Metabolic regulation of T lymphocytes.

T cell activation leads to dramatic shifts in cell metabolism to protect against pathogens and to orchestrate the action of other immune cells. Quiescent T cells require predominantly ATP-generating processes, whereas proliferating effector T cells require high metabolic flux through growth-promoting pathways. Further, functionally distinct T cell subsets require distinct energetic and biosynthetic pathways to support their specific functional needs. Pathways that control immune cell function and metabolism are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell functions. As a result of these findings, cell metabolism is now appreciated as a key regulator of T cell function specification and fate. This review discusses the role of cellular metabolism in T cell development, activation, differentiation, and function to highlight the clinical relevance and opportunities for therapeutic interventions that may be used to disrupt immune pathogenesis.

Slow phosphorylation of a tyrosine residue in LAT optimizes T cell ligand discrimination.

Self-non-self discrimination is central to T cell-mediated immunity. The kinetic proofreading model can explain T cell antigen receptor (TCR) ligand discrimination; however, the rate-limiting steps have not been identified. Here, we show that tyrosine phosphorylation of the T cell adapter protein LAT at position Y132 is a critical kinetic bottleneck for ligand discrimination. LAT phosphorylation at Y132, mediated by the kinase ZAP-70, leads to the recruitment and activation of phospholipase C-γ1 (PLC-γ1), an important effector molecule for T cell activation. The slow phosphorylation of Y132, relative to other phosphosites on LAT, is governed by a preceding glycine residue (G131) but can be accelerated by substituting this glycine with aspartate or glutamate. Acceleration of Y132 phosphorylation increases the speed and magnitude of PLC-γ1 activation and enhances T cell sensitivity to weaker stimuli, including weak agonists and self-peptides. These observations suggest that the slow phosphorylation of Y132 acts as a proofreading step to facilitate T cell ligand discrimination.

A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity.

14-3-3 proteins regulate biological processes by binding to phosphorylated serine or phosphorylated threonine motifs of cellular proteins. Among the 14-3-3 proteins, 14-3-3ɛ serves a crucial function in antiviral immunity by mediating the cytosol-to-mitochondrial membrane translocation of the pathogen sensor RIG-I. Here we found that the NS3 protein of dengue virus (DV) bound to 14-3-3ɛ and prevented translocation of RIG-I to the adaptor MAVS and thereby blocked antiviral signaling. Intriguingly, a highly conserved phosphomimetic RxEP motif in NS3 was essential for the binding of 14-3-3ɛ. A recombinant mutant DV deficient in binding to 14-3-3ɛ showed impairment in antagonism of RIG-I and elicited a markedly augmented innate immune response and enhanced T cell activation. Our work reveals a novel phosphomimetic-based mechanism for viral antagonism of 14-3-3-mediated immunity, which might guide the rational design of therapeutics.

Phosphatase PP2A is requisite for the function of regulatory T cells.

Homeostasis of the immune system depends on the proper function of regulatory T cells (T(reg) cells). Compromised suppressive activity of T(reg) cells leads to autoimmune disease and graft rejection and promotes anti-tumor immunity. Here we report a previously unrecognized requirement for the serine-threonine phosphatase PP2A in the function of T(reg) cells. T(reg) cells exhibited high PP2A activity, and T(reg) cell-specific ablation of the PP2A complex resulted in a severe, multi-organ, lymphoproliferative autoimmune disorder. Mass spectrometry revealed that PP2A associated with components of the mTOR metabolic-checkpoint kinase pathway and suppressed the activity of the mTORC1 complex. In the absence of PP2A, T(reg) cells altered their metabolic and cytokine profile and were unable to suppress effector immune responses. Therefore, PP2A is required for the function of T(reg) cells and the prevention of autoimmunity.

K33-linked polyubiquitination of Zap70 by Nrdp1 controls CD8(+) T cell activation.

The key molecular mechanisms that control signaling via T cell antigen receptors (TCRs) remain to be fully elucidated. Here we found that Nrdp1, a ring finger-type E3 ligase, mediated Lys33 (K33)-linked polyubiquitination of the signaling kinase Zap70 and promoted the dephosphorylation of Zap70 by the acidic phosphatase-like proteins Sts1 and Sts2 and thereby terminated early TCR signaling in CD8(+) T cells. Nrdp1 deficiency significantly promoted the activation of naive CD8(+) T cells but not that of naive CD4(+) T cells after engagement of the TCR. Nrdp1 interacted with Zap70 and with Sts1 and Sts2 and connected K33 linkage of Zap70 to Sts1- and Sts2-mediated dephosphorylation. Our study suggests that Nrdp1 terminates early TCR signaling by inactivating Zap70 and provides new mechanistic insights into the non-proteolytic regulation of TCR signaling by E3 ligases.

