The imaging and biochemical data presented
The imaging and biochemical data presented here demonstrated that in T cells, AEP played a specific function in cleaving Foxp3 but not Tbet. These results are in accordance with previous AEP studies where it has been shown that AEP substrates in part are not amenable to other protease activity. In addition, AEP is well known for its substrate and cleavage specificity and often AEP-mediated cleavage results in a functional immunological outcome in vivo (Manoury et al., 1998, Manoury et al., 2002). In T cells, AEP adheres to this phenotype, whereby it specifically targets and cleaves Foxp3 at a single site, which results in the instability of the protein. Therefore, our study implicates the occurrence of a proteolytic-mediated regulation of Foxp3 in iTreg and Tbet+iTreg cells. The data presented here postulate a post-translational mechanism of Foxp3 protein regulation in addition to the previously described proteosomal pathway that is operational in Foxp3 regulation. However, deleting AEP was sufficient for maintaining Foxp3 protein in iTreg and Tbet+iTreg Mupirocin in vivo. In contrast to the proteosomal degradation study where an shRNA approach was used (Chen et al., 2013, van Loosdregt et al., 2013, Zhao et al., 2015), we have utilized a genetic loss-of-function model (Aep mice) to demonstrate the stability of Foxp3 in vivo during acute inflammation. Furthermore, mutating AEP-specific sites in Foxp3 protected mice from GvHD-mediated lethality. Therefore, our data demonstrate that either AEP deficiency or Foxp3 mutated at AEP-specific sites can enhance Treg cell function and is a primary pathway in modulating post-translational stability of Foxp3 protein. Our results suggest that AEP inhibitor can be used to generate large-scale Treg cells that are stable and are functionally robust in vivo. The use of PDL-1 to grow Tbet+iTreg cells may be efficacious but a substantial decrease in cell numbers can occur given the role of PD-1 in inhibiting T cell proliferation. Using AEP inhibitors, this hurdle can be overcome in order to generate large numbers of antigen-primed Tbet+iTreg cells that maintain regulatory function in vivo. In addition, donor-derived AEP-deficient Treg cells can also be generated for the treatment of GvHD. Data from the acute and chronic LCMV-infected mice further identified a role for PDL-1 in inducing Foxp3 in primed “antigen-specific” Tbet+ Th1 cells. Although insufficient cell numbers prevented us from isolating antigen-specific T cells prior to ex vivo iTreg cell culture, the results presented here imply that PDL-1 can be used to generate iTreg cells from previously antigen-primed Tbet+Th1 cells. Consistent with previous studies (Hall et al., 2012, Koch et al., 2009, Koch et al., 2012, Levine et al., 2017), we have also identified a unique population of Tbet+iTreg cells that expands in both acute and chronic LCMV. In summary, these experiments highlight many aspects of Tbet+iTreg cells: (1) primed CD44hiTbethiFoxp3+ cells can arise during acute and chronic LCMV infection, (2) primed CD4+CD44hiTbethiFoxp3− T cells can give rise to Tbet+Foxp3+ T cells in ex vivo cultures, and finally (3) blocking PDL-1 in vivo dampened Tbet+pTreg cell conversion, therefore confirming PDL-1 as a critical mediator of Tbet+pTreg cell generation in chronic viral infection. In melanoma, Treg cell-mediated tolerance has largely been attributed to the migration of Treg cells from the periphery to the tumor site (Spranger et al., 2013), while Tbet+Th1 cell conversion is largely unexplored. Since the microenvironment in melanoma provides an abundance of PDL-1 that can result in activation of PD-1 signaling on Tbet+ TILs, we explored and found that Tbet+Th1 cell conversion indeed occurred. However, this study does not address the contribution of hematopoietic versus non-hematopoietic versus tumor tissue-derived PDL-1 in inducing Treg cell conversion within the tumor microenvironment. The results presented here raise the possibility that the PDL-1-driven Treg cell generation within the TILs is dependent on AEP expression and that individuals who overexpress AEP within their Treg TIL populations may be more responsive to PD-1- and/or PDL-1-based immunotherapeutics.