by Juan Chen, Jocelyn A. Schroeder, Xiaofeng Luo, and Qizhen Shi The impact of von Willebrand factor on factor VIII memory immune responses by Juan Chen, Jocelyn A. Schroeder, Xiaofeng Luo, and Qizhen Shi BloodAdv Volume 1(19):1565-1574 August 22, 2017 © 2017 by The American Society of Hematology

Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology

rhF8 dose response on FVIII-sensitized CD4+ T-cell proliferation. rhF8 dose response on FVIII-sensitized CD4+T-cell proliferation. Splenocytes isolated from rhF8-immunized FVIIInull mice were labeled with CellTrace Violet and cultured with various doses of rhF8 or rhVWF for 96 hours. Concanavalin A was used as a positive control for T-cell proliferation. Cells were stained with anti–mouse CD4 and anti–mouse TCRβ antibodies and analyzed by flow cytometry for daughter CD4+ T cells. (A) Representative flow cytometry histograms. (B) rhF8 dose–response curve. Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology

The impact of rhVWF on FVIII-sensitized CD4+ T cell proliferation in response to rhF8 restimulation. The impact of rhVWF on FVIII-sensitized CD4+T cell proliferation in response to rhF8 restimulation. Splenocytes isolated from rhF8-immunized FVIIInull mice were labeled with CellTrace Violet and cultured with a low (1 U/mL) or a high (10 U/mL) dose of rhF8 with or without 1 U/mL rhVWF for 96 hours. Cells were stained with anti–mouse CD4 and anti–mouse TCRβ antibodies and analyzed by flow cytometry for daughter CD4+ T cells. (A) Representative flow cytometry histograms. (B-C) The impact of rhVWF on CD4+ T cell proliferation in response to a low dose of rhF8 (B) and a high dose of rhF8 (C). The paired Student t test was used to compare data sets. (D-E) Normalized daughter cell data. The percentage of daughter CD4+ T cells in the condition without VWF was defined as 1. The Mann-Whitney U test was used to compare normalized data sets. These data demonstrate that VWF can attenuate FVIII-sensitized CD4+ T-cell proliferation in response to rhF8 restimulation. Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology

The impact of rhVWF on subsets of FVIII-sensitized CD4+ T-cell proliferation in response to rhF8 restimulation. The impact of rhVWF on subsets of FVIII-sensitized CD4+T-cell proliferation in response to rhF8 restimulation. Splenocytes isolated from rhF8-immunized FVIIInull mice were labeled with CellTrace Violet and cultured with rhF8 with or without rhVWF for 96 hours. Cells were stained with anti–mouse CD4, TCRβ, CD44, and CD62L and analyzed by flow cytometry. A subset of CD4+ T cells was gated and analyzed for daughter cells. (A) CD4+ T-cell proliferation. (B) Activated CD4+ (CD4+CD44+) T-cell proliferation. (C) Central memory (CD4+CD44+CD62L+) T cell proliferation. (D) Effector CD4+ (CD4+CD44+CD62L−) T-cell proliferation. The paired Student t test was used to compare data sets. These data demonstrate that VWF can attenuate FVIII-sensitized effector as well as central memory CD4+ T-cell proliferation in response to rhF8 restimulation. Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology

Cytokine profile analysis. Cytokine profile analysis. Splenocytes isolated from rhF8-immunized FVIIInull mice were cultured with rhF8 with or without rhVWF for 96 hours. The conditioned media was used to analyze the cytokine expression using the Bio-Plex cytokine assay. (Ai-Aii) Impact of VWF on IFN-γ expression in rhF8-sensitized splenocytes in response to rhF8 restimulation. (Bi-Bii) Impact of VWF on IL-10 expression in rhF8-sensitized splenocytes in response to rhF8 restimulation. The paired Student t test was used to compare data sets. These data demonstrate that VWF can attenuate IFN-γ production in FVIII-sensitized splenocytes in response to rhF8 restimulation. Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology

Memory B-cell–mediated ELISPOT assay. Memory B-cell–mediated ELISPOT assay. Splenocytes were isolated from rhF8-immunized FVIIInull mice, and CD138+ plasma cells were depleted. The remaining cells (CD138−) were used as memory B-cell pools. CD138− memory B-cell pools were cultured with various doses of rhF8 with or without 1 U/mL rhVWF for 6 days to induce differentiation of memory B cells into ASCs. ASCs were determined by ELISPOT assay. Each spot represents a single ASC. (A) The rhF8 dose response on inducing differentiation of memory B cells into ASCs and anti–FVIII antibody production. (B) Representative ELISPOT images showing the impact of VWF on memory B-cell differentiation and antibody secretion. (C) ASC counts in various culture conditions. The paired Student t test was used to compare data sets. These results demonstrate that VWF can mitigate FVIII-specific differentiation of memory B cells into ASCs and anti–FVIII antibody production. Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology

The impact of VWF on FVIII memory immune responses in vivo. The impact of VWF on FVIII memory immune responses in vivo. Splenocytes were isolated from rhF8-immunized FVIIInull mice, and CD138+ plasma cells were depleted. The remaining cells (CD138−) were used as memory B-cell pools. CD138− memory B-cell pools were infused into age- and sex-matched paired immunocompromised NSGF8KO littermates followed by rhF8 immunization with or without rhVWF. One week after immunization, plasma was collected for Bethesda (to determine inhibitor titers) and ELISA assays (to determine anti-FVIII total IgG titers). (A-B) Inhibitor titers. (C-D) Anti-FVIII total IgG titers. Data are normalized by defining the titer in the group immunized with rhF8 only as 100%. The paired Student t test was used to compare the actual titer data sets, and the Mann-Whitney U test was used to compare the normalized data sets. These data demonstrate that VWF can mitigate anti-FVIII memory immune responses in vivo. Juan Chen et al. Blood Adv 2017;1:1565-1574 © 2017 by The American Society of Hematology