Second donor type iPS DCregs activated donor

Second, donor-type iPS-DCregs activated donor-specific Tregs. Donor-type DCregs expressing donor MHC molecules traveled to the recipient\'s secondary lymphoid tissues and interacted with T cells through the “direct pathway” of allorecognition. The direct pathway of allorecognition is considered to be the most powerful mechanism to instigate early acute graft rejection (Morelli and Thomson, 2007). Figuratively speaking, pretreatment of donor-type iPS-DCregs acted as an immune suppressive vaccine, which led to a primary immune response. According to the “two-signal” hypothesis of T cell activation (Mueller et al., 1989), recipient naive T cells interacted with allogeneic MHC molecules on DCregs through a direct pathway, followed by the delivery of potent signal 1 plus poor signal 2 by DCregs to naive T cells. This resulted in the generation of donor-specific Tregs (Bakdash et al., 2013) and anergy of donor-specific Teffs (Sato et al., 2003a). On the other hand, Tiao et al. (2005) pretreated recipients with recipient-type immature BM-DCs pulsing with donor antigens, which prolonged the allograft MST by 40 days. However, in our study, recipient-type BM-DCregs with or without pulsing with donor buy Ramelteon could not protect allografts from acute rejection (MST 7 days). In Tiao et al.’s (2005) study, 2 × 106 immature BM-DCs were intravenously injected into recipients, while we only used 1 × 106 BM-DCregs. The different DC dose may be the reason why recipient-type DCregs treatment did not work in our preliminary study. Third, Tregs generated by iPS-DCregs were vital in allograft tolerance (Joffre et al., 2008; Kitazawa et al., 2007; Sakaguchi, 2004; Zhang et al., 2009b), especially in the maintenance phase (1–3 months post operation). Interestingly, donor-type iPS-DCregs served as the “primary vaccination,” which “prepared” the inhibited immune situation for the allografts. Based on this analogy, the alloantigen loaded by allografts served as the “secondary vaccination.” We hypothesized that after transplantation, the alloantigen interacted with the Tregs and precursor Tregs induced by the primary vaccination and then further activated alloantigen-specific Tregs. The number of Tregs and the expression of CTLA-4 in Tregs were significantly higher on POD100 compared with POD7 (Figure S4), which indicated that donor-specific Tregs expanded and activated unceasingly (Schubert et al., 2014; Wing et al., 2008). This may have caused the alloantigen loaded on the allograft to work as a stimulator to promote the clonal expansion of donor-specific Tregs, which we referred to as the secondary vaccination. Fourth, IFN-γ, a key inflammatory cytokine produced by Teffs, was higher in the iPS-DCregs-treated group than in the non-treated group on POD7, but became lower on POD14. Some groups reported that IFN-γ knockout (KO) and IFN-γ receptor (IFN-γR) KO recipients rejected allografts much more quickly compared with wild-type, because IFN-γ plays a crucial role in Teff apoptosis through several pathways (Morita et al., 2015; Ring et al., 1999). Thus, we speculate that IFN-γ is essential in DCregs-induced tolerance via Teff apoptosis. Fifth, TGF-β1 was certainly required in the “primary vaccination” but was not essential for the early stage of “secondary vaccination.” We have revealed here that the blockage of TGF-β1 during the period between iPS-DCregs treatment and allotransplantation prevented iPS-DCregs-induced allotolerance. However, the blockage of TGF-β1 post transplantation could not break the tolerance. Early studies demonstrated that TGF-β1 could generate Tregs through several pathways, for example, the inhibition of IL-2, the upregulation of cyclin-dependent kinase (CDK) inhibitors (p15, p21, and p27), and the downregulation of cell-cycle-promoting factors (c-myc, cyclin D2, CDK2, and cyclin E) (Wan and Flavell, 2007). We identified that the blockage of TGF-β1 not only decreased the number of Tregs induced by iPS-DCregs (CD4+CD25+%), but also downregulated the activity and transmigration ability of Tregs (CD4+CD25+FOXP3+CCR4+Ki-67hi% and CD4+CD25+FOXP3+CCR7+Ki-67hi%). The protective effects of Tregs on allograft survival were abrogated if they failed to migrate to the graft due to CCR4 and CCR7 deficiency. Logistically, the protection was enhanced when Tregs were delivered locally into the grafts (Sugiyama et al., 2013; Zhang et al., 2009a). We believe that this is the reason why the blockage of TGF-β1 during the period between iPS-DCregs immunization and allotransplantation could prevent iPS-DCregs-induced allotolerance. Although our data showed that donor-type iPS-DCregs treatment induced donor-specific Tregs by upregulating TGF-β1, it remains possible that these Tregs and TGF-β1 may participate in non-specific immune suppression. Nonetheless, although our examination indicated that Tregs and TGF-β1 are two key factors in allotolerance induced by iPS-DCregs, there could be other mechanisms involved in this therapy. In addition, further research is needed before this therapy can be adapted for clinical application, including dose, timing, dosage, and/or combination with low-dose IS drugs.