Driving Rel-iant Tregs toward an Identity Crisis
Authors: Amy Li, Tyler Jacks
Journal: Immunity (2017)

Inhibiting Treg cell function in tumors is an attractive strategy to improve anti-cancer immunity. In a pair of papers in Immunity and Cell, Ghosh and colleagues show that the canonical NF-κB subunits p65 and c-Rel have non-redundant, critical roles in promoting Treg cell development and function (Oh et al., 2017Oh H. Grinberg-Bleyer Y. Liao W. Maloney D. Wang P. Wu Z. Wang J. Bhatt D.M. Nicole Heise N. Schmid R.M. Immunity. 2017; 47 (this issue): 450-465Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Targeting c-Rel blunts Treg cell immunosuppressive activity in the tumor microenvironment and enhances anti-tumor T cell responses (Grinberg-Bleyer et al., 2017Grinberg-Bleyer Y.G. Oh H. Desrichard A. Bhatt D.M. Caron R. Chan T.A. Schmid R.M. Klein U. Hayden M.S. Ghosh S. Cell. 2017; 170: 1-13Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Inhibiting Treg cell function in tumors is an attractive strategy to improve anti-cancer immunity. In a pair of papers in Immunity and Cell, Ghosh and colleagues show that the canonical NF-κB subunits p65 and c-Rel have non-redundant, critical roles in promoting Treg cell development and function (Oh et al., 2017Oh H. Grinberg-Bleyer Y. Liao W. Maloney D. Wang P. Wu Z. Wang J. Bhatt D.M. Nicole Heise N. Schmid R.M. Immunity. 2017; 47 (this issue): 450-465Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Targeting c-Rel blunts Treg cell immunosuppressive activity in the tumor microenvironment and enhances anti-tumor T cell responses (Grinberg-Bleyer et al., 2017Grinberg-Bleyer Y.G. Oh H. Desrichard A. Bhatt D.M. Caron R. Chan T.A. Schmid R.M. Klein U. Hayden M.S. Ghosh S. Cell. 2017; 170: 1-13Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Regulatory T (Treg) cells are critical for maintaining peripheral immune tolerance and preventing autoimmunity, and recent evidence suggests that Treg cells can also promote tumor growth by suppressing anti-tumor immune responses (Tanaka and Sakaguchi, 2017Tanaka A. Sakaguchi S. Cell Res. 2017; 27: 109-118Crossref PubMed Scopus (862) Google Scholar). The presence of Treg cells is associated with poor prognosis in a number of cancers (Shang et al., 2015Shang B. Liu Y. Jiang S.-J. Liu Y. Sci. Rep. 2015; 5: 15179Crossref PubMed Scopus (566) Google Scholar) and mouse models suggest that "checkpoint" antibodies directed against the inhibitory receptor CTLA-4, which is expressed on Treg cells and other T cells, act in part by depleting Treg cells in the tumor microenvironment (Simpson et al., 2013Simpson T.R. Li F. Montalvo-Ortiz W. Sepulveda M.A. Bergerhoff K. Arce F. Roddie C. Henry J.Y. Yagita H. Wolchok J.D. et al.J. Exp. Med. 2013; 210: 1695-1710Crossref PubMed Scopus (995) Google Scholar). Therefore, inhibiting the function of Treg cells in tumors is an attractive strategy to alleviate tumor immunosuppression and improve immune responses against cancer. In these studies, Ghosh and colleagues establish the importance of canonical NF-κB signaling for Treg cell function and demonstrate that c-Rel inhibition preferentially targets tumor Treg cells without causing systemic autoimmunity. The NF-κB signaling network is well known to control aspects of T cell development (Gerondakis and Siebenlist, 2010Gerondakis S. Siebenlist U. Cold Spring Harb. Perspect. Biol. 2010; 2: a000182Crossref PubMed Scopus (217) Google Scholar). Previously, Ghosh and colleagues and other groups (Isomura et al., 2009Isomura I. Palmer S. Grumont R.J. Bunting K. Hoyne G. Wilkinson N. Banerjee A. Proietto A. Gugasyan R. Wu L. et al.J. Exp. Med. 2009; 206: 3001-3014Crossref PubMed Scopus (194) Google Scholar, Messina et al., 2016Messina N. Fulford T. O'Reilly L. Loh W.X. Motyer J.M. Ellis D. McLean C. Naeem H. Lin A. Gugasyan R. et al.J. Autoimmun. 2016; 70: 52-62Crossref PubMed Scopus (38) Google Scholar) demonstrated that both p65 and c-Rel, subunits that are involved in canonical NF-κB signaling, promote the development of Treg cells in the thymus. However, it has remained unclear whether these subunits have overlapping roles in controlling the function and maintenance of mature Treg cells, and how their activity might be distinct in Treg cells versus conventional CD4+ T (Tconv) cells. The authors also sought to determine whether activated Treg cells at sites of inflammation might be particularly dependent on one Rel subunit, thus providing a means to preferentially target tumor Treg cells. The study by Oh et al. determines the effect of canonical NF-κB signaling on mature Treg cell function by generating mice with Treg cell-specific loss of p65, c-Rel, or both (Rela−/−Rel−/−) (Oh et al., 2017Oh H. Grinberg-Bleyer Y. Liao W. Maloney D. Wang P. Wu Z. Wang J. Bhatt D.M. Nicole Heise N. Schmid R.M. Immunity. 