The phosphatidylinositol-transfer protein Nir3 promotes PI(4,5)P2 replenishment in response to TCR signaling during T cell development and survival.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C-γ (PLCγ1) represents a critical step in T cell antigen receptor (TCR) signaling and subsequent thymocyte and T cell responses. PIP2 replenishment following its depletion in the plasma membrane (PM) is dependent on delivery of its precursor phosphatidylinositol (PI) from the endoplasmic reticulum (ER) to the PM. We show that a PI transfer protein (PITP), Nir3 (Pitpnm2), promotes PIP2 replenishment following TCR stimulation and is important for T cell development. In Nir3-/- T lineage cells, the PIP2 replenishment following TCR stimulation is slower. Nir3 deficiency attenuates calcium mobilization in double-positive (DP) thymocytes in response to weak TCR stimulation. This impaired TCR signaling leads to attenuated thymocyte development at TCRß selection and positive selection as well as diminished mature T cell fitness in Nir3-/- mice. This study highlights the importance of PIP2 replenishment mediated by PITPs at ER-PM junctions during TCR signaling.

Pharmacological Inhibition of MALT1 Ameliorates Autoimmune Pathogenesis and Can Be Uncoupled From Effects on Regulatory T-Cells.

MALT1 forms part of a central signaling node downstream of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, across a broad range of immune cell subsets, and regulates NF-κB driven transcriptional responses via dual scaffolding-protease activity. Allosteric inhibition of MALT1 activity has demonstrated benefit in animal models of inflammation. However, development of MALT1 inhibitors to treat autoimmune and inflammatory diseases (A&ID) has been hindered by reports linking MALT1 inhibition and genetic loss-of-function to reductions in regulatory T-cell (Treg) numbers and development of auto-inflammatory syndromes. Using an allosteric MALT1 inhibitor, we investigated the consequence of pharmacological inhibition of MALT1 on proinflammatory cells compared to regulatory T-cells. Consistent with its known role in ITAM-driven responses, MALT1 inhibition suppressed proinflammatory cytokine production from activated human T-cells and monocyte-derived macrophages, and attenuated B-cell proliferation. Oral administration of a MALT1 inhibitor reduced disease severity and synovial cytokine production in a rat collagen-induced arthritis model. Interestingly, reduction in splenic Treg numbers was less pronounced in the context of inflammation compared with naïve animals. Additionally, in the context of the disease model, we observed an uncoupling of anti-inflammatory effects of MALT1 inhibition from Treg reduction, with lower systemic concentrations of inhibitor needed to reduce disease severity compared to that required to reduce Treg numbers. MALT1 inhibition did not affect suppressive function of human Tregs in vitro. These data indicate that anti-inflammatory efficacy can be achieved with MALT1 inhibition without impacting the number or function of Tregs, further supporting the potential of MALT1 inhibition in the treatment of autoimmune disease.

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis.

Successful metastasis requires the co-evolution of stromal and cancer cells. We used stable isotope labeling of amino acids in cell culture coupled with quantitative, label-free phosphoproteomics to study the bidirectional signaling in ovarian cancer cells and human-derived, cancer-associated fibroblasts (CAFs) after co-culture. In cancer cells, the interaction with CAFs supported glycogenolysis under normoxic conditions and induced phosphorylation and activation of phosphoglucomutase 1, an enzyme involved in glycogen metabolism. Glycogen was funneled into glycolysis, leading to increased proliferation, invasion, and metastasis of cancer cells co-cultured with human CAFs. Glycogen mobilization in cancer cells was dependent on p38α MAPK activation in CAFs. In vivo, deletion of p38α in CAFs and glycogen phosphorylase inhibition in cancer cells reduced metastasis, suggesting that glycogen is an energy source used by cancer cells to facilitate metastatic tumor growth.

CD28 Costimulation: From Mechanism to Therapy.

Ligation of the CD28 receptor on T cells provides a critical second signal alongside T cell receptor (TCR) ligation for naive T cell activation. Here, we discuss the expression, structure, and biochemistry of CD28 and its ligands. CD28 signals play a key role in many T cell processes, including cytoskeletal remodeling, production of cytokines, survival, and differentiation. CD28 ligation leads to unique epigenetic, transcriptional, and post-translational changes in T cells that cannot be recapitulated by TCR ligation alone. We discuss the function of CD28 and its ligands in both effector and regulatory T cells. CD28 is critical for regulatory T cell survival and the maintenance of immune homeostasis. We outline the roles that CD28 and its family members play in human disease and we review the clinical efficacy of drugs that block CD28 ligands. Despite the centrality of CD28 and its family members and ligands to immune function, many aspects of CD28 biology remain unclear. Translation of a basic understanding of CD28 function into immunomodulatory therapeutics has been uneven, with both successes and failures. Such real-world results might stem from multiple factors, including complex receptor-ligand interactions among CD28 family members, differences between the mouse and human CD28 families, and cell-type specific roles of CD28 family members.

