Role of Adaptive Regulatory T Cells in the Induction of Tolerance to FVIII

Individuals who lack a given protein may never properly develop self tolerance. Thus, the administration of protein therapeutics can lead to undesirable immune responses. A classic example is that patients with Hemophilia A can develop an inhibitory immune response to therapeutic treatments with coagulation factor VIII (fVIII). Several years ago, we developed a B-cell delivered gene therapy based approach to prevent this response and a mouse model to study the mechanisms of the induction and maintenance of immunological tolerance to fVIII inhibitors. This model takes advantage of a knock-in mouse with the transcription factor, FoxP3, in frame with green fluorescent protein (GFP). FoxP3 is considered to be a marker for regulatory T cells (Tregs). The FoxP3-GFP knock-in allows us to follow existing (natural) Tregs and the induction of adaptive Tregs. We have backcrossed this mouse, which expresses GFP in all FoxP3+ Tregs, with the hemophilic mouse, which has an existing deletion in fVIII (E16), for 10 generations. Establishing a mouse that is transgenic for both the FoxP3-GFP fusion and a deletion in fVIII will enhance future clinically oriented experiments to understand how tolerogenic B cells interact with Tregs in a hemophilia system. Lymphocytes from these mice have now been characterized using flow cytometry and confocal microscopy, and analyzed during treatment with tolerogenic B cells. These cells express Treg markers that include, CD25, CTLA-4, GITR and reduced amounts of CD127. However, the population of fVIII antigen-specific cells is small in this mouse model. Thus, it is difficult to detect changes over the natural variation found between mice in a colony, although small changes have been detected. To explore the underlying role of Tregs in tolerance induction, we have bred an ovalbumin (OVA)-specific, T-cell receptor (TCR) transgenic mouse that contains the FoxP3-GFP fusion (FoxP3GFP/DO11.10 and FoxP3GFP/DO11.10/Rag2^−/−). Using our B-cell delivered tolerance protocol, we can prevent immunization when an immunologically competent mouse is immunized with OVA in adjuvant. When FoxP3GFP/DO11.10 TCR transgenic mice are treated with OVA-Ig transduced B cells, we found a significant increase in antigen-specific Tregs (p<0.05). Finally, when the same treatment is performed on FoxP3GFP/DO11.10/Rag2^−/−, which completely lack natural regulatory cells, we found that FoxP3 was expressed in 4% (p<0.001) of T-cells above background. These cells express markers typical of Tregs. These data strongly support the hypothesis that transduced B cell treatment induces a regulatory phenotype in the antigen-specific T cell population. To extend this system to the hemophilia model, fVIII C2 tetramers will be used to label and isolate antigen-specific T cells during tolerance induction. Additionally, this model opens up avenues of analysis to intra-vital microscopy which gives a true representation of interactions in a live organism. (Supported by NIH RO1 HL061883, NIH T32 HL007698, and a predoctoral fellowship from the American Heart Association.)