Lipids are small hydrophobic molecules that perform a variety of cellular functions. Though best known for their role in maintaining cell structure and storing energy, lipids have gained in importance over the past few decades as signaling mediators (1, 2). While lipids that participate in signaling are thought to be much less abundant in the cell as compared to structural lipids, their levels vary dynamically in response to external signals.

In this review, we discuss the signaling functions of two key lipid second messengers, diacylglycerol (DAG) and phosphatidic acid (PA), in the context of T cell development and function. DAGs are esters of glycerol in which two of its hydroxyl groups are esterified with long-chain fatty acids. One manner of PA generation in cells is via phosphorylation of the free hydroxyl group in DAG by a family of enzymes called diacylglycerol kinases (DGKs) (3, 4). DGKs therefore act as molecular switches that simultaneously turn off DAG-mediated signaling and turn on PA-mediated signals.

While all 10 mammalian DGK isoforms contain a kinase domain and at least two cysteine-rich C1 domains, they can be grouped into five types based on the homology of their other structural features. α and ζ are the major isoforms expressed in T cells (5). DGKα is a type I DGK and contains an N-terminal recoverin homology domain and two Ca2⁺-binding EF hands. DGKζ is a type IV DGK and contains a myristoylated alanine rich C kinase substrate (MARCKS) motif, four ankyrin repeats and a C-terminal PDZ-binding domain. DGKζ undergoes alternative splicing, producing a 130 kDa ζ2 isoform that is highly expressed in immature thymocytes and a 115 kDa ζ1 isoform that is predominant in mature thymocytes and peripheral T cells (6). Functional differences between the two splice variants remain unclear.

Here, we begin by discussing the various effector molecules that transduce DAG-mediated signals and PA-mediated signals. We then switch gears to review our current understanding of the role of DGKs in T cell development and function with an emphasis on recent advances that have revealed hitherto unknown functions for these enzymes. The roles of DGK activity in other immune cell lineages are also discussed briefly.

Several enzymes contribute toward DAG production upon receptor stimulation in immune cells (7) (Figure 1). Phosphatidylinositol-dependent phospholipases hydrolyze membrane phosphatidylinositol bisphosphate (PIP₂) to DAG and inositol triphosphate, and phosphatidylcholine (PC)-dependent phospholipases hydrolyze PC to DAG and phosphoryl choline. In addition, sphingomyelin synthase generates DAG and sphingomyelin from PC and ceramide, while PA phosphatases such as lipins dephosphorylate PA to DAG. On the other hand, DAG is primarily removed by the activity of DGKs, which catalyze its phosphorylation to PA. DAG can recruit a variety of downstream effector molecules through their C1 domains, and thereby trigger multiple signaling pathways.

The protein kinase C (PKC) family of serine/threonine kinases consists of 10 isozymes that are activated by a number of distinct mechanisms (8, 9). Upon engagement of the TCR, production of DAG by activated PLCγ1 recruits PKCθ to the plasma membrane in T cells. Co-stimulation via CD28 also plays an important role in the recruitment and spatial segregation of PKCθ at the immunological synapse (10, 11). Activation of PKCθ is indispensable for TCR-mediated NF-κB activation in mature T cells (12, 13). A role for PKCθ has also been identified in an array of key processes (14) including invariant NKT (iNKT) cell development and activation (15, 16), T cell survival (17), IL-2 production (18), T_H2 responses (19, 20), and T_H17 responses (21). Thus, by recruiting PKCθ, DAG regulates multiple aspects of T cell function.

Another important protein that is brought to the plasma membrane by DAG upon TCR stimulation is Ras guanyl-releasing protein 1 (RasGRP1) (22). RasGRP1 is a member of the RasGRP family of factors that help activate Ras by exchanging bound GDP for GTP (23), and is selectively expressed in T cells and a few other cell types (24). RasGRP1 plays an essential role in thymocyte development (25), and is particularly required for the selection of thymocytes that express weakly selecting TCRs (26). RasGRP1 is not critical for the development of γδ T cells, but is important for their proliferation and IL-17 production (27). Other studies have shown that RasGRP1 may play a role in promoting antigen-induced CD8 cell expansion by lowering the threshold of T cell activation (28). RasGRP1 is therefore a key effector downstream of DAG that plays a multitude of critical roles in T cell development and function.

