In the current study we have elucidated an
In the current study, we have elucidated an intrinsic mechanism by which PD-1 signaling maintains Foxp3 in Tbet+iTreg and iTreg cells. The data presented here demonstrate that PD-1 can inhibit a functional nuclear pool of active asparaginyl endopeptidase (AEP), an endo-lysosomal protease previously implicated in antigen processing in dendritic Ebastine (Dall and Brandstetter, 2016, Manoury et al., 1998, Manoury et al., 2002). We show that AEP is responsible for destabilizing Foxp3 in both iTreg and Tbet+iTreg cells. We found that PD-1 activation significantly enhanced Foxp3 expression in primed anti-viral and anti-tumor Tbet+Th1 cells, which was reversed in the presence of a blocking antibody to PDL-1. Of note, PDL-1 blockade did not reverse Tbet+Th1 cell conversion and iTreg cell induction in the absence of AEP. Therefore, this study demonstrates that downregulation of AEP is necessary for PD-1-generated Foxp3 stability.
Discussion The regulation of Foxp3 in Treg cells and T helper cell subsets is an active area of investigation and may help in understanding dysregulation of the immune system in disease processes. In this report, we have identified a proteolytic regulation of Foxp3 protein in iTreg cells, Tbet+iTreg cells, and pTbet+Treg cells. We demonstrated that (1) PD-1 signaling maintains Foxp3 protein stability through regulating AEP, (2) AEP directly cleaves Foxp3 and results in Foxp3 instability in iTreg cells and Tbet+iTreg cells, and (3) inhibiting AEP resulted in enhanced Treg cell function. These data elucidate a basic mechanism that is operational in Treg cells and paves a path to the development of translational approaches for developing Treg cell-based cell therapies. The results outlined in this paper are in agreement with the existence of these Tbet+Foxp3+ T cells in vivo. However, reports on Tbet+Treg cells (Hall et al., 2012, Koch et al., 2009, Koch et al., 2012, Levine et al., 2017) propose that tTreg cells are the likely precursors of Tbet+Treg cells. The data presented here extend these observations and clearly demonstrate that an alternate pathway is involved in the upregulation of Foxp3 expression by Tbet+Foxp3− T cells. In vivo cell tracing experiments performed in the long-term colitis model highlights a mechanism by which PDL-1 imparts regulatory function to Tbet+iTregPDL-1 cells. During colitis, Tbet+iTregPDL-1 cells had sustained Foxp3 expression after 60 days in an inflammatory environment unlike their counterpart Tbet+iTreg cells. These data support a regulatory mechanism whereby differentiation of Tbet+ cells in the presence of PDL-1 can result in sustained Foxp3 expression in vivo and led us to explore the molecular mechanisms by which PD-1 signaling regulated Foxp3 stability. We found a proteolytic pathway that was operational in maintaining Foxp3 protein stability in iTreg cells and Tbet+iTreg cells involving direct inhibition of the activity of AEP. PD-1 inhibition of AEP was independent of CD28 signaling (Hui et al., 2017, Kamphorst et al., 2017). The notion that a specific protease can perform an essential specific proteolytic function is controversial as cells express many proteases that exhibit considerable functional redundancy (van Kasteren and Overkleeft, 2014). However, cell type-specific differences in protease function have been previously reported. For example, AEP breaks down self-antigens in DCs (Manoury et al., 2002) and cathepsin G performs this function in B cells (Burster et al., 2004). AEP activity has been reported both in lysosomes and in the nucleus of tumor cells (Haugen et al., 2013). Functionally AEP can induce tumor cell proliferation and migration and process antigens for optimal presentation by DCs (Andrade et al., 2011, Lin et al., 2014, Manoury et al., 1998). In contrast to other lysosomal proteases, AEP is expressed in the cytosol and nucleus and the activity of AEP across both neutral and acidic pH has been previously reported (Haugen et al., 2013). These observations enable a mechanism by which Foxp3 in the nucleus can be targeted by AEP.