was viable and platelet-FVIII expression was sustained on whole bone marrow transplantation in FVIII-sensitized hemophilia A mice under Bu preconditioning alone. ATG, antithymocyte globulin; BMT, bone marrow transplantation.

Article Snippet: Recombinant human full-length FVIII (rhfFVIII, Kogenate FS) was purchased from Bayer Pharma.

Techniques: Transgenic Assay, Transplantation Assay, Expressing, Recombinant, Isolation, Chromogenic Assay, Modification, Comparison

Journal: Molecular Therapy. Nucleic Acids

Article Title: Platelet gene therapy induces robust immune tolerance even in a primed model via peripheral clonal deletion of antigen-specific T cells

doi: 10.1016/j.omtn.2020.12.026

Figure Lengend Snippet: The development of anti-OVA immune tolerance in 2bOVA-transduced OVA-primed mice (the WT/WT model) To investigate how the primed immune system responds after platelet-targeted gene transfer, we monitored anti-OVA total IgG titers in treated animals by ELISA. (A) Anti-OVA titers in recipients before and 5 weeks after HSCT. (B) Anti-OVA titers during the study course. The titer at the pre-HSCT time point was normalized to 1. (C) Changes in anti-OVA titers after OVA rechallenge. To investigate whether immune tolerance was induced in 2bOVA-transduced recipients, recipients were immunized with OVA (20 μg/mouse by i.p. injection) twice. One week after each rechallenge, plasmas were collected for ELISA to determine anti-OVA total IgG titers. The titer at the pre-HSCT time point was normalized to 100%. (D) Titers of anti-FVIII antibodies. To investigate whether the immune tolerance developed in 2bOVA-transduced recipients is OVA-specific, mice were further challenged with the unrelated antigen recombinant human FVIII (rhF8) at 200 U/kg/week intravenously (i.v.) (four times), and anti-FVIII inhibitory antibodies (inhibitors) were determined by a Bethesda assay. (E and F) Skin graft transplantation. To further confirm the antigen-specific immune tolerization developed in 2bOVA-transduced OVA-primed recipients, tail skin grafts from Act-mOVA transgenic mice were transplanted onto recipients. Complete graft rejection was recorded. 2bGFP-transduced and untransduced transplanted controls were performed in parallel. Representative skin graft on the 2bOVA-transduced recipient (8 months after skin transplantation) is shown in (E). Skin graft survival rate is shown in (F). These data demonstrate that antigen-specific immune tolerance is achieved after platelet-specific gene therapy in a primed model. ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001. n.s., not significant (for difference between the two groups).

Article Snippet: Recombinant human full-length FVIII (rhfF8; Kogenate FS) was from Bayer Healthcare (Whippany, NJ, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Recombinant, Transplantation Assay, Transgenic Assay

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: FVIII immunization induces activated TFH cells in spleen. FVIIInull mice were given 5 rounds of weekly IV FVIII immunization injection. Five days after the last immunization, the mice were euthanized, and splenocytes and plasma were collected for analysis. Age-matched naive FVIIInull mice were used as controls. Anti-FVIII inhibitor titers were determined by chromogenic-based Bethesda assay. Splenocytes were stained for CD3, CD4, CD19, CD44, CD62L, Foxp3, CXCR5, PD-1, and Ki-67 and analyzed by flow cytometry. (A) Gating strategy for the identification of activated TFH cells. CD4+ Th cells were identified by excluding Foxp3+ CD4+ cells inside the singlet live CD19−CD3+CD4+ cell population. Effector CD4+ Th cells were identified by CD44 and CD62L expression (CD44+CD62L−). Effector CD4+ cells were analyzed for CXCR5 and PD-1 coexpression to identify TFH cells. (B) shows representative histograms and (C) shows quantification of BCL6, ICOS, and CD40L expression on naive, CXCR−PD-1− effector (non-TFH effector cells), and TFH cells (n = 6 mice per group). (D) Ki-67 cell cycling staining on CXCR5−PD-1− effector and TFH cells (n = 6 mice per group). **P < .01; ***P < .001.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Injection, Clinical Proteomics, Staining, Flow Cytometry, Expressing

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: FVIII immunization increases the frequency of activated TFH cells in mice that produce inhibitors. FVIIInull mice were given 5 rounds of weekly IV FVIII immunizations. Five days after the last immunization, inhibitor titers were measured by Bethesda assay. Representative animals with inhibitors and no inhibitors were euthanized for TFH analysis. Age-matched naive FVIIInull mice were used as controls. Splenocytes were stained for CD3, CD4, CD19, CD44, CD62L, Foxp3, CXCR5, PD-1, and Ki-67, and analyzed by flow cytometry. (A) Percentage of effector helper cells (CD44+CD62L−) among Foxp3−CD4 T cells in saline-injected control mice, non-inhibitor–producing mice, and inhibitor-producing mice. (B) Percentage of CXCR5+PD-1+ TFH cells among effector CD4 helper cells. (C) Percentage of activated TFH cells among total CD4 T cells. (D) Total number of activated TFH cells per spleen. (E) Percentage of Ki-67+ in activated TFH cells. (F) Representative samples and summary data (n = 4 mice per group) of apoptotic cells in naive, non-TFH effector CD4 T cells and TFH cells from FVIII-immunized mice. *P < .05; **P < .01; ***P < .001.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Staining, Flow Cytometry, Saline, Injection, Control

