Danger signal-dependent activation of human immune cells by factor VIII products

L. Miller, K. M. Kistner, E. Ringler, Z. Waibler, on behalf of the ABIRISK consortium (Langen, Germany)

Bleeding Disorders - Clinical Studies
Date: 18.02.2017,
Time: 08:30 - 09:45

Objective: The most severe side effect in hemophilia A treatment is the development of anti-factor VIII (FVIII) antibodies, also called inhibitors. Why inhibitors develop in a proportion of treated patients and how this can be prevented still remains unanswered. Among numerous theories, the presence of immunological danger signals, usually associated with events such as surgery or infection, has been proposed to play a role. Our objective was to investigate whether those danger signals in combination with FVIII products might synergize and boost the maturation of dendritic cells (DC) resulting in effective T cell activation.

Methods: For that we first developed a DC assay where human monocyte-derived DC of healthy donors were treated with FVIII alone, with a danger signal alone or with a combination of both. Subsequently, we assessed the activation status of treated DC by analyzing the expression of co-stimulatory molecules by flow cytometry and by measuring secreted cytokines in cell culture supernatants. Next, we developed a DC:T cell co-cultivation assay, where autologous T cells were added to DC that were pretreated as described above. Following co-culture, T cell proliferation was assessed.

Results: We showed that plasma-derived FVIII (pdFVIII) products and the bacterial danger signal lipopolysaccharide (LPS) synergize in increasing DC activation, as characterized by increased expression of co-stimulatory molecules CD83 and CD86 and secretion of pro-inflammatory cytokines. These activated DC are further able to induce T cell proliferation that is abrogated by adding neutralizing monoclonal antibodies against MHCII and CD86 as well as by treating DC with bafilomycin prior to co-stimulation. We could further show that proliferating T cells are CD4+CD25+ and consist of both naive as well as memory T cells. Of note, no T cell proliferation was observed for naive T cells when stimulated in absence of other T cell populations.

Conclusion: We conclude that pdFVIII products and LPS lead to an increased DC activation and subsequent induction of an adaptive immune response. The observed immune response is dependent on antigen processing and presentation on MHCII.