Xiao-Ping Zhong

Clinical efficacy and immune regulation with peanut oral immunotherapy

BACKGROUND Oral immunotherapy (OIT) has been thought to induce clinical desensitization to allergenic foods, but trials coupling the clinical response and immunologic effects of peanut OIT have not been reported. OBJECTIVE The study objective was to investigate the clinical efficacy and immunologic changes associated with OIT. METHODS Children with peanut allergy underwent an OIT protocol including initial day escalation, buildup, and maintenance phases, and then oral food challenge. Clinical response and immunologic changes were evaluated. RESULTS Of 29 subjects who completed the protocol, 27 ingested 3.9 g peanut protein during food challenge. Most symptoms noted during OIT resolved spontaneously or with antihistamines. By 6 months, titrated skin prick tests and activation of basophils significantly declined. Peanut-specific IgE decreased by 12 to 18 months, whereas IgG(4) increased significantly. Serum factors inhibited IgE-peanut complex formation in an IgE-facilitated allergen binding assay. Secretion of IL-10, IL-5, IFN-gamma, and TNF-alpha from PBMCs increased over a period of 6 to 12 months. Peanut-specific forkhead box protein 3 T cells increased until 12 months and decreased thereafter. In addition, T-cell microarrays showed downregulation of genes in apoptotic pathways. CONCLUSION Oral immunotherapy induces clinical desensitization to peanut, with significant longer-term humoral and cellular changes. Microarray data suggest a novel role for apoptosis in OIT.

Disruption of diacylglycerol metabolism impairs the induction of T cell anergy

Anergic T cells have altered diacylglycerol metabolism, but whether that altered metabolism has a causative function in the induction of T cell anergy is not apparent. To test the importance of diacylglycerol metabolism in T cell anergy, we manipulated diacylglycerol kinases (DGKs), which are enzymes that terminate diacylglycerol-dependent signaling. Overexpression of DGK-α resulted in a defect in T cell receptor signaling that is characteristic of anergy. We generated DGK-α-deficient mice and found that DGK-α-deficient T cells had more diacylglycerol-dependent T cell receptor signaling. In vivo anergy induction was impaired in DGK-α-deficient mice. When stimulated in anergy-producing conditions, T cells lacking DGK-α or DGK-ζ proliferated and produced interleukin 2. Pharmacological inhibition of DGK-α activity in DGK-ζ-deficient T cells that received an anergizing stimulus proliferated similarly to wild-type T cells that received CD28 costimulation and prevented anergy induction. Our findings suggest that regulation of diacylglycerol metabolism is critical in determining whether activation or anergy ensues after T cell receptor stimulation.

Enhanced T cell responses due to diacylglycerol kinase ζ deficiency

Much is known about how T cell receptor (TCR) engagement leads to T cell activation; however, the mechanisms terminating TCR signaling remain less clear. Diacylglycerol, generated after TCR ligation, is essential in T cells. Its function must be controlled tightly to maintain normal T cell homeostasis. Previous studies have shown that diacylglycerol kinase ζ (DGKζ), which converts diacylglycerol to phosphatidic acid, can inhibit TCR signaling. Here we show that DGKζ-deficient T cells are hyperresponsive to TCR stimulation both ex vivo and in vivo. Furthermore, DGKζ-deficient mice mounted a more robust immune response to lymphocytic choriomeningitis virus infection than did wild-type mice. These results demonstrate the importance of DGKζ as a physiological negative regulator of TCR signaling and T cell activation.

The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy

Background: Diacylglycerol kinases (DGKs) synthesize phosphatidic acid (PA), and PA can activate growth-regulatory mTOR signaling. Results: The ζ isoform of DGK is necessary for a mechanically induced increase in PA-mTOR signaling, and overexpression of DGKζ induces skeletal muscle hypertrophy. Conclusion: PA synthesized by DGKζ regulates the mechanical activation of mTOR signaling and hypertrophy. Significance: DGKζ is a potential target for treating muscle atrophy/wasting. The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

Regulation of T-cell survival and mitochondrial homeostasis by TSC1.

The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and metabolism. It associates with multiple proteins and forms two distinct signaling complexes, mTORC1 and mTORC2. Accumulating evidence has revealed critical roles for intact mTOR signaling during T‐cell activation and responses to microbial infection. However, the importance of mTOR regulation in T cells has yet to be explored. The TSC1/TSC2 complex has been shown to inhibit mTORC1 signaling in cell line models. We show here that deletion of TSC1 in the murine T‐cell lineage results in a dramatic reduction of the peripheral T‐cell pool, correlating with increased cell death. While mTORC1 is constitutively activated, mTORC2 signaling, reflected by Akt phosphorylation and activity, is decreased in TSC1‐deficient T cells. Furthermore, TSC1‐deficient T cells contain elevated reactive oxygen species (ROS) and exhibit decreased mitochondrial content and membrane potential, which is correlated with the activation of the intrinsic death pathway. Overall, our results demonstrate that TSC1 differentially regulates mTORC1 and mTORC2 activity, promotes T‐cell survival, and is critical for normal mitochondrial homeostasis in T cells.

Negative regulation of mTOR activation by diacylglycerol kinases

The engagement of TCR induces T-cell activation, which initiates multiple characteristic changes such as increase in cell size, cell division, and the production of cytokines and other effector molecules. The mammalian target of rapamycin (mTOR) regulates protein synthesis, transcription, cell survival, and autophagy. Critical roles of mTOR in T-cell activation and effector/memory differentiation have been revealed using chemical inhibitors or by genetic ablation of mTOR in T cells. However, the connection between mTOR signaling and other signaling cascades downstream of TCR is unclear. We demonstrate that diacylglycerol (DAG) and TCR engagement activate signaling in both mTOR complexes 1 and 2 through the activation of the Ras-mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (Mek1/2)-extracellular signal-regulated kinase 1/2 (Erk1/2)-activator protein 1 (AP-1), known collectively as the Ras-Mek1/2-Erk1/2-AP-1 pathway. Deficiency of RasGRP1 or inhibition of Mek1/2 activity drastically decreases TCR-induced mTOR activation, whereas constitutively active Ras or Mek1 promotes mTOR activation. Although constitutively active Akt promotes TCR-induced mTOR activation, such activation is attenuated by Mek1/2 inhibition. We demonstrated further that DAG kinases (DGKs) α and ζ, which terminate DAG-mediated signaling, synergistically inhibit TCR-induced mTOR activation by inhibiting the Ras-Mek1/2-Erk/12 pathway. These observations provide novel insights into the regulation of mTOR activation.

Diacylglycerol kinases in immune cell function and self-tolerance.

Summary: Both diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers involved in signal transduction from many immune cell receptors and can be generated and metabolized through multiple mechanisms. Recent studies indicate that diacylglycerol kinases (DGKs), the enzymes that catalyze phosphorylation of DAG to produce PA, play critical roles in regulating the functions of multiple immune cell lineages. In T cells, two DGK isoforms, α and ζ, inhibit DAG‐mediated signaling following T‐cell receptor engagement and prevent T‐cell hyperactivation. DGK α and ζ synergistically promote T‐cell anergy and are critical for T‐cell tolerence. In mast cells, DGKζ plays differential roles in their activation by promoting degranulation but attenuating cytokine production following engagement of the high affinity receptor for immunoglobulin E. In dendritic cells and macrophages, DGKζ positively regulates Toll‐like receptor‐induced proinflammatory cytokine production through its product PA and is critical for host defense against Toxoplama gondii infection. These studies demonstrate pivotal roles of DGKs in regulating immune cell function by acting both as signal terminator and initiator.

Developmental regulation of V(D)J recombination at the TCR a/5 locus

Summary: The T‐cell receptor (TCR) α/σ locus includes a large number of V, D, J and C gene segments that are used lo produce functional TCR 8 and TCR a chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T‐lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis‐acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR α/σ locus imparts developmental control to VDJσ and VJα rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR α/σ locus, highly localized and region‐specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

Synergistic control of T cell development and tumor suppression by diacylglycerol kinase α and ζ

Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK α and ζ synergistically promote T cell maturation in the thymus. Absence of both DGKα and ζ (DGKα−/−ζ−/−) results in a severe decrease in the number of CD4+CD8− and CD4−CD8+ single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKα−/−ζ−/− mice. The developmental blockage in DGKα−/−ζ−/− mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKzeta initiates RhoGDI release and Rac1 activation. In DGKzeta-deficient fibroblasts PAK1 phosphorylation and Rac1-RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKzeta, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKzeta stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKzeta, and PA to the activation of Rac1: the PA generated by DGKzeta activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.