CD36-mediated metabolic adaptation supports regulatory T cell survival and function in tumors.

Depleting regulatory T cells (Treg cells) to counteract immunosuppressive features of the tumor microenvironment (TME) is an attractive strategy for cancer treatment; however, autoimmunity due to systemic impairment of their suppressive function limits its therapeutic potential. Elucidating approaches that specifically disrupt intratumoral Treg cells is direly needed for cancer immunotherapy. We found that CD36 was selectively upregulated in intrautumoral Treg cells as a central metabolic modulator. CD36 fine-tuned mitochondrial fitness via peroxisome proliferator-activated receptor-ß signaling, programming Treg cells to adapt to a lactic acid-enriched TME. Genetic ablation of Cd36 in Treg cells suppressed tumor growth accompanied by a decrease in intratumoral Treg cells and enhancement of antitumor activity in tumor-infiltrating lymphocytes without disrupting immune homeostasis. Furthermore, CD36 targeting elicited additive antitumor responses with anti-programmed cell death protein 1 therapy. Our findings uncover the unexplored metabolic adaptation that orchestrates the survival and functions of intratumoral Treg cells, and the therapeutic potential of targeting this pathway for reprogramming the TME.

Publisher Correction: Uncoupling protein 2 reprograms the tumor microenvironment to support the anti-tumor immune cycle.

In the version of this article initially published, the bars were not aligned with the data points or horizontal axis labels in Fig. 5d, and the labels along each horizontal axis of Fig. 5j-l indicating the presence (+) or absence (-) of doxycycline (Dox) were incorrectly included with the labels below that axis. Also, the right vertical bar above Fig. 7b linking 'P = 0.0001' to the key was incorrect; the correct comparison is αPD-1 versus Dox + αPD-1. Similarly, the right vertical bar above Fig. 7e linking 'P = 0.0002' to the key was incorrect; the correct comparison is αPD-1 versus Rosig + αPD-1. The errors have been corrected in the HTML and PDF versions of the article.

Uncoupling protein 2 reprograms the tumor microenvironment to support the anti-tumor immune cycle.

Immune checkpoint blockade therapy has shifted the paradigm for cancer treatment. However, the majority of patients lack effective responses due to insufficient T cell infiltration in tumors. Here we show that expression of mitochondrial uncoupling protein 2 (UCP2) in tumor cells determines the immunostimulatory feature of the tumor microenvironment (TME) and is positively associated with prolonged survival. UCP2 reprograms the immune state of the TME by altering its cytokine milieu in an interferon regulatory factor 5-dependent manner. Consequently, UCP2 boosts the conventional type 1 dendritic cell- and CD8+ T cell-dependent anti-tumor immune cycle and normalizes the tumor vasculature. Finally we show, using either a genetic or pharmacological approach, that induction of UCP2 sensitizes melanomas to programmed cell death protein-1 blockade treatment and elicits effective anti-tumor responses. Together, this study demonstrates that targeting the UCP2 pathway is a potent strategy for alleviating the immunosuppressive TME and overcoming the primary resistance of programmed cell death protein-1 blockade.

Phosphoenolpyruvate Is a Metabolic Checkpoint of Anti-tumor T Cell Responses.

Activated T cells engage aerobic glycolysis and anabolic metabolism for growth, proliferation, and effector functions. We propose that a glucose-poor tumor microenvironment limits aerobic glycolysis in tumor-infiltrating T cells, which suppresses tumoricidal effector functions. We discovered a new role for the glycolytic metabolite phosphoenolpyruvate (PEP) in sustaining T cell receptor-mediated Ca(2+)-NFAT signaling and effector functions by repressing sarco/ER Ca(2+)-ATPase (SERCA) activity. Tumor-specific CD4 and CD8 T cells could be metabolically reprogrammed by increasing PEP production through overexpression of phosphoenolpyruvate carboxykinase 1 (PCK1), which bolstered effector functions. Moreover, PCK1-overexpressing T cells restricted tumor growth and prolonged the survival of melanoma-bearing mice. This study uncovers new metabolic checkpoints for T cell activity and demonstrates that metabolic reprogramming of tumor-reactive T cells can enhance anti-tumor T cell responses, illuminating new forms of immunotherapy.