2017; 47 (this issue): 450-465Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Surprisingly, although mice lacking c-Rel had a more dramatic defect in Treg cell development than p65-deficient animals, Treg cell-specific loss of c-Rel resulted in only mild autoimmunity in aged mice (Figure 1). Meanwhile, Treg cell-specific p65 deletion resulted in lymphoproliferative disease affecting multiple tissues. Despite their varying phenotypes, loss of either Rel subunit in Treg cells resulted in greater expression of interferon-γ (IFN-γ) and impaired activity in in vivo suppression assays. Rela−/−Rel−/− mice died at 2–4 weeks old with a scurfy-like phenotype characterized by severe inflammation and runting, indicative of extreme Treg cell dysfunction. To test whether the Rel subunits are necessary to maintain Treg cell function, the authors generated mice in which Treg cell-specific deletion of p65, c-Rel, or both could be induced conditionally through the administration of tamoxifen. Interestingly, only mice that had loss of c-Rel had fewer Treg cells and lower expression of hallmark Treg cell genes, including Foxp3, Gitr, and Cd25. Taken together, these findings demonstrate that c-Rel and p65 have non-redundant, cell-intrinsic roles in mediating Treg cell function. While p65 appears to have a greater role in preventing lymphoproliferative disease, c-Rel might be more important for maintaining Treg cell activity. To more fully characterize the effects of mutation of p65, c-Rel, or both, the authors performed RNA sequencing on Treg cells of different mutant genotypes. Notably, Treg cells with constitutive and induced loss of both p65 and c-Rel expressed lower levels of Treg cell hallmark genes like Cd83, Lrrc32, and Foxp3, but higher levels of genes associated with activated Tconv cells like Il4, Il13, and Ifng. The observation that Rela−/−Rel−/− Treg cells assume a Tconv cell phenotype led the authors to propose that p65 may have distinct transcriptional targets in Treg cells versus Tconv cells. Chromatin immunoprecipitation revealed a number of p65 binding sites that are enriched in Treg cells, several of which were associated with the Treg cell signature genes. Further analyses showed that these sites are associated with greater chromatin accessibility. These data suggest that the chromatin structure in Treg cells enhances p65 binding at Treg cell-specific loci, resulting in NF-κB-dependent regulation of the Treg cell transcriptional program. Given the particular importance of c-Rel in maintaining Treg cell function, future studies on the differences in c-Rel occupancy between Tconv cells and Treg cells might offer further insight on how canonical NF-κB signaling maintains Treg cell identity. The related study from Grinberg-Bleyer et al. focuses on the specificity of the effects of particular Rel subunits in different Treg cell populations (Grinberg-Bleyer et al., 2017Grinberg-Bleyer Y.G. Oh H. Desrichard A. Bhatt D.M. Caron R. Chan T.A. Schmid R.M. Klein U. Hayden M.S. Ghosh S. Cell. 2017; 170: 1-13Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). They hypothesized that c-Rel is particularly important in activated Treg (aTreg) cells, which are active in inflamed tissues and tumors, as compared to resting Treg (rTreg) cells, which reside in lymphoid organs and prevent lymphoproliferative disease (Li and Rudensky, 2016Li M.O. Rudensky A.Y. Nat. Rev. Immunol. 2016; 16: 220-233Crossref PubMed Scopus (301) Google Scholar). Indeed, Treg cell-specific c-Rel deletion, but not p65 deletion, caused a reduction in aTreg cells, while rTreg cell numbers were unaffected by c-Rel loss. c-Rel mutation also had a greater impact on the expression of aTreg cell hallmark genes than p65 loss. The authors thus proposed that c-Rel might be critical for Treg cell activity in tumors. Remarkably, while Treg cell-specific p65 deletion had no effect on the growth of B16F1 melanoma cells, tumor growth was significantly delayed in mice with Treg cell-specific loss of c-Rel. Moreover, these mice had fewer tumor-infiltrating Treg cells, which had decreased expression of Treg cell signature genes and increased expression of effector cytokines and transcription factors, including Ifng and Tbx21. Furthermore, anti-CD8 treatment rescued tumor growth, suggesting that the altered activity of c-Rel-deficient Treg cells results in enhanced CD8+ T cell responses and impaired tumor growth. The authors next explored whether specific inhibition of c-Rel could recapitulate the effect of Treg cell-specific c-Rel loss on tumor growth. Pentoxifylline (PTXF) is an FDA-approved xanthine derivative that has been shown to inhibit c-Rel and activate Treg cells. Indeed, PTXF-treated Treg cells had lower expression of c-Rel and genes associated with the aTreg cell phenotype, and were less