CAR- and TRuC-redirected regulatory T cells differ in capacity to control adaptive immunity to FVIII.

Regulatory T cells (Tregs) control immune responses in autoimmune disease, transplantation, and enable antigen-specific tolerance induction in protein-replacement therapies. Tregs can exert a broad array of suppressive functions through their T cell receptor (TCR) in a tissue-directed and antigen-specific manner. This capacity can now be harnessed for tolerance induction by "redirecting" polyclonal Tregs to overcome low inherent precursor frequencies and simultaneously augment suppressive functions. With the use of hemophilia A as a model, we sought to engineer antigen-specific Tregs to suppress antibody formation against the soluble therapeutic protein factor (F)VIII in a major histocompatibility complex (MHC)-independent fashion. Surprisingly, high-affinity chimeric antigen receptor (CAR)-Treg engagement induced a robust effector phenotype that was distinct from the activation signature observed for endogenous thymic Tregs, which resulted in the loss of suppressive activity. Targeted mutations in the CD3ζ or CD28 signaling motifs or interleukin (IL)-10 overexpression were not sufficient to restore tolerance. In contrast, complexing TCR-based signaling with single-chain variable fragment (scFv) recognition to generate TCR fusion construct (TRuC)-Tregs delivered controlled antigen-specific signaling via engagement of the entire TCR complex, thereby directing functional suppression of the FVIII-specific antibody response. These data suggest that cellular therapies employing engineered receptor Tregs will require regulation of activation thresholds to maintain optimal suppressive function.

Reprogrammed CD4+ T Cells That Express FoxP3+ Control Inhibitory Antibody Formation in Hemophilia A Mice.

Coagulation Factor VIII (FVIII) replacement therapy in hemophilia A patients is complicated by the development of inhibitory antibodies, which often render the treatment ineffective. Previous studies demonstrated a strong correlation between induction of regulatory T cells (Treg) and tolerance to the therapeutic protein. We, therefore, set out to evaluate whether the adoptive transfer of FVIII-specific CD4+ Treg cells prevents inhibitor response to FVIII protein therapy. To this end, we first retrovirally transduced FoxP3+ into FVIII-specific CD4+ cells, which resulted in cells that stably express FoxP3, are phenotypically similar to peripherally induced Tregs and are antigen specific suppressors, as judged by in vitro assays. Upon transfer of the FVIII-specific CD4+ FoxP3+ cells into hemophilia A mice, development of inhibitory antibodies in response to administering FVIII protein was completely suppressed. Suppression was extended for 2 months, even after transferred cells were no longer detectable in the secondary lymphoid organs of recipient animals. Upon co-transfer of FoxP3+-transduced cells with the B cell depleting anti-CD20 into mice with pre-existing inhibitory antibodies to FVIII, the escalation of inhibitory antibody titers in response to subsequent FVIII protein therapy was dramatically reduced. We conclude that reprogramed FoxP3 expressing cells are capable of inducing the in vivo conversion of endogenous FVIII peripheral Tregs, which results in sustained suppression of FVIII inhibitors caused by replacement therapy in recipient hemophilia A animals.