Prostaglandin E2 and programmed cell death 1 signaling coordinately impair CTL function and survival during chronic viral infection.

More than 10% of the world's population is chronically infected with HIV, hepatitis C virus (HCV) or hepatitis B virus (HBV), all of which can cause severe disease and death. These viruses persist in part because continuous antigenic stimulation causes the deterioration of virus-specific cytotoxic T lymphocyte (CTL) function and survival. Additionally, antiviral CTLs autonomously suppress their responses to limit immunopathology by upregulating inhibitory receptors such as programmed cell death 1 (PD-1). Identification and blockade of the pathways that induce CTL dysfunction may facilitate the clearance of chronic viral infections. We found that the prostaglandin E2 (PGE2) receptors EP2 and EP4 were upregulated on virus-specific CTLs during chronic lymphocytic choriomeningitis virus (LCMV) infection and suppressed CTL survival and function. We show that the combined blockade of PGE2 and PD-1 signaling was therapeutic in terms of improving viral control and augmenting the numbers of functional virus-specific CTLs. Thus, PGE2 inhibition is both an independent candidate therapeutic target and a promising adjunct therapy to PD-1 blockade for the treatment of HIV and other chronic viral infections.

Chronic viral infection promotes sustained Th1-derived immunoregulatory IL-10 via BLIMP-1.

During the course of many chronic viral infections, the antiviral T cell response becomes attenuated through a process that is regulated in part by the host. While elevated expression of the immunosuppressive cytokine IL-10 is involved in the suppression of viral-specific T cell responses, the relevant cellular sources of IL-10, as well as the pathways responsible for IL-10 induction, remain unclear. In this study, we traced IL-10 production over the course of chronic lymphocytic choriomeningitis virus (LCMV) infection in an IL-10 reporter mouse line. Using this model, we demonstrated that virus-specific T cells with reduced inflammatory function, particularly Th1 cells, display elevated and sustained IL-10 expression during chronic LCMV infection. Furthermore, ablation of IL-10 from the T cell compartment partially restored T cell function and reduced viral loads in LCMV-infected animals. We found that viral persistence is needed for sustained IL-10 production by Th1 cells and that the transcription factor BLIMP-1 is required for IL-10 expression by Th1 cells. Restimulation of Th1 cells from LCMV-infected mice promoted BLIMP-1 and subsequent IL-10 expression, suggesting that constant antigen exposure likely induces the BLIMP-1/IL-10 pathway during chronic viral infection. Together, these data indicate that effector T cells self-limit their responsiveness during persistent viral infection via an IL-10-dependent negative feedback loop.

Immune-based antitumor effects of BRAF inhibitors rely on signaling by CD40L and IFNγ.

B-Raf(V600E) inhibitors have been suggested to promote tumor regression with the help of host immunity, but this hypothesis has not been examined directly in detail. In this study, we profiled immunologic changes in the tumor microenvironment and tumor-infiltrating lymphocytes (TIL) in a B-RafV600E/Pten-driven murine model of melanoma after administration of the B-Raf(V600E) small molecule inhibitor PLX4720. In this model, we found that as tumors developed, they gradually acquired immunosuppressive features, including accumulation of regulatory T cells (Treg) and CD11b(+)/Gr-1(+) myeloid cells and loss of Th1 effector functions on CD4(+) TILs, such as CD40L and IFNγ expression. PLX4720 administration promoted development of a more immune stimulatory microenvironment associated with a relative increase in CD40L and IFNγ expression on intratumoral CD4(+) TILs and a reduced accumulation of Tregs and CD11b(+)/Gr-1(+) myeloid cells. Strikingly, CD40L or IFNγ blockade compromised the ability of PLX4720 to inhibit melanoma growth. Supporting this result, agonistic CD40 antibody was sufficient to evoke antitumor immunity and suppress tumor growth in tumor-bearing mice. Taken together, our results establish the critical role of immune-related changes, with key contributions for CD40L and IFNγ signaling in the antitumor responses triggered in vivo by B-Raf(V600E) inhibitors.

