Lequn Li

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade.

Selective Effects of PD-1 on Akt and Ras Pathways Regulate Molecular Components of the Cell Cycle and Inhibit T Cell Proliferation

The inhibitory receptor PD-1 blocks T cell proliferation by preventing cells from leaving the G1 phase of the cell cycle. PD-1 Inhibits the Cell Cycle Machinery Effector T cells are stimulated through the T cell receptor (TCR) to become activated and proliferate. However, inhibitory receptors, such as PD-1, counteract these stimulatory signals and block T cell proliferation. Patsoukis et al. found that PD-1–mediated inhibition of Akt and Ras signaling pathways affected a number of components of the cell cycle machinery to “lock” T cells in the G1 phase and prevent their proliferation. These mechanistic details may help in the development of therapeutics designed to block PD-1 function and enable T cell activation in the context of antiviral or antitumor responses. The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4+ T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCFSkp2 degrades p27kip1, an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G1 phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G1 phase inhibitor p15INK4 and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase–Akt and Ras–mitogen-activated and extracellular signal–regulated kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle.

The Nore1B/Mst1 complex restrains antigen receptor-induced proliferation of naïve T cells

The Mst1 and Mst2 protein kinases are the mammalian homologs of hippo, a major inhibitor of cell proliferation in Drosophila. Mst1 is most abundant in lymphoid tissues. Mice lacking Mst1 exhibit markedly reduced levels of the Mst1 regulatory protein Nore1B/RAPL in lymphoid cells, whereas Mst2 abundance is unaltered. Mst1-null mice exhibit normal T cell development but low numbers of mature naïve T cells with relatively normal numbers of effector/memory T cells. In vitro, the Mst1-deficient naïve T cells exhibit markedly greater proliferation in response to stimulation of the T cell receptor whereas the proliferative responses of the Mst1-null effector/memory T cell cohort is similar to wild type. Thus, elimination of Mst1 removes a barrier to the activation and proliferative response of naïve T cells. The levels of Mst1 and Nore1B/RAPL in wild-type effector/memory T cells are approximately 10% those seen in wild-type naïve T cells, which may contribute to the enhanced proliferative responses of the former. Freshly isolated Mst1-null T cells exhibit high rates of ongoing apoptosis, a likely basis for their low numbers in vivo; they also exhibit defective clustering of LFA-1, as previously observed for Nore1B/RAPL-deficient T cells. Among known Mst1 substrates, only the phosphorylation of the cell cycle inhibitory proteins MOBKL1A/B is lost entirely in TCR-stimulated, Mst1-deficient T cells. Mst1/2-catalyzed MOBKL1A/B phosphorylation slows proliferation and is therefore a likely contributor to the anti-proliferative action of Mst1 in naïve T cells. The Nore1B/RAPL-Mst1 complex is a negative regulator of naïve T cell proliferation.

IL-1β–Mediated Signals Preferentially Drive Conversion of Regulatory T Cells but Not Conventional T Cells into IL-17–Producing Cells

Regulatory T cells (Tregs) are committed to suppressive functions. Recently, it was proposed that Tregs could produce IL-17 under proinflammatory, polarizing conditions. We studied the role of Tregs on IL-17 production in the absence of exogenous cytokines and insults. Using in vitro and in vivo approaches, we determined that under neutral conditions, simultaneous activation of Tregs and naive CD4+ conventional T cells in the presence of APCs resulted in conversion of Tregs into IL-17–producing cells, and endogenous IL-1β was mandatory in this process. Mechanistic analysis revealed that the IL-1R1 was highly expressed on Tregs and that IL-1β induced marked activation of p38 and JNK, which were involved in IL-17 production. These observations could have important implications on therapeutic strategies using Tregs.

PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

ABSTRACT Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.

Biochemical signaling of PD-1 on T cells and its functional implications.

AbstractMaintenance of peripheral tolerance is essential for homeostasis of the immune system. While central tolerance mechanisms result in deletion of the majority of self-reactive T cells, T lymphocytes specific for self-antigens also escape this process and circulate in the periphery. To control the development of autoimmunity, multiple mechanisms of peripheral tolerance have evolved, including T cell anergy, deletion, and suppression by regulatory T (Treg) cells. The pathway consisting of the programmed cell death 1 (PD-1) receptor (CD279) and its ligands PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC; CD273) plays a vital role in the induction and maintenance of peripheral tolerance. This pathway also regulates the balance between stimulatory and inhibitory signals needed for effective immunity and maintenance of T cell homeostasis. In contrast to this important beneficial role in maintaining T cell homeostasis, PD-1 mediates potent inhibitory signals that prevent the expansion and function of T effector cells and have detrimental effects on antiviral and antitumor immunity. Despite the compelling studies on the significant functional role of PD-1 in mediating inhibition of activated T cells, little is known about how PD-1 blocks T cell activation. Here, we will provide a brief overview of the signaling events that are regulated by PD-1 triggering, and we will discuss their implications on cell intrinsic and extrinsic mechanisms that determine the fate and function of T effector cells.