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: FVII inhibitor-producing mice have increased GC formation and increased GC TFH cells in response to F8 immunization. FVIIInull mice were given 5 weekly IV FVIII injections. Five days after the last immunization, inhibitor titers were measured by Bethesda assay. Representative animals with inhibitors and no inhibitors were euthanized for splenocyte analysis. Splenocytes were stained for CD3, CD4, CD19, CD44, CD62L, B220, Foxp3, CXCR5, PD-1, Fas, GL-7, ICOS, and CD40L and analyzed by flow cytometry. (A) Percentage of B220+ B cells bearing a GC phenotype (Fas+ GL-7+) in saline-injected control mice, non–inhibitor-producing mice and inhibitor-producing mice. (B) Percentage and total number of activated TFH cells expressing GL-7 in in spleen. (C) Representative histograms and quantification (n = 6 per group) of expression of ICOS and CD40L on naive, CXCR5−PD-1− effector, GL-7− TFH, and GL-7+ TFH cells. *P < .05. MFI, mean fluorescence intensity.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Staining, Flow Cytometry, Saline, Injection, Control, Expressing, Fluorescence

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: Emergence of PD-1+CXCR5+TFH cells correlates with the development of FVIII inhibitors. FVIII and VWF double-knockout mice were given weekly IV FVIII immunizations. One week after the last immunization, the mice were euthanized. Splenocytes and plasma were collected for analysis. Age-matched saline-treated mice were used as a control. Plasma inhibitor titers were determined by Bethesda assay. Splenocytes were analyzed by flow cytometry. (A) Percentage of CXCR5+PD-1+ TFH cells among effector CD4-helper cells. Anti-FVIII inhibitor titers greater than 250 BU/mL were defined as the high-titer inhibitor group. (B) Percentage of activated TFH cells among whole CD4 T cells. Anti-FVIII inhibitor titers greater than 250 BU/mL were defined as the high-titer inhibitor group. (C) Dot-plot shows the correlation between the titer of FVIII inhibitor and the percentage of activated TFH cells among CD4 T cells. Statistical significance was analyzed by linear regression. The r and P values are indicated in the figure. *P < .05; **P < .01; ***P < .001.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Double Knockout, Clinical Proteomics, Saline, Control, Flow Cytometry

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: FVIII-specific proliferating CD4 T cells have a TFH phenotype. Splenocytes from rhF8-immunized FVIIInull mice were labeled with CellTrace Violet and stimulated with rhF8 for 96 hours. Nonstimulated or unrelated protein rhF9-stimulated splenocytes were used as controls in parallel. Cells were stained for mouse CD3, CD4, CXCR5, BCL6, GATA3, and Tbet. Proliferating daughter cells were analyzed by flow cytometry. (A) CD4+ T-cell proliferation is depicted. Representative plots from 4 experiments are shown. (B) CXCR5+ and CXCR5− T-cell proliferation is depicted. Representative plots from 4 experiments are shown. (C) BCL6 and CXCR5 expression levels on proliferating CD4 T cells (blue line) induced by ex vivo FVIII stimulation were compared with nonproliferating CD4 T cells (red line). (D) Transcription factor BCL6, GATA3, and Tbet expression on FVIII-induced proliferating CD4 T cells (top row). Corresponding isotype control antibody staining was used to define gating (bottom row). PI, proliferation index.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Labeling, Staining, Flow Cytometry, Expressing, Ex Vivo, Control

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: CD4 T-cell–specific CXCR5 deficiency impairs anti-FVIII inhibitor induction. (A) Schematic diagram for generating mixed BM chimeras. Mixed BM chimera in irradiated CD4−/− mice reconstituted with 80% BM from CD4−/− mice plus 20% from WT mice or 80% from CD4−/− mice plus 20% from CXCR5−/− mice. (B) Flow cytometry analysis of CD4 and CD8 T-cell reconstitution in blood at 10 weeks after BM transplantation. Mixed-BM chimeric mice were immunized with rhF8, as shown in the experimental schematic in panel C. (D) One week after the fourth injection with rhF8, plasma samples were collected for a Bethesda assay to determine the FVIII inhibitor titers. The data shown are for 6 mice per group. **P < .01.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Irradiation, Flow Cytometry, Transplantation Assay, Injection, Clinical Proteomics

Journal: Blood Advances

Article Title: Induction of activated T follicular helper cells is critical for anti-FVIII inhibitor development in hemophilia A mice

doi: 10.1182/bloodadvances.2019000650

Figure Lengend Snippet: Rechallenge with FVIII antigen induces recall responses of TFH cells. Eight to 10 weeks after the last round of IV FVIII immunization, pairs of mice bearing similar inhibitor titers were rechallenged with 50 U/kg rhF8 or saline. Five days after boost, the mice were euthanized. Splenocytes were stained for CD3, CD4, CD19, CD44, CD62L, Foxp3, CXCR5, and PD-1 and analyzed by flow cytometry. (A) FVIII inhibitor titers. (B) Percentage of CXCR5+PD-1+ TFH cells among effector CD4 helper cells. (C) Percentage of activated TFH cells among total CD4 T cells. (D) Total number of activated TFH cells per spleen was compared between saline-treated and rhF8-boosted animals. Paired mice of similar inhibitor titers are represented by the same symbol. *P < .05; **P < .01.

Article Snippet: 31 , 32 C57BL/6J WT and mixed bone marrow (BM) chimeric mice, which had normal levels of endogenous murine FVIII, were immunized with recombinant human full-length FVIII (rhfF8, Kogenate FS; Bayer Pharma, Whippany, NJ) at a dose of 200 U/kg weekly for 4 weeks.

Techniques: Saline, Staining, Flow Cytometry