NF-κB-mediated degradation of the coactivator RIP140 regulates inflammatory responses and contributes to endotoxin tolerance.

Tolerance to endotoxins that is triggered by prior exposure to Toll-like receptor (TLR) ligands provides a mechanism with which to dampen inflammatory cytokines. The receptor-interacting protein RIP140 interacts with the transcription factor NF-κB to regulate the expression of genes encoding proinflammatory cytokines. Here we found lipopolysaccharide stimulation of kinase Syk-mediated tyrosine phosphorylation of RIP140 and interaction of the NF-κB subunit RelA with RIP140. These events resulted in more recruitment of the E3 ligase SCF to tyrosine-phosphorylated RIP140, which degraded RIP140 to inactivate genes encoding inflammatory cytokines. Macrophages expressing nondegradable RIP140 were resistant to the establishment of endotoxin tolerance for specific 'tolerizable' genes. Our results identify RelA as an adaptor with which SCF fine tunes NF-κB target genes by targeting the coactivator RIP140 and show an unexpected role for RIP140 degradation in resolving inflammation and endotoxin tolerance.

Endothelin-1 promotes cytoplasmic accumulation of RIP140 through a ET(A)-PLCß-PKCε pathway.

The physiological signal activating cytoplasmic accumulation of nuclear receptor interacting protein 140 (RIP140) in adipocytes was unclear. We uncover that endothelin-1 (ET-1) promotes cytoplasmic accumulation of RIP140 in 3T3-L1 adipocytes. We determine ET-1's signal transduction pathway in adipocytes, which is by activating ET(A) receptor-PLCß-nuclear PKCε. Blocking this pathway in 3T3-L1 adipocyte cultures, by treating cells with an ET(A) antagonist, inhibiting PLCß, or silencing PKCε, reduces ET-1-stimulated cytoplasmic accumulation of RIP140. In a HFD-fed obese mouse model, administration of a selective ET(A) antagonist, ambrisentan, effectively dampens cytoplasmic accumulation of RIP140 in the epididymal adipose tissue and reduces HFD-caused adipocyte dysfunctions. Importantly, ambrisentan improves blood glucose control and reduces the severity of hepatic steatosis in HFD-fed mice. This study reports a physiological signal that stimulates nuclear export of RIP140 in adipocytes and provides evidence for a strategy using selective ET(A) antagonist to treat obesity-induced insulin resistance and, possibly, other metabolic disorders.

Dual action of epidermal growth factor: extracellular signal-stimulated nuclear-cytoplasmic export and coordinated translation of selected messenger RNA.

We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear-cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator. Using the kappa-opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2-containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.

Kappa opioid receptor contributes to EGF-stimulated neurite extension in development.

Epidermal growth factor (EGF), a mitogen, also stimulates neurite extension during development, but the underlying mechanism is elusive. This study reveals a functional role for kappa opioid receptor (KOR) in EGF-stimulated neurite extension, and the underlying mechanism. EGF and activated EGF receptor (EGFR) levels are elevated in embryonic spinal cords during late gestation stages, with concurrent rise in protein levels of KOR and axon extension markers, growth-associated protein 43 (GAP43), and transient axonal glycoprotein-1 (TAG-1). Both GAP43 and TAG-1 levels are significantly lower in KOR-null (KOR(-/-)) spinal cords, and EGFR inhibitors effectively reduce the levels of KOR, GAP43, and TAG-1 in wild-type embryonic spinal cords. For KOR(-/-) or KOR-knockdown dorsal root ganglion (DRG) neurons, EGF can no longer effectively stimulate axon extension, which can be rescued by introducing a constitutive KOR expressing vector but not by a regulated KOR vector carrying its 5' untranslated region, which can be bound and repressed by growth factor receptor-bound protein 7 (Grb7). Furthermore, blocking KOR activation by application of anti-dynorphin, KOR antagonist, or EGFR inhibitor effectively reduces axon extension of DRG neurons. Thus, EGF-stimulated axon extension during development is mediated, at least partially, by specific elevation of KOR protein production at posttranscriptional level, as well as activation of KOR signaling. The result also reveals an action of EGF to augment posttranscriptional regulation of certain mRNAs during developmental stages.

