Naturally Occurring Regulatory T Cells Modulate Immune Responses Against Exogenous Factor VIII in Hemophilic Balb/c Mice but Not in C57BL/6J Mice.
Authors: Hausl Christina , Ilas Josenato , Lubich Christian , Ahmad Rafi U. , Muchitsch Eva M. , Ehrlich Hartmut , Schwarz Hans Peter , Reipert Birgit M. 
Published: October 2019

Abstract
Antibody responses against factor VIII (FVIII) are the major complication that arises when patients with hemophilia A are treated with factor VIII products. Therefore, understanding regulation of anti-FVIII immune responses is of outmost importance. Antibody responses are well established to result from differentiation of B cells into antibody-secreting plasma cells. B cells need help from activated CD4+ T cells to develop high-affinity antibody responses against protein antigens such as FVIII. Recently, naturally occurring CD4+CD25+ regulatory T cells have been shown to modulate antibody responses by either suppressing the function of CD4+ T helper cells or by directly acting on B cells. However, the potential importance of CD4+CD25+ T cells in regulating antibody responses to foreign protein antigens is controversial. Furthermore, the extent to which naturally occurring CD4+CD25+ T cells regulate antibody responses against exogenous proteins such as FVIII when these proteins are given to previously untreated patients is unclear. To obtain information on how important naturally occurring CD4+CD25+ T cells are under such conditions, we asked whether these cells regulate anti-FVIII antibody responses in murine hemophilia A. We studied E17 hemophilic mice with two different genetic backgrounds (C57BL/6J and Balb/c) and treated them with four intravenous doses of human FVIII given at weekly intervals. Before the first dose of FVIII, CD4+CD25+ T cells were depleted in vivo using an anti-CD25 antibody that has been shown to deplete naturally occurring CD4+CD25+ T cells in mice. In vivo depletion of regulatory T cells using the same antibody has been successfully applied in a variety of mouse studies to evaluate the significance of naturally occurring CD4+CD25+ T cells in different immunological systems. An isotype-matched control antibody was used as a negative control. A week after the second and the fourth dose of FVIII, plasma samples were taken and tested for anti-FVIII antibodies. We found differences in titers of anti-FVIII antibodies between mice treated with anti-CD25 antibodies and control mice in Balb/c mice but not in C57BL/6J mice. Hemophilic Balb/c mice that had been pre-treated with anti-CD25 antibodies developed higher titers of anti-FVIII antibodies than mice that had been pre-treated with an isotype-matched control antibody. Differences were seen as a statistical trend (p=0.091) after two doses of FVIII and reached statistical significance (p=0.024) after four doses of FVIII. No differences in antibody titers were observed in hemophilic C57BL/6J mice. Our results strongly indicate that the ability of naturally occurring regulatory T cells to modulate anti-FVIII antibody responses in hemophilic mice depends on the genetic background of these mice. Immunoregulatory factors such as cytokines or chemokines as well as differences in the number and functional activity of naturally occurring regulatory T cells that are found in secondary lymphoid organs are likely to determine the regulatory capacity of these cells. Based on our results we conclude that differences in number and functional activity of naturally occurring regulatory T cells should be considered in the search for risk factors associated with the development of FVIII inhibitors in patients.