The role of IL-17-producing Foxp3+ CD4+ T cells in inflammatory bowel disease and colon cancer.

The intestinal epithelium and underlying lamina propria contain T cells that play important roles in maintaining colonic homeostasis. These T cells mediate substantial and specific regulation to ensure that pathogenic microorganisms are eliminated while commensal bacteria are tolerated. There is considerable evidence supporting the notion that the altered ratio between Foxp3(+)CD4(+) T regulatory cells and T effector cells in the colonic microenvironment might contribute to the initiation and progression of inflammation and eventually development of colon cancer. Recent findings on the heterogeneity and plasticity of T regulatory cells, such as the identification of IL-17(+)Foxp3(+)CD4(+) and the RORγt(+)Foxp3(+)CD4(+) subsets, in patients with colorectal inflammation and cancer have provided a new twist in our understanding of the pathogenesis of colonic diseases. Phenotypic and functional properties of IL-17-producing Foxp3(+)CD4(+) T cells as well as the significant implications of these cells in the initiation and progression of colorectal diseases are discussed in this review.

Rap1-GTP Is a Negative Regulator of Th Cell Function and Promotes the Generation of CD4+CD103+ Regulatory T Cells In Vivo

The small GTPase Rap1 is transiently activated during TCR ligation and regulates integrin-mediated adhesion. To understand the in vivo functions of Rap1 in regulating T cell immune responses, we generated transgenic (Tg) mice, which express the active GTP-bound mutant Rap1E63 in their T lymphocytes. Although Rap1E63-Tg T cells exhibited increased LFA-1-mediated adhesion, ERK1/2 activation and proliferation of Rap1E63-Tg CD4+ T cells were defective. Rap1E63-Tg T cells primed in vivo and restimulated with specific Ag in vitro, exhibited reduced proliferation and produced reduced levels of IL-2. Rap1E63-Tg mice had severely deficient T cell-dependent B cell responses, as determined by impaired Ig class switching. Rap1E63-Tg mice had an increased fraction of CD4+CD103+ regulatory T cells (Treg), which exhibited enhanced suppressive efficiency as compared with CD4+CD103+ Treg from normal littermate control mice. Depletion of CD103+ Treg significantly restored the impaired responses of Rap1E63-Tg CD4+ T cells. Thus Rap1-GTP is a negative regulator of Th cell responses and one mechanism responsible for this effect involves the increase of CD103+ Treg cell fraction. Our results show that Rap1-GTP promotes the generation of CD103+ Treg and may have significant implications in the development of strategies for in vitro generation of Treg for the purpose of novel immunotherapeutic approaches geared toward tolerance induction.

Intrinsic and Extrinsic Regulation of T Lymphocyte Quiescence

The outcome of an immune response is dependent on the interplay and complex interactions of positive (stimulatory) and negative (inhibitory) pathways and a major goal of modern immunology is to dissect these physiologic interactions in order to apply these physiologic mechanisms therapeutically. The balance of stimulatory and inhibitory signals is critical for maximizing the ability of the adoptive immune response to defend the host while maintaining immunologic tolerance and preventing autoimmunity. Cellular quiescence is a state characterized by decreased cell size and metabolic activity. The quiescent state of unstimulated T lymphocytes is thought to be due to the lack of activation signals. However, recent studies have shown that quiescence in lymphocytes is not a default state, but an actively maintained gene program. This program regulates intrinsic expression of quiescence factors in T lymphocytes. In addition to intrinsic mechanisms regulating T cell quiescence, CD4 + CD25 + regulatory T cells (Treg) naturally arising in the thymus, engage in the maintenance of immunological self-tolerance by preventing autoimmunity in vivo in a non cell-autonomous manner. Although there is still only a rudimentary knowledge of the molecular mechanisms that govern the activity of the intrinsic quiescence factors and the development of Treg, it is now clear that immune quiescence is regulated by constitutively ongoing active mechanisms.

Physiologic regulation of central and peripheral T cell tolerance: lessons for therapeutic applications

Immunologic tolerance is a state of unresponsiveness that is specific for a particular antigen. The immune system has an extraordinary potential for making T cell and B cell that recognize and neutralize any chemical entity and microbe entering the body. Certainly, some of these T cells and B cells recognize self-components; therefore, cellular mechanisms have evolved to control the activity of these self-reactive cells and achieve immunological self-tolerance. The most important in vivo biological significance of mechanisms regulating self-tolerance is to prevent the immune system from mounting an attack against the host’s own tissues resulting in autoimmunity. This review summarizes recent developments in our understanding of T-helper cell tolerance and discusses how the new findings can be exploited to prevent and treat autoimmune diseases, allergy, cancer, and chronic infection, or establish donor-specific transplantation tolerance.