A negative regulatory pathway of GLUT4 trafficking in adipocyte: new function of RIP140 in the cytoplasm via AS160.

Receptor-interacting protein 140 (RIP140), a nuclear receptor corepressor, is important for lipid and glucose metabolism. In adipocytes, RIP140 can be phosphorylated by protein kinase C epsilon (PKCvarepsilon), followed by arginine methylation, and exported to the cytoplasm. This study demonstrates for the first time a cytoplasmic function for RIP140: to counteract insulin-stimulated glucose transporter 4 (GLUT4) membrane partitioning and glucose uptake in adipocytes. Cytoplasmic RIP140 interacts with the Akt substrate AS160, thereby impeding AS160 phosphorylation by Akt; this in turn reduces GLUT4 trafficking. This signal transduction pathway can be recapitulated in the epididymal adipocytes of diet-induced obese mice: nuclear PKCvarepsilon is activated, cytoplasmic RIP140 increases, and GLUT4 trafficking and glucose uptake are reduced. The data reveal a new, cytoplasmic function for RIP140 as a negative regulator of GLUT4 trafficking and glucose uptake, and shed insight into the regulation of basal and insulin-stimulated glucose disposal by a nuclear-initiated counteracting mechanism.

Structural basis of inhibition specificities of 3C and 3C-like proteases by zinc-coordinating and peptidomimetic compounds.

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3C(pro)) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CL(pro)) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CL(pro). As shown in the present study, some of these compounds were also found to be active against 3C(pro) of CV strain B3 (CVB3). Several crystal structures of 3C(pro) from CVB3 and 3CL(pro) from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His(40)-Cys(147) catalytic dyad of CVB3 3C(pro). The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Modulation of lysine acetylation-stimulated repressive activity by Erk2-mediated phosphorylation of RIP140 in adipocyte differentiation.

Receptor-interacting protein 140 is a co-regulator for many transcription factors. Previous mass spectrometry studies showed that either phosphorylation or lysine acetylation of RIP140 directly enhanced its trans-repressive activity. In this study, we first identified p300 as a specific lysine acetyltransferase, and extracellular-signal-related kinase 2 (Erk2) as a specific kinase for threonine phosphorylation, of RIP140 in vivo. We further determined two specific acetylated lysine residues (Lys(158)/Lys(287)) and phosphorylated threonine residues (Thr(202)/Thr(207)) that were critical for its gene-repressive activity. We then delineated signal transduction from Erk2-mediated phosphorylation of RIP140 that enhanced its recruiting p300 for subsequent lysine acetylation, and demonstrated the kinetics of activation of this signal transduction pathway in differentiating adipocytes. Finally, the physiological significance of this cell signal transduction pathway was illustrated in rescuing experiments where the defect in fat accumulation of RIP140-null cultures was rescued by re-expressing the wild type RIP140 or its phospho-mimetic mutant, but not its acetylation deficient mutant. These results demonstrate the signal transduction pathway, initiated from Erk2 activation for specific threonine phosphorylation, followed by p300 recruitment for lysine acetylation, which ultimately enhances the gene-repressive activity of RIP140 and its functional role in fat accumulation in differentiated adipocytes.