Xiao Hu

Sterol metabolism controls TH17 differentiation by generating endogenous RORγ agonists

Retinoic acid receptor-related orphan receptor γ (RORγt) controls the differentiation of naive CD4(+) T cells into the TH17 lineage, which are critical cells in the pathogenesis of autoimmune diseases. Here we report that during TH17 differentiation, cholesterol biosynthesis and uptake programs are induced, whereas their metabolism and efflux programs are suppressed. These changes result in the accumulation of the cholesterol precursor, desmosterol, which functions as a potent endogenous RORγ agonist. Generation of cholesterol precursors is essential for TH17 differentiation as blocking cholesterol synthesis with chemical inhibitors at steps before the formation of active precursors reduces differentiation. Upon activation, metabolic changes also lead to production of specific sterol-sulfate conjugates that favor activation of RORγ over the TH17-inhibiting sterol receptor LXR. Thus, TH17 differentiation is orchestrated by coordinated sterol synthesis, mobilization and metabolism to selectively activate RORγ.

Inhibiting Oxidative Phosphorylation In Vivo Restrains Th17 Effector Responses and Ameliorates Murine Colitis

Integration of signaling and metabolic pathways enables and sustains lymphocyte function. Whereas metabolic changes occurring during T cell activation are well characterized, the metabolic demands of differentiated T lymphocytes are largely unexplored. In this study, we defined the bioenergetics of Th17 effector cells generated in vivo. These cells depend on oxidative phosphorylation (OXPHOS) for energy and cytokine production. Mechanistically, the essential role of OXPHOS in Th17 cells results from their limited capacity to increase glycolysis in response to metabolic stresses. This metabolic program is observed in mouse and human Th17 cells, including those isolated from Crohn disease patients, and it is linked to disease, as inhibiting OXPHOS reduces the severity of murine colitis and psoriasis. These studies highlight the importance of analyzing metabolism in effector lymphocytes within in vivo inflammatory contexts and suggest a therapeutic role for manipulating OXPHOS in Th17-driven diseases.

Inhibition of RORγT Skews TCRα Gene Rearrangement and Limits T Cell Repertoire Diversity

Recent studies have elucidated the molecular mechanism of RORγT transcriptional regulation of Th17 differentiation and function. RORγT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP) thymocytes. While RORγ antagonists are currently being developed to treat autoimmunity, it remains unclear how RORγT inhibition may impact thymocyte development. In this study, we show that in addition to regulating DP thymocytes survival, RORγT also controls genes that regulate thymocyte migration, proliferation, and Txa0cell receptor (TCR)α selection. Strikingly, pharmacological inhibition of RORγ skews TCRα gene rearrangement, limits Txa0cell repertoire diversity, and inhibits development of autoimmune encephalomyelitis. Thus, targeting RORγT not only inhibits Th17 cell development and function but also fundamentally alters thymic-emigrant recognition of self and foreign antigens. The analysis of RORγ inhibitors has allowed us to gain a broader perspective of the diverse function of RORγT and its impact on Txa0cell biology.

Synthetic RORγ agonists regulate multiple pathways to enhance antitumor immunity

ABSTRACT RORγt is the key transcription factor controlling the development and function of CD4+ Th17 and CD8+ Tc17 cells. Across a range of human tumors, about 15% of the CD4+ T cell fraction in tumor-infiltrating lymphocytes are RORγ+ cells. To evaluate the role of RORγ in antitumor immunity, we have identified synthetic, small molecule agonists that selectively activate RORγ to a greater extent than the endogenous agonist desmosterol. These RORγ agonists enhance effector function of Type 17 cells by increasing the production of cytokines/chemokines such as IL-17A and GM-CSF, augmenting expression of co-stimulatory receptors like CD137, CD226, and improving survival and cytotoxic activity. RORγ agonists also attenuate immunosuppressive mechanisms by curtailing Treg formation, diminishing CD39 and CD73 expression, and decreasing levels of co-inhibitory receptors including PD-1 and TIGIT on tumor-reactive lymphocytes. The effects of RORγ agonists were not observed in RORγ−/− T cells, underscoring the selective on-target activity of the compounds. In vitro treatment of tumor-specific T cells with RORγ agonists, followed by adoptive transfer to tumor-bearing mice is highly effective at controlling tumor growth while improving T cell survival and maintaining enhanced IL-17A and reduced PD-1 in vivo. The in vitro effects of RORγ agonists translate into single agent, immune system-dependent, antitumor efficacy when compounds are administered orally in syngeneic tumor models. RORγ agonists integrate multiple antitumor mechanisms into a single therapeutic that both increases immune activation and decreases immune suppression resulting in robust inhibition of tumor growth. Thus, RORγ agonists represent a novel immunotherapy approach for cancer.