immunosuppressive in an in vivo assay. Mice treated with PTXF prior to tumor induction and throughout tumor development had significantly delayed tumor growth. Tumors from PTXF-treated mice had enhanced T cell infiltration, which was characterized by a higher CD8+:CD4+ T cell ratio and greater production of IFN-γ. Conversely, PTXF-treated mice had fewer Treg cells, of which a greater percentage were IFN-γ+. PTXF treatment had no effect on mice treated with anti-CD8 antibodies, suggesting that delayed tumor growth was the result of enhanced CD8+ T cell activity. The authors proposed that combination therapy with PD-1 blockade, which is thought to work by reducing T cell exhaustion, would demonstrate even greater anti-tumor activity. Indeed, while monotherapy with either anti-PD-1 or PTXF following tumor induction had no effect on tumor growth, combined therapy resulted in drastic reduction of tumor growth. Combined treatment further improved CD8+ T cell numbers and IFN-γ production, as well as reduced Treg cell numbers and enhanced Treg cell production of IFN-γ. Taken together, the data suggest that PTXF and c-Rel inhibition can be used in combination with existing immunotherapies to undermine Treg function and improve anti-tumor immunity. Diverse strategies for depleting or impairing tumor Treg cells are currently under investigation. While many of these efforts target highly expressed markers on tumor Treg cells, the studies from Ghosh and colleagues and others suggest that "control switches" that regulate the Treg cell transcriptome can be manipulated to subvert Treg cell identity. Treg cell-specific loss of the transcription factor Helios results in an "unstable" phenotype characterized by expression of IFN-γ and TNF-α, and mice with Helios-deficient Treg cells have delayed growth of transplanted tumor cells (Nakagawa et al., 2016Nakagawa H. Sido J.M. Reyes E.E. Kiers V. Cantor H. Kim H.-J. Proc. Natl. Acad. Sci. USA. 2016; 113: 6248-6253Crossref PubMed Scopus (100) Google Scholar). Interestingly, anti-GITR antibodies reduce Helios expression on Treg cells, thus providing a link between the anti-tumor efficacy of GITR blockade with the generation of an unstable Treg cell phenotype. Another recent study showed that a subset of neuropilin 1 (Nrp1)-deficient Treg cells secretes IFN-γ, which abrogates the immunosuppressive activity of neighboring wild-type Treg cells (Overacre-Delgoffe et al., 2017Overacre-Delgoffe A.E. Chikina M. Dadey R.E. Yano H. Brunazzi E.A. Shayan G. Horne W. Moskovitz J.M. Kolls J.K. Sander C. et al.Cell. 2017; 169: 1130-1141.e11Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). These "fragile" Treg cells are no longer able to promote tumor growth, which might provide an explanation for why even small increases in IFN-γ production by Helios- and c-Rel-deficient Treg cells result in profound impairment of Treg cell activity. It remains to be explored how Helios, Nrp1, c-Rel, and other factors might act in concert to maintain Treg cell identify and function. The studies by Ghosh and colleagues also remind us that pathways that have striking effects on Treg cell function might also have distinct, even opposing, roles in other immune cells, as is the case for canonical NF-κB signaling in Tconv cells versus Treg cells. Consequently, the evaluation of strategies targeting tumor Treg cells must take into account the effects of manipulating these pathways in non-Treg cells. However, careful examination of the role of specific signaling components in particular cell types as demonstrated in these studies can offer critical insights on novel ways to target a population of interest. By taking advantage of the pleiotropic and partially redundant functions of p65 and c-Rel, the authors have shown that c-Rel inhibition and PTXF appear to be effective and well-tolerated means to enhance anti-tumor activity through alteration of the tumor Treg cell phenotype. An NF-κB Transcription-Factor-Dependent Lineage-Specific Transcriptional Program Promotes Regulatory T Cell Identity and FunctionOh et al.ImmunitySeptember 7, 2017In BriefNF-κB regulates genes essential for conventional T cell activation and function, but its role in Treg cell function remains unclear. Oh et al. use Treg cell-specific conditional deletions of canonical NF-κB subunits to demonstrate central roles for p65 and c-Rel in orchestrating Treg cell development, suppressive function, and molecular identity. Full-Text PDF Open ArchiveNF-κB c-Rel Is Crucial for the Regulatory T Cell Immune Checkpoint in CancerGrinberg-Bleyer et al.CellSeptember 07, 2017In BriefNF-κB c-Rel is critical for the function of activated Tregs and serves as a target to reduce Treg function in the tumor microenvironment without compromising systemic tolerance or causing autoimmunity. Full-Text PDF Open Archive