Members of the protein kinase D (PKD) family have been identified more recently as DAG effectors (29). A unique characteristic of PKDs is that they are targets of both DAG and DAG-activated PKCs (30). PKDs are thought to be activated by a multi-step mechanism. Upon cell stimulation, inactive PKD translocates from the cytosol to the plasma membrane in response to membrane DAG production, where it is then activated by novel PKCs that are also recruited to the membrane by DAG. During T cell development, PKD has been shown to exert different effects on VDJ recombination at the TCRβ locus and on CD4 and CD8 expression, based on its localization at the cytosol or plasma membrane (31). Subsequent work has revealed that regulation of thymocyte development by membrane-localized PKD, but not cytosol-localized PKD, is dependent on the GTPase RhoA (32). Bringing PKD to the plasma membrane therefore represents another important mechanism by which DAG regulates T cell development.

Munc13 proteins are mammalian homologs of the C. elegans Unc13 that is localized to pre-synaptic active zones of neurons and important for neurotransmitter secretion (33). Munc13-1, Munc13-2, and Munc13-3 isoforms bind to DAG with high affinity, and translocate to the plasma membrane in response to receptor stimulation. In the immune system, the Munc13-4 isoform which lacks a C1 domain (34, 35) has been shown to be important for granule maturation and exocytosis in NK cells and cytotoxic T lymphocytes (CTLs) (36, 37), and for phagosomal maturation and killing of intracellular bacteria in neutrophils (38, 39). Further studies are required to investigate parallel roles for DAG-binding Munc13 isoforms in NK cells, CTLs, neutrophils, and other types of immune cells. Over-expressing human Munc13 in opossum renal epithelial cell lines enhanced their susceptibility to apoptosis after DAG treatment, suggesting that Munc13 proteins may transduce apoptosis-inducing signals downstream of DAG in some cell types (40). The role of Munc13 proteins in T cell development and function remain poorly understood.

Chimaerins, a family of proteins that possess Rac-specific GTPase Activating Protein (GAP) activity, contain C1 domains that bear about 40 percent homology to those of PKCs (41, 42). Chimaerin isoforms α2 and β2 are expressed at different levels in T cells and have been shown to participate in TCR signaling (43). Results from the study suggest that these chimaerin isoforms translocate to the immunological synapse upon T cell activation, but in a manner that is independent of canonical DAG-binding by the C1 domains. Catalytic activity of these chimaerins was found to play an important role in inhibiting TCR-mediated NFAT activation. Other studies have delineated a role for β2 chimaerin in mediating DAG-dependent changes in T cell adhesion and chemotaxis (44). In this study, expression of GFP-tagged β2 chimaerin revealed that active Rac and C1-dependent PMA binding could co-operate to induce sustained localization of β2 chimaerin to the plasma membrane in Jurkat T cells. Overexpression of GFP-β2 chimaerin was associated with decreased CXCL12-induced static adhesion but enhanced CXCL12-induced migration. Chimaerin proteins therefore represent an important class of DAG effectors in T cells, but further work is required to dissect aspects of their function that are dependent on and independent of DAG-binding.

Diacylglycerol kinases and enzymes of the phospholipase D (PLDs) family act as key mediators of PA production in immune cells by phosphorylating DAG and hydrolyzing PC, respectively (7, 45) (Figure 1). On the other hand, enzymes such as lipins that possess PA phosphatase activity play a critical role in turning off PA-mediated signaling by removing PA (46). Cellular levels of PA have been shown to change dynamically in response to environmental stimuli, and a wealth of data has revealed a diverse array of functions for this bioactive lipid (47).