The phosphatase DUSP2 controls the activity of the transcription activator STAT3 and regulates TH17 differentiation.

Deregulation of the TH17 subset of helper T cells is closely linked with immunological disorders and inflammatory diseases. However, the mechanism by which TH17 cells are regulated remains elusive. Here we found that the phosphatase DUSP2 (PAC1) negatively regulated the development of TH17 cells. DUSP2 was directly associated with the signal transducer and transcription activator STAT3 and attenuated its activity through dephosphorylation of STAT3 at Tyr705 and Ser727. DUSP2-deficient mice exhibited severe susceptibility to experimental colitis, with enhanced differentiation of TH17 cells and secretion of proinflammatory cytokines. In clinical patients with ulcerative colitis, DUSP2 was downregulated by DNA methylation and was not induced during T cell activation. Our data demonstrate that DUSP2 is a true STAT3 phosphatase that modulates the development of TH17 cells in the autoimmune response and inflammation.

The kinase p38 activated by the metabolic regulator AMPK and scaffold TAB1 drives the senescence of human T cells.

In T lymphocytes, the mitogen-activated protein kinase (MAPK) p38 regulates pleiotropic functions and is activated by canonical MAPK signaling or the alternative activation pathway downstream of the T cell antigen receptor (TCR). Here we found that senescent human T cells lacked the canonical and alternative pathways for the activation of p38 but spontaneously engaged the metabolic master regulator AMPK to trigger recruitment of p38 to the scaffold protein TAB1, which caused autophosphorylation of p38. Signaling via this pathway inhibited telomerase activity, T cell proliferation and the expression of key components of the TCR signalosome. Our findings identify a previously unrecognized mode for the activation of p38 in T cells driven by intracellular changes such as low-nutrient and DNA-damage signaling (an 'intrasensory' pathway). The proliferative defect of senescent T cells was reversed by blockade of AMPK-TAB1-dependent activation of p38.

The transcription factor IRF1 dictates the IL-21-dependent anticancer functions of TH9 cells.

The TH9 subset of helper T cells was initially shown to contribute to the induction of autoimmune and allergic diseases, but subsequent evidence has suggested that these cells also exert antitumor activities. However, the molecular events that account for their effector properties are elusive. Here we found that the transcription factor IRF1 enhanced the effector function of TH9 cells and dictated their anticancer properties. Under TH9-skewing conditions, interleukin 1ß (IL-1ß) induced phosphorylation of the transcription factor STAT1 and subsequent expression of IRF1, which bound to the promoters of Il9 and Il21 and enhanced secretion of the cytokines IL-9 and IL-21 from TH9 cells. Furthermore, IL-1ß-induced TH9 cells exerted potent anticancer functions in an IRF1- and IL-21-dependent manner. Our findings thus identify IRF1 as a target for controlling the function of TH9 cells.

T cell regulation mediated by interaction of soluble CD52 with the inhibitory receptor Siglec-10.

Functionally diverse T cell populations interact to maintain homeostasis of the immune system. We found that human and mouse antigen-activated T cells with high expression of the lymphocyte surface marker CD52 suppressed other T cells. CD52(hi)CD4(+) T cells were distinct from CD4(+)CD25(+)Foxp3(+) regulatory T cells. Their suppression was mediated by soluble CD52 released by phospholipase C. Soluble CD52 bound to the inhibitory receptor Siglec-10 and impaired phosphorylation of the T cell receptor-associated kinases Lck and Zap70 and T cell activation. Humans with type 1 diabetes had a lower frequency and diminished function of CD52(hi)CD4(+) T cells responsive to the autoantigen GAD65. In diabetes-prone mice of the nonobese diabetic (NOD) strain, transfer of lymphocyte populations depleted of CD52(hi) cells resulted in a substantially accelerated onset of diabetes. Our studies identify a ligand-receptor mechanism of T cell regulation that may protect humans and mice from autoimmune disease.

The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Phosphorylation of the adaptor ASC acts as a molecular switch that controls the formation of speck-like aggregates and inflammasome activity.

The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks.

The Cytokine GM-CSF Drives the Inflammatory Signature of CCR2+ Monocytes and Licenses Autoimmunity.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has emerged as a crucial cytokine produced by auto-reactive T helper (Th) cells that initiate tissue inflammation. Multiple cell types can sense GM-CSF, but the identity of the pathogenic GM-CSF-responsive cells is unclear. By using conditional gene targeting, we systematically deleted the GM-CSF receptor (Csf2rb) in specific subpopulations throughout the myeloid lineages. Experimental autoimmune encephalomyelitis (EAE) progressed normally when either classical dendritic cells (cDCs) or neutrophils lacked GM-CSF responsiveness. The development of tissue-invading monocyte-derived dendritic cells (moDCs) was also unperturbed upon Csf2rb deletion. Instead, deletion of Csf2rb in CCR2(+)Ly6C(hi) monocytes phenocopied the EAE resistance seen in complete Csf2rb-deficient mice. High-dimensional analysis of tissue-infiltrating moDCs revealed that GM-CSF initiates a combination of inflammatory mechanisms. These results indicate that GM-CSF signaling controls a pathogenic expression signature in CCR2(+)Ly6C(hi) monocytes and their progeny, which was essential for tissue damage.