Vav1 Regulates T-Cell Activation through a Feedback Mechanism and Crosstalk between the T-Cell Receptor and CD28.

Vav1, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T-cell receptor (TCR) signaling cascade is tyrosine phosphorylated rapidly in response to T-cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca(2+) responses, and actin cytoskeleton regulation; however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck and ZAP-70, is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways.

Protein networks and activation of lymphocytes.

The signal transduction pathways initiated by lymphocyte activation play a critical role in regulating host immunity. High-resolution mass spectrometry has accelerated the investigation of these complex and dynamic pathways by enabling the qualitative and quantitative investigation of thousands of proteins and phosphoproteins simultaneously. In addition, the unbiased and wide-scale identification of protein-protein interaction networks and protein kinase substrates in lymphocyte signaling pathways can be achieved by mass spectrometry-based approaches. Critically, the integration of these discovery-driven strategies with single-cell analysis using mass cytometry can facilitate the understanding of complex signaling phenotypes in distinct immunophenotypes.

Mitogen-activated protein kinase phosphatase 3 (MKP-3)-deficient mice are resistant to diet-induced obesity.

Mitogen-activated protein kinase phosphatase 3 (MKP-3) is a negative regulator of extracellular signal-related kinase signaling. Our laboratory recently demonstrated that MKP-3 plays an important role in obesity-related hyperglycemia by promoting hepatic glucose output. This study shows that MKP-3 deficiency attenuates body weight gain induced by a high-fat diet (HFD) and protects mice from developing obesity-related hepatosteatosis. Triglyceride (TG) contents are dramatically decreased in the liver of MKP-3(-/-) mice fed an HFD compared with wild-type (WT) controls. The absence of MKP-3 also reduces adiposity, possibly by repressing adipocyte differentiation. In addition, MKP-3(-/-) mice display increased energy expenditure, enhanced peripheral glucose disposal, and improved systemic insulin sensitivity. We performed global phosphoproteomic studies to search for downstream mediators of MKP-3 action in liver lipid metabolism. Our results revealed that MKP-3 deficiency increases the phosphorylation of histone deacetylase (HDAC) 1 on serine 393 by 3.3-fold and HDAC2 on serine 394 by 2.33-fold. Activities of HDAC1 and 2 are increased in the livers of MKP-3(-/-) mice fed an HFD. Reduction of HDAC1/2 activities is sufficient to restore TG content of MKP-3(-/-) primary hepatocytes to a level similar to that in WT cells.

ERK positive feedback regulates a widespread network of tyrosine phosphorylation sites across canonical T cell signaling and actin cytoskeletal proteins in Jurkat T cells.

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser(59) Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr(598) of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK's role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.

Identification of sucrose non-fermenting-related kinase (SNRK) as a suppressor of adipocyte inflammation.

In this study, the role of sucrose non-fermenting-related kinase (SNRK) in white adipocyte biology was investigated. SNRK is abundantly expressed in adipose tissue, and the expression level is decreased in obese mice. SNRK expression is repressed by inflammatory signals but increased by insulin sensitizer in cultured adipocytes. In vivo, adipose tissue SNRK expression can be decreased by lipid injection but enhanced by macrophage ablation. Knocking down SNRK in cultured adipocytes activates both JNK and IKKß pathways as well as promotes lipolysis. Insulin-stimulated Akt phosphorylation and glucose uptake are impaired in SNRK knockdown adipocytes. Phosphoproteomic analysis with SNRK knockdown adipocytes revealed significantly decreased phosphorylation of 49 proteins by 25% or more, which are involved in various aspects of adipocyte function with a clear indication of attenuated mTORC1 signaling. Phosphorylation of 43 proteins is significantly increased by onefold or higher, among which several proteins are known to be involved in inflammatory pathways. The inflammatory responses in SNRK knockdown adipocytes can be partially attributable to defective mTORC1 signaling, since rapamycin treatment activates IKKß and induces lipolysis in adipocytes. In summary, SNRK may act as a suppressor of adipocyte inflammation and its presence is necessary for maintaining normal adipocyte function.