Corrigendum: Sterol metabolism controls TH17 differentiation by generating endogenous RORγ agonists.

Nat. Chem. Biol. 11, 141–147 (2015); published online 5 January 2015; corrected after print 15 July 2015 In the version of this article initially published, the zymosterol and zymostenol structures shown in Figure 3a were depicted with a double bond at C14-C15, where there should have been a single bond.

Abstract 3762: Novel RORγt agonist induces antitumor immune effect through enhancement of tumor antigen-specific CD8+ T-cell infiltration into the TME

RORγt is a master transcription factor, which regulates the proliferation and functionality of Type 17 T-cells (T H 17 and Tc17). Recent nonclinical studies have shown that in addition to promoting effector differentiation, stemness and plasticity, RORγt also inhibits Treg differentiation, an important component of suppressive tumor microenvironment (TME). Therefore, stimulation of RORγt by synthetic, small-molecule agonists holds promise as an immunotherapy. We tested the effects of a RORγ agonist (LYC-54143; 100 mg/kg BID given continuously till the end of study) on the tumor growth and survival in mice in combination with a tumor antigen-specific vaccine (3 doses, one week apart), which is required for proper priming in this model. We found that the RORγ agonist resulted in a highly significant enhancement of the antitumor effect delaying the tumor growth (p≤0.001 at day 21 compared to vaccine alone treatment) and prolonging survival. At day 36 after tumor implantation, 80% of mice survived in the RORγ agonist + vaccine treatment group compared to 0% survival following RORγ agonist or vaccine alone treatments. Immunologically, we found that RORγ agonist treatment led to a significant decrease in the numbers of Tregs and a significant increase in IL-17 + IFNγ + T cells in the TME. Moreover, RORγ agonist treatment led to a significant increase in the numbers of T cells including increased antigen-specific CD8 + T-cells in the TME. Interestingly, scheduling RORγ agonist before or with the vaccine demonstrated comparable antitumor activity, suggesting that the scheduling of RORγ agonist treatment does not affect the therapeutic outcomes. These results show that an RORγ agonist can enhance the antitumor immune response by enhancing effector functions and by decreasing immune suppression in the TME by increasing cytokine production and switching the differentiation of immune-suppressive Tregs to effector Th17 cells and support that RORγ agonists are promising immune-modulatory agents in cancer. Citation Format: Pankaj Gaur, Vivek Verma, Rahul Nandre, Pooja Vir, Hua Wang, Baolin Kang, Laura Carter, Xiao Hu, Xikui Liu, Seema Gupta, Samir Khleif. Novel RORγt agonist induces antitumor immune effect through enhancement of tumor antigen-specific CD8 + T-cell infiltration into the TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3762.

In vitro priming of adoptively transferred T cells with a RORγ agonist confers durable memory and stemness in vivo

Adoptive T-cell transfer therapy is an FDA- approved treatment for leukemia that relies on the ex vivo expansion and reinfusion of a patients immune cells, which can be engineered with a chimeric antigen receptor (CAR) for more efficient tumor recognition. Type 17 T cells, controlled transcriptionally by RORγ, have been reported to mediate potent antitumor effects superior to those observed with conventionally expanded T cells. Here, we demonstrate that addition of a synthetic, small-molecule RORγ agonist during ex vivo expansion potentiates the antitumor activity of human Th17 and Tc17 cells redirected with a CAR. Likewise, ex vivo use of this agonist bolstered the antitumor properties of murine tumor-specific CD4+ and CD8+ T cells. Expansion in the presence of the RORγ agonist enhanced IL17A production without compromising IFNγ secretion in vitroIn vivo, cytokine neutralization studies revealed that IFNγ and IL17A were required to regress murine melanoma tumors. The enhanced antitumor effect of RORγ agonist treatment was associated with recovery of more donor T cells in the tumor and spleen; these cells produced elevated levels of cytokines months after infusion and expressed markers of long-lived stem and central memory cells such as Tcf7 and CD62L. Conversely, untreated cells mainly exhibited effector phenotypes in the tumor. Cured mice previously treated with agonist-primed T cells were protected from tumor rechallenge. Collectively, our work reveals that in vitro treatment with a RORγ agonist generates potent antitumor Type 17 effector cells that persist as long-lived memory cells in vivoSignificance: RORγ agonists can be used in vitro during T-cell expansion to enhance the efficacy of adoptive cell therapy (e.g., CAR-T) and to provide long-term protection against tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3888/F1.large.jpg Cancer Res; 78(14); 3888-98. ©2018 AACR.