Functional monovalency amplifies the pathogenicity of anti-MuSK IgG4 in myasthenia gravis.

Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed "Fab-arm exchange" in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severe myasthenic muscle weakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects on MuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders.

Altered increase in STAT1 expression and phosphorylation in severe COVID-19.

The interferon pathway, a key antiviral defense mechanism, is being considered as a therapeutic target in COVID-19. Both, substitution of interferon and JAK/STAT inhibition to limit cytokine storms have been proposed. However, little is known about possible abnormalities in STAT signaling in immune cells during SARS-CoV-2 infection. We investigated downstream targets of interferon signaling, including STAT1, STAT2, pSTAT1 and 2, and IRF1, 7 and 9 by flow cytometry in 30 patients with COVID-19, 17 with mild, and 13 with severe infection. We report upregulation of STAT1 and IRF9 in mild and severe COVID-19 cases, which correlated with the IFN-signature assessed by Siglec-1 (CD169) expression on peripheral monocytes. Interestingly, Siglec-1 and STAT1 in CD14+ monocytes and plasmablasts showed lower expression among severe cases compared to mild cases. Contrary to the baseline STAT1 expression, the phosphorylation of STAT1 was enhanced in severe COVID-19 cases, indicating a dysbalanced JAK/STAT signaling that fails to induce transcription of interferon stimulated response elements (ISRE). This abnormality persisted after IFN-α and IFN-γ stimulation of PBMCs from patients with severe COVID-19. Data suggest impaired STAT1 transcriptional upregulation among severely infected patients may represent a potential predictive biomarker and would allow stratification of patients for certain interferon-pathway targeted treatments.

STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development.

Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca(2+)-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide-exchange factor RasGRP and depended on the concentration of the Ca(2+) sensor STIM1, which controls the magnitude of Ca(2+) entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca(2+)-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells.

Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity.

Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but mechanisms for its activation and regulation remain unknown. We found here that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1; this resulted in activation of IRAK1 and facilitated its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-deficient cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant activation of IRAK1 and type I interferons in various immune diseases, controlling IRAK1 activation via inhibition of Pin1 may represent a useful therapeutic approach.

Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments.

Binding of antigen to the B cell antigen receptor (BCR) triggers both BCR signaling and endocytosis. How endocytosis regulates BCR signaling remains unknown. Here we report that BCR signaling was not extinguished by endocytosis of BCRs; instead, BCR signaling initiated at the plasma membrane continued as the BCR trafficked intracellularly with the sequential phosphorylation of kinases. Blocking the endocytosis of BCRs resulted in the recruitment of both proximal and downstream kinases to the plasma membrane, where mitogen-activated protein kinases (MAPKs) were hyperphosphorylated and the kinase Akt and its downstream target Foxo were hypophosphorylated, which led to the dysregulation of gene transcription controlled by these pathways. Thus, the cellular location of the BCR serves to compartmentalize kinase activation to regulate the outcome of signaling.

Toxoplasma gondii ROP18I inhibits host innate immunity through cGAS-STING signaling.

Toxoplasma gondii is an opportunistic protozoan, which widely infects humans and other warm-blooded animals. The type I interferon (IFN) such as IFN-α/ß is involved in cGAS-STING signaling to resist T. gondii infection. We found in RAW264.7 cells, that T. gondii virulence factor TgROP18I , inhibited IFN-ß production through interacting with interferon regulatory factor 3 (IRF3). Besides, TgROP18I interacted with p62 and Tumor Necrotic Factor Receptor Associated Factor 6 (TRAF6), which resulted in the inhibition of TRAF6-p62 interaction, and phosphorylation of p62. Furthermore, TgROP18I restricted the recruitment of ubiquitin, p62 and microtubule-associated protein light chain 3 (LC3) to the parasitophorous vacuole membrane (PVM) in IFN-γ-stimulated murine cell line L929 cells. In IFN-γ-stimulated human cells, TgROP18I restricted the decoration of PVM with ubiquitin, p62, and LC3, and bound with TRAF2, TRAF6, and p62, respectively. As a result, TgROP18I led to a successful parasitic replication in murine and human cells. Collectively, our study revealed the function of TgROP18I in suppressing host type I interferon responses in T. gondii infection for parasitic immune escape.

Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase-mediated antiviral response.

Type I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.