The impact of von Willebrand factor on factor VIII memory immune responses

Abstract

Immune tolerance induction (ITI) with aggressive infusion of factor VIII (FVIII) is the current strategy used to eradicate FVIII inhibitors and restore normal FVIII pharmacokinetics in inhibitor patients. Whether the use of FVIII products containing von Willebrand factor (VWF) will affect the efficacy of ITI is still controversial. In this study, we explored the impact of VWF on FVIII memory immune responses in hemophilia A (HA) mice. A T-cell proliferation assay and cytokine profile analysis were used to study FVIII-primed CD4⁺ T cells. When CD4⁺ T cells from primed FVIII^null mice were restimulated with recombinant human FVIII (rhF8) plus recombinant human VWF (rhVWF) in vitro, the percentages of daughter CD4⁺ T cells were significantly decreased compared with the groups cultured with rhF8 only. Levels of interferon-γ and interleukin 10 were significantly lower in the rhF8 plus rhVWF groups than in the rhF8 groups. When memory B-cell pools from primed FVIII^null mice were cultured with rhF8 with or without rhVWF to induce differentiation of memory B cells into antibody-secreting cells (ASCs), the number of ASCs was significantly lower in the rhF8 plus VWF group than in the rhF8 group. When memory B-cell pools were transferred into NSGF8KO mice followed by rhF8 immunization with or without rhVWF, the titers of anti-F8 inhibitors and total immunoglobulin G were significantly higher in the rhF8 group than in the rhF8 plus rhVWF group, with an average difference of 2.23- and 2.04-fold. Together, our data demonstrate that VWF attenuates FVIII memory immune responses in HA mice.

Graphical abstract

Figure 1.

rhF8 dose response on FVIII-sensitized CD4⁺T-cell proliferation. Splenocytes isolated from rhF8-immunized FVIII^null 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.

Figure 2.

The impact of rhVWF on FVIII-sensitized CD4⁺T cell proliferation in response to rhF8 restimulation. Splenocytes isolated from rhF8-immunized FVIII^null 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.

Figure 3.

The impact of rhVWF on subsets of FVIII-sensitized CD4⁺T-cell proliferation in response to rhF8 restimulation. Splenocytes isolated from rhF8-immunized FVIII^null 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.

Figure 4.

Cytokine profile analysis. Splenocytes isolated from rhF8-immunized FVIII^null 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.

Figure 5.

Memory B-cell–mediated ELISPOT assay. Splenocytes were isolated from rhF8-immunized FVIII^null 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.

Figure 6.

The impact of VWF on FVIII memory immune responses in vivo. Splenocytes were isolated from rhF8-immunized FVIII^null 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.