Abstract 565: RORγ agonists regulate immune checkpoint receptors to enhance anti-tumor immunity

As the master transcription factor for Type 17 T cells, RORγt activates a program of gene expression associated with enhancing effector function and overcoming immune suppression. Lycera is developing synthetic, small molecule RORγ agonists for immunotherapy of cancer. We have previously reported that RORγγ agonists increase type 17 cytokine and chemokine production, enhance cell survival and have single agent anti-tumor activity in syngeneic tumor models. Interestingly, transcriptional profiling and cytometry studies revealed that treatment of murine and human Th17 or Tc17 cells with RORγ agonists increases the expression of costimulatory receptors such as CD137 and decreased expression of coinhibitory receptors like PD-1. Given the importance of PD-1 in anti-tumor immunity, we further characterized the effects of RORγ agonist on this pathway. In vitro treatment with RORγ agonists significantly decreases mean fluorescent intensity and percent PD-1+ cells after resting and repetitive restimulation of Type 17 T cells. RORγ agonists do not modulate PD-1 expression in RORγ-/- T cells. In co-cultures of wild type and RORγ-/- T cells in the presence of the agonist, RORγ-/- T cells have reduced PD-1 expression suggesting that RORγ agonists induce a transmissible effect on RORγ-/- T cells. However, ChIP-seq data indicates that RORγt does not directly bind to the promoter or enhancer element of PD-1. Transcriptional and epigenetic profiling experiments have identified several pathways modulated by RORγ agonists that may regulate PD-1 expression. Reduced PD-1 expression following RORγ agonist treatment has a functional impact as treated cells also resist PD-L1-mediated inhibition of cytokine production and proliferation. Importantly, the decreased PD-1 expression observed after in vitro treatment with RORγ agonists is maintained following adoptive transfer of tumor specific T cells. These cells are highly effective at controlling tumor growth without further agonist treatment in vivo, suggesting that in vitro RORγ agonist treatment results in durable epigenetic changes. In summary, RORγ agonists have been shown to decrease checkpoint receptor expression while enhancing cytokine production and promoting long term survival and self-renewal of T cells. These results provide rationales for combining an RORγ agonist with checkpoint inhibitor such as anti-CTLA4 or anti-PD-1. By integrating effects on different effector pathways, RORγ agonists represent a promising immunotherapy approach for the treatment of cancer. Citation Format: Xiao Hu, Xikui Liu, Jacques Moisan, Chrystal Paulos, Yahong Wang, Chauncey Spooner, Charles Lesch, Rodney Morgan, David Mertz, Dick Bousley, Clarke Taylor, Chad Van Huis, Don Skalitzky, Thomas Aicher, Peter Toogood, Laura Carter. RORγ agonists regulate immune checkpoint receptors to enhance anti-tumor immunity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 565.

Erratum: Sterol metabolism controls TH17 differentiation by generating endogenous RORγ agonists (Nat. Chem. Biol. (2015) 11:141-147)

Nat. Chem. Biol. 11, 141–147 (2015); published online 5 January 2015; corrected after print 15 July 2015 In the version of this article initially published, the zymosterol and zymostenol structures shown in Figure 3a were depicted with a double bond at C14-C15, where there should have been a single bond.

Corrigendum: Sterol metabolism controls TH17 differentiation by generating endogenous ROR[gamma] agonists

Nat. Chem. Biol. 11, 141–147 (2015); published online 5 January 2015; corrected after print 15 July 2015 In the version of this article initially published, the zymosterol and zymostenol structures shown in Figure 3a were depicted with a double bond at C14-C15, where there should have been a single bond.