Phosphatidic acid performs its signaling functions primarily by associating with a growing number of effector molecules that include kinases such as mammalian/mechanistic target of rapamycin (mTOR) and phosphatidylinositol-4-phosphate 5-kinase (PIP5K), and phosphatases such as Src homology region 2 domain-containing phosphatase 1 (SHP1) (48). In mammalian HEK293 cells, treatment with exogenous PA was found to promote the phosphorylation of S6K1 and 4E-BP1, which are substrates of mTOR complex 1 (49). This phosphorylation was abolished by rapamycin, a bacterial macrolide that inhibits mTOR activity. Results from this study showed that mitogenic stimulation of HEK293 cells led to cellular PA accumulation within 5 min. Small unilamellar vesicles containing PA could also directly bind to the FKBP12-rapamycin binding (FRB) domain on mTOR in a manner that competed with FKBP12-rapamycin. Together, these results suggest a role for PA as a critical mediator that connects mitogenic stimuli to mTOR activation in mammalian cells (50). More recent work has revealed that PA may activate mTOR by a distinctive two-step mechanism that involves the displacement of the endogenous mTOR-inhibitor FKBP38 and allosteric activation of the kinase (51). On the other hand, studies with Rat2 fibroblasts suggest that PA may indirectly activate mTOR complex 1 via the MEK-ERK pathway (52). In this study, two structurally distinct MEK inhibitors were found to inhibit PA-mediated activation of mTOR complex 1. Other studies with human renal cell adenocarcinoma cell lines have shown that suppression of cellular PA production by treatment with a PLD inhibitor may inhibit the association of mTOR with both Raptor and Rictor, suggesting that PLD-derived PA may act as a key stabilizer of mTOR complexes 1 and 2 (53).

Diacylglycerol kinase-derived PA has also been shown to modulate mTOR complex 1 activity in HEK293 cell lines (54). Overexpression of DGKζ, but not DGKα, led to increased mTOR complex 1-dependent phosphorylation of S6K1 both in the presence and absence of serum, suggesting that DGKζ-derived PA may activate mTOR complex 1. DGKζ-induced S6K1 phosphorylation was dose-dependent, as cells expressing higher levels of DGKζ showed more intense S6K1 phosphorylation. DGKζ-mediated increase in S6K1 phosphorylation was abolished when a mutant form of mTOR (that lacked the capacity to bind PA) was co-expressed, suggesting that DGKζ-generated PA enhances S6K1 phosphorylation by directly binding to mTOR.

While it is tempting to extend these observations to suggest that PA may directly bind and activate mTOR in immune cells, only a few studies have rigorously examined the relationship between PA and mTOR in these cell types. RAW264.7 macrophage cell lines showed enhanced secretion of pro-inflammatory cytokines such as IL-6, TNF-α, and IL-1β in response to PA stimulation (55). This was specifically abrogated by rapamycin, suggesting that PA-induced production of pro-inflammatory cytokines in macrophages may occur through the mTOR complex 1 pathway. Treatment of mice with PA also led to an increase in serum levels of these cytokines, suggesting that PA may promote systemic inflammatory responses in a manner that is dependent on mTOR complex 1 activation.

As we discuss later in this review, a bevy of studies provide clear genetic evidence of critical roles for DGKα and DGKζ in multiple aspects of T cell maturation and function. However, much work remains to be done in understanding the role of DGK-derived and PLD-derived PA in activating mTOR complex 1 during T cell development and function.

Another key signaling pathway that is activated by PA is the Ras/MEK/ERK signaling cascade (56). The first line of evidence for PA-mediated modulation of MAP kinase signaling came from a study which showed that a C-terminal domain of Raf1 could bind to PA in a canine kidney cell line (57). Subsequent work showed that growth factor-mediated activation of PLD and the concomitant production of PA were directly linked to the activation of the Raf1-MAP kinase pathway (58). Based on findings that insulin-dependent PLD activation was critically dependent on ADP ribosylation factor (ARF) activation, the researchers used an ARF inhibitor (brefeldin A) to block PLD activation (59). Blocking PLD activity and PA production with brefeldin A blocked insulin-induced activation of the MAPK pathway in Rat-1 fibroblasts. Results from the study also showed that PA did not directly activate Raf1, but instead enhanced its recruitment to the plasma membrane to allow for its activation by Ras. Mutations in Raf1 that disrupted Raf1-PA interaction prevented plasma membrane recruitment of Raf1 in response to insulin stimulation (60).

