293. Optimal In Vivo Treg Induction and Suppression of Immune Responses By Synergistic Use of Rapamycin and FLT3 Ligand
Authors: Moanaro Biswas, Debalina Sarkar, Sushrusha Nayak, Sandeep R.P. Kumar, Geoffrey L. Rogers, David M. Markusic, Gongxian Liao, Cox Terhorst, Roland W. Herzog
Journal: Molecular Therapy (2015)

Immune-mediated rejection of therapeutic proteins is a major hurdle for gene and protein replacement therapies for genetic disease. However, optimal induction and expansion of CD4+CD25+FoxP3+ regulatory T cells (Treg) counteract effector responses. We have previously shown that co-administration of antigen with rapamycin simultaneously promotes deletion of conventional CD4+ T cells and induction of Treg, so that we were able to induce tolerance to factors VIII and IX in treatment of the X-linked bleeding disorders hemophilia A and B. Here, we found in DO11.10 Rag−/− mice (homozygous for ovalbumin-peptide specific TCR, deficient in endogenous mature B, T or Treg cells) that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin via selective expansion of plasmacytoid dendritic cells (pDC), resulting in substantially improved induction of OVA-specific Treg. This regimen, consisting of co-administration of rapamycin/Flt3L/OVA 3x/week for 1 month, also prevented OVA-specific CD8+ T cell responses in immune competent mice after intrasmuscular OVA gene transfer with an AAV1 vector. Flt3L signaling is critical to the generation and steady state expansion of both the conventional (CD11c+, CD8+CD11c+) and plasmacytoid (CD11cmid-loPDCA-1+) subsets of DC, which indirectly expands naturally occurring Treg. Interestingly, Flt3L-induced signaling in DC and DC precursors occurs through the mTOR pathway and can therefore be blocked by rapamycin, posing the question of why we are able to use these drugs synergistically for Treg induction. We find that limited doses of rapamycin selectively block Flt3L-induced expansion of conventional DC (cDC) but not pDC. Induced mTOR signaling is effectively blocked in cDC, while pDC have increased mTOR activity and are thus more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDC. During Flt3L/rapamycin/antigen co-administration, Treg induction is abrogated upon pDC depletion, thus confirming that Treg induction is pDC dependent. Similarly, Flt3L/antigen by itself fails to induce Treg, indicating that both pDC and rapamycin are required for Treg induction in this regimen. Finally, we are able to demonstrate in a therapeutic model for FVIII protein replacement therapy that combined Flt3L/rapamycin/FVIII treatment is superior in prevention of inhibitory antibody responses in hemophilia A mice (BALB/c F8e16−/−) when compared to rapamycin/FVIII only. In contrast, Flt3L/antigen administration in the absence of rapamycin, while enhancing activation induced cell death (AICD) of conventional CD4+ T cells, fails to induce Treg and also fails to induce tolerance to OVA or to FVIII. Thus, the differential effect of rapamycin on DC subsets can be exploited to improve tolerance induction via enhanced Treg induction, creating new opportunities for development of immune tolerance therapies with wide applicability.