In vivo induction and expansion of Treg is a powerful tool to limit unwanted immune responses and promote tolerance. For example, we have been successful inducing tolerance to factors VIII and FIX in hemophilic mice when the coagulation factor antigen was administered with the mTOR inhibitor rapamycin (J Thromb Haemost 7:1523 and 9:1524, Front Microbiol 2:244). Rapamycin, a macrocyclic triene antibiotic, is an immunosuppressant used to avoid transplant rejection. It suppresses the mTOR1 (and upon prolonged exposure also mTOR2) signaling pathway. Importantly, while mTOR blockage results in deletion of Teff, Treg can be induced and expanded because they are able to utilize alternative (STAT) signaling pathways. Others have shown that existing Treg can be expanded in vivo upon administration of Fms-like tyrosine kinase ligand-3 (Flt3L), a cytokine that drives generation of dendritic cells (DC) from hematopoietic progenitor cells and DC proliferation. This link between DC homeostasis and Treg is evident from the low Treg numbers found in Flt3L-deficient mice and from prevention of graft vs host disease upon treatment with Flt3L. This raises the question of whether a combined approach of rapamycin administration and Flt3L-induced DC generation would result in an optimal immune tolerance protocol. Interestingly, it has been reported that rapamycin blocks Flt3L-induced differentiation of progenitor cells into DC, indicating that Flt3L signaling in DC occurs through the mTOR pathway. However, we find in mice transgenic for an ovalbumin-specific CD4+ T cell receptor (but deficient in recombinase activating gene, rag-2) that ova peptide antigen administration results in substantially enhanced deletion of Teff and in induction of CD4+CD25+FoxP3+CD62L+GITR+ Treg when combined with these two drugs. This was accomplished by repeated administration (twice per week) of a cocktail of the 3 components. Antigen plus either drug causes Teff deletion, while rapamycin is required for Treg induction (which is further enhanced by Flt3L). Antigen, rapamycin, and Flt3L all impact changes in the numbers and frequencies of DC subsets in the spleen during the regimen. The combination all 3 components most potently directs a substantial (3–5 fold, P<0.001) increase in CD11cloPDCA+ plasmacytoid DC numbers (but not of conventional CD11chiPDCA− DCs). While pDCs are known to provide innate anti-viral responses, they also play an important role in immune tolerance. Consequently, when pDC were partially depleted with anti-PDCA, Treg induction was significantly impaired. Furthermore, the protocol caused an increase in the frequency of Indoleamine-pyrrole 2,3-dioxygenase (IDO)-expressing pDCs (which is known to activate resting Treg for suppressor activity). Finally, FLt3L-induced expansion of Treg (but not of DCs) is less effective in GITR-L −/− mice. Combined, these data demonstrate that i) Flt3L and rapamycin can be used synergistically for induction of T cell tolerance, ii) pDCs can be expanded within a rapamycin regimen, iii) and Fl3tL-induced pDC expansion facilitates Treg induction, which is partially dependent on GITR-L (a co-stimulatory molecule primarily expressed by pDCs that promotes cross talk to Treg by engagement of the GITR receptor). In order to establish relevance of this protocol for treatment of disease, we intravenously injected a F.VIII protein/rapamycin/Flt3L cocktail into hemophilia A mice (C57BL6/129 F8e16 −/−) twice per week for 1 month. Subsequently, mice received 1 month of factor replacement therapy (1 IU human FVIII, IV, once per week). Control mice without prior immune modulatory regiment or that received non-specific immune suppression (rapamycin and Flt3L only) formed high-titer inhibitors against FVIII (70–80 BU), which was significantly suppressed to ∼10 BU (P<0.001, n=5 per group). Importantly, inhibitor titers were only mildly reduced (to ∼40 BU) when Flt3L was omitted from the tolerogenic cocktail, thereby confirming the synergistic effect of flt3L and rapamycin in tolerance induction. This approach combines expansion of regulatory antigen presenting and T cells and should be of broad relevance for cell and organ transplantation as well as for treatment of inherited protein deficiencies and of autoimmune diseases.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.