Kinase suppressor of Ras 1 (KSR1) is a scaffolding protein that interacts with several components of the Raf-MEK-ERK cascade to co-ordinate the formation of localized multi-protein complexes that enable efficient signal transduction (61). More recent work has shown that KSR1 contains a sequence homologous to Raf1’s PA-binding domain that allows it to directly bind PA and be recruited to the plasma membrane in response to insulin stimulation in fibroblasts (62). The recruitment of KSR1 by PA was also found to be essential for its scaffolding function, as mutations in its PA-binding motif impaired insulin-induced MEK and ERK phosphorylation. Studies have shown that PA also recruits Sos, an activator of Ras, to the plasma membrane through its plextrin homology domain (63). PLD2-derived PA was found to play essential role in EGF-induced Ras activation, as mutations of Sos that impaired PA-binding prevented its membrane recruitment and subsequent activation of Ras.

Studies with Jurkat T cells suggest that PLD2 may act upstream of RasGRP1 upon TCR crosslinking and co-stimulation via the integrin lymphocyte function-associated antigen 1 (LFA1) (64). In this case, production of PA by PLD2 and subsequent dephosphorylation of PA to DAG by PA phosphatase were shown to be critical for plasma membrane Ras activation. Others have demonstrated a role for PLD-derived PA in ERK1/2 activation downstream of Galectin-8 engagement in Jurkat T cells (65). Galectins are a family of widely expressed carbohydrate-recognizing proteins, and Gal-8 has previously been shown to bind certain integrins on the T cell surface to provide co-stimulatory and proliferative signals (66). In this study, Gal-8 induced PA accumulation in Jurkat T cells within 15 min in a manner that was inhibited by treatment with a PLD inhibitor (1-butanol). Such PLD-inhibition also abrogated ERK1/2 phosphorylation, suggesting that PLD-derived PA may play an essential role in ERK activation downstream of Gal-8 stimulation in Jurkat T cells. However, this result must be interpreted with caution, as PLD-derived PA may also activate ERK in an indirect manner involving the lipin-mediated conversion of PA to DAG.

Type I PIP5K enzymes catalyze the production of PIP₂ from phosphatidylinositol-4-phosphate (PIP). During T cell activation, PIP₂ is involved in modulating T cell rigidity but is primarily hydrolyzed to produce key second messengers DAG and inositol triphosphate (IP₃) (51). A number of studies have established that PA can bind to and activate PIP5K (67). Modulation of PIP5K activity by PA was originally shown using PIP5K purified from bovine brain membranes, where PA was shown to enhance enzymatic activity by up to 20-fold (68). Subsequent studies demonstrated that type I PIP5Ks (which phosphorylate PIP to PIP₂), but not type II PIP5Ks (which phosphorylate phosphatidylinositol 5 phosphate to PIP₂), are specifically regulated by PA (69). More recent work has suggested that PA may regulate the affinity of murine PIP5K-1β for its substrate PIP (70). In this study, PA was shown to bind specifically to the C-terminal region of PIP5K-1β and kinetic analysis revealed that the addition of PA increased the affinity of PIP-binding to the enzyme’s active site by nearly 70-fold. Other studies have elegantly demonstrated a role for DGKζ-derived PA in stimulating PIP5K-1α activity to increase local PIP₂ levels and promote actin polymerization in cell lines (71). Expression of DGKζ enhanced PIP5K-1α activity in thrombin-stimulated HEK293 cells, and DGKζ and PIP5K-1α were found to co-localize and co-immunoprecipitate with each other. DGKζ and PIP5K-1α were also found to co-localize with actin at lamellipodial protrusions in epithelial cells. While there is evidence to suggest that the activity of PIP5K-1α and γ isoforms may be critical for normal human NK cell cytotoxicity (72, 73), the role of PLD-derived and DGK-derived PA in regulating PIP5K activity in immune cells remains quite poorly understood.

Protein phosphatase 1 (PP1) is a eukaryotic serine/threonine phosphatase that regulates the function of a variety of proteins in the cell. The PP1 catalytic subunit is able to interact with more than 50 different regulatory subunits in a mutually exclusive manner and this allows the enzyme to target different substrates in diverse subcellular locations depending on its binding partner (74). Initial studies identified that PA acted as a highly specific tight-binding inhibitor of the γ isoform of human PP1 in vitro (75). Further studies used a deletion mutagenesis approach to reveal that residues 286–296 of PP1γ were necessary and sufficient for PA-binding (76). Results from one study suggest that PP1 activity may play a role in suppressing T cell function in a rat model of alcohol intoxication and burn injury (77). While this suggests that PA-mediated inhibition of PP1 function may facilitate T cell activation, further experiments are required to better understand the role of PLD-derived and DGK-derived PA in suppressing PP1 activity in T cells.

Src homology region 2 domain-containing phosphatase 1 is a tyrosine phosphatase that plays a critical role in T cell function (78). “Moth-eaten” mice carry a spontaneous frame-shift mutation in the SHP1 gene and lack detectable SHP1 protein (79). Studies with these mice revealed a role for SHP1 in negatively regulating positive and negative thymocyte selection (80), while the use of conditional SHP1 knockout mice showed that SHP1 limits the number of short-lived effector CD8 cells produced in response to viral infection (81). Early studies showed that PA could increase the phosphatase activity of SHP1 toward the EGF receptor when the two proteins were transiently co-expressed in 293 cells (82). Subsequently, PA was shown to directly bind to recombinant SHP1, and two distinct PA-binding sites (a high affinity site on the C-terminal end and a low affinity site on the N-terminal end) were identified on SHP1 (83). Future studies are required to determine if PA modulates SHP1 activity in immune cells and if PA may serve as an effective therapeutic agent to modulate immune responses.

Bone marrow-derived early progenitor cells must go through an elaborate process of development in the thymus to become mature T cells (84, 85). Thymocytes at different stages of maturation are readily distinguished by a combination of CD4 and CD8 co-receptors expressed on their cell surface, proceeding from the earliest stage with neither CD4 nor CD8 (double negative/DN) through an intermediate stage expressing both CD4 and CD8 (double positive/DP) to a mature stage marked by the expression of either CD4 or CD8 (CD4 single positive/CD4SP or CD8 single positive/CD8SP) (86, 87). While progenitor cells enter the thymus at the cortico-medullary junction, a number of sequential chemokine/chemokine-receptor interactions help guide a developing thymocyte through the thymic cortex and medulla, facilitating its progressive relocation to appropriate micro-environments within the thymus (88, 89).

With the expression of RAG proteins, DN cells undergo VDJ recombination at the TCRβ locus, expressing a pre-TCR on the cell membrane. DN cells with a productive TCRβ rearrangement pass through the β-selection developmental checkpoint, undergoing multiple rounds of proliferation and upregulating expression of CD4 and CD8 to become DP cells. DP cells subsequently rearrange V and J genes at the TCRα locus, expressing a unique TCR on the cell surface. Subsequently, cells bearing TCRs that recognize self-peptide-MHC complexes on thymic epithelial cells receive survival signals during the so-called positive selection process, while others that fail to recognize these complexes die of “neglect” (90, 91). On the other hand, DP cells with TCRs that recognize self-peptide-MHC complexes with high affinity are eliminated by apoptosis-inducing signals during negative selection (92, 93). Together, positive and negative selection processes ensure the generation of a T cell repertoire that is both functional and self-tolerant (94, 95). DP cells that survive these selection processes mature into CD4SP and CD8SP cells that eventually migrate to secondary lymphoid organs as naïve CD4 and CD8 T cells (96, 97).

A plethora of studies have implicated DAG-dependent signaling pathways in β, positive and negative selection. For instance, early studies showed that signaling via the pre-TCR activates ERK1/2 (98), while more recent ones have demonstrated an essential role for RasGRP1 and ERK activation in efficient β-selection (27, 99). Mice with a T cell-specific deficiency of PLCγ1 show dramatically reduced numbers of mature CD4SP and CD8SP thymocytes, and defects in both positive and negative selection when crossed with HY TCR transgenic mice (100). Impairment of thymic selection in the absence of PLCγ1 suggests that its product DAG may play an important role in the process. Lending further credence to this notion, RasGRP1-deficient mice show impaired Ras-ERK signaling in thymocytes and defective thymic selection with a 70–90% reduction of mature SP cells (25, 26). Transgenic mice expressing a dominant negative form of Ras present with defects in positive selection, but not negative selection, when crossed with HY TCR transgenic mice (101). Similar observations were made with transgenic mice that expressed a catalytically inactive form of MEK1 (K97A) under the control of the thymocyte-specific Lck proximal promoter (102). ERK1 deficiency results in a severe developmental block at the DP stage (103). Conditional deletion of ERK2 using proximal Lck-Cre partially blocked DN3 to DN4 progression, while deletion with CD4-Cre led to defective positive selection. Mice with a combined deficiency of ERK1 and ERK2 showed that ERK activity is required for proliferation and differentiation associated with β-selection, and for positive selection (104). The MAP kinase-interacting serine/threonine kinases (Mnks) 1 and 2 lie downstream of ERK1/2 and p38 (105, 106). Recent studies have shown that TCR triggering can activate Mnk1/2 via the Ras-ERK pathway in a manner that is negatively regulated by DGK αζ activity (107). Although Mnk1/2 phosphorylate EIF4E, which is thought to promote translation initiation, combined deficiency of Mnk1/2 did not lead to obvious changes in thymocyte development. The mechanisms by which ERK1/2 regulate thymocyte selection remain to be clearly defined. Together, these studies suggest that the DAG-RasGRP1-Ras-ERK pathway plays a critical role in thymocyte development.

The role of the DAG-mediated PKCθ-IKK-NF-κB pathway in T cell development has also been studied extensively. While initial studies found no obvious developmental defects in PKCθ deficient thymocytes, more recent ones have suggested that PKCθ may be required for efficient positive selection (108, 109). T cell-specific deletion of IKKγ or replacement of IKKβ with a dominant kinase-dead form results in a reduction of mature CD8SP cells (110), while transgenic models that allow for activation or inhibition of NF-κB have revealed its role in the establishment of signaling thresholds for positive and negative selection (111).

The importance of these DAG-mediated pathways suggests that their tight regulation by DGK may be critical for normal thymocyte development. Studies with mice that lack both DGKα and DGKζ (DGKαζDKO) have confirmed this hypothesis (112). DGKαζDKO thymocytes experience excessive DAG accumulation and enhanced DAG-mediated signaling after TCR engagement. This is associated with a severe developmental block at the DP stage and a marked paucity of mature CD4SP and CD8SP cells. Defects in positive, but not negative, selection were revealed using a HY TCR transgenic system. Addition of exogenous PA to fetal thymic organ cultures increased the frequency of SP cells in DGKαζDKO thymi without obvious effects on control thymi, suggesting that DGKα and DGKζ play a synergistic role in T cell development not just by dampening DAG-mediated signals but also by promoting PA-mediated signals. This DGK-induced switch from DAG-driven to PA-driven signals may also play a critical role in preventing prolonged activation of the highly oncogenic Ras-ERK and NF-κB pathways in developing thymocytes. Indeed, HY TCR transgenic mice with decreased DGK activity showed significantly enhanced thymic lymphomagenesis, suggesting an important role for DGK activity in tumor suppression (112).

More recent work from our group has uncovered a novel role for DGKs as negative regulators of mTOR activity in thymocytes (113). Results from the study showed that low concentrations of phorbol 12-myristate 13-acetate (PMA), a functional analog of DAG, were able to induce phosphorylation of mTOR complex 1 substrates S6K1 and 4E-BP1 and mTOR complex 2 substrate Akt (S473), suggesting that DAG-mediated signaling is sufficient to induce activation of both mTOR complexes in thymocytes. DGKαζDKO thymocytes showed enhanced phosphorylation of S6K1, 4E-BP1, and Akt (S473) upon TCR engagement as compared to WT counterparts, suggesting that DGK activity inhibits TCR-induced activation of mTORc1 and mTORc2. Further studies are required to determine if dysregulated mTOR signaling might contribute to the defects in T cell development and function observed in DGKαζDKO mice.

Emerging evidence also suggests that tight regulation of DAG-mediated signaling by DGK activity may be critical for the development of iNKT cells. iNKT cells are a rare but distinct lineage of αβ T cells that express a highly restricted TCR repertoire and recognize glycolipids presented on CD1d. Sometimes called the “Swiss-Army knife” of the immune system, iNKT cells bridge innate and adaptive immunity by performing an array of functions that include killing of infected cells and secretion of cytokines and chemokines (114). Despite their relative rarity, an important role for iNKT cells has been demonstrated in immune responses to pathogens, allergens, self-antigens, and cancer.

Previous work has revealed a critical role for signaling via the PKCθ-IKK-NF-κB pathway in the ontogeny of iNKT cells (15, 115–117). More recent studies have identified that RasGRP1-Ras-ERK signaling may also be indispensable for iNKT development. In one study, the absence of RasGRP1 was associated with a severe reduction of iNKT cell numbers in the thymus, spleen, and liver (118). The generation of bone marrow chimeras showed that the iNKT cell developmental defects were cell-intrinsic, and the remaining RasGRP1-deficient iNKT cell population displayed both a selective absence of CD4⁺ cells and defects in TCR-induced proliferation. In another study, the expression of a dominant negative form of Ras dramatically hindered iNKT development (118, 119).

While lack of IKK-NF-κB and Ras-ERK signaling is detrimental to iNKT cell development, recent findings indicate that hyperactive signaling via these DAG-mediated pathways may also perturb iNKT development (120). In this study, T lineage specific expression of a constitutively active form of Ras resulted in a late stage block in iNKT cell maturation, and constitutively IKKβ activity was associated with increased cell death at multiple developmental stages. Since the maintenance of optimal levels of DAG-mediated signaling appears to be essential for normal iNKT development, we hypothesized that tight regulation of DAG-mediated signals by DGK activity might be essential for this process. Results from our studies showed that while iNKT cell numbers were unaltered in mice lacking either DGKα or DGKζ, they were dramatically diminished in DGKαζDKO counterparts, suggesting that these DGK isoforms may play a redundant role in regulating iNKT cell development (120). Defective DGKαζDKO iNKT development was associated with enhanced cell death and co-incident with enhanced activation of the Ras-ERK and NF-κB pathways. Taken together, these results suggest that DGKα and DGKζ work synergistically to maintain an optimal level of DAG-mediated signaling that is essential for normal iNKT development.

Apart from T cells, DAG and PA are critical signaling intermediates in several cell types including mast cells, dendritic cells (DCs), and macrophages. It is therefore not surprising that tight regulation of DAG and PA levels by DGK activity is essential for normal functioning of these cell types. Mast cells are abundant at the host’s interface with the environment, such as the skin and mucose (156). While best known for their role in the pathogenesis of asthma, allergy, and anaphylaxis, mast cells also play a critical role in pathogen surveillance and defense against parasites (157). In contrast to observations with T cells, mast cell function in vivo was diminished in the absence of the DGKζ, impairment of local anaphylactic responses (158). Bone marrow-derived mast cells that lacked DGKζ showed impaired degranulation but enhanced production of cytokines such as IL-6 when stimulated ex vivo. Other studies have shown that both the ζ1 and ζ2 isoforms of DGKζ expressed in bone marrow-derived macrophages and DCs, with ζ1 being predominant (159). Considered “professional phagocytes,” macrophages, and DCs express a diverse array of pattern recognition receptors (including toll-like receptors or TLRs) that enable them to detect the presence of pathogens and cell debris. Upon stimulation with Toxoplasma gondii – stable tachyzoite antigen (STAg, which activates multiple TLRs), DGKζ-deficient splenic DCs and bone marrow-derived macrophages (BMMϕ) produced less TNFα and IL-12 p40 than WT counterparts. Consistent with this impairment in cytokine production, both resistance to endotoxin shock and susceptibility to T. gondii infection were increased in DGKζ KO mice. While these findings provide tantalizing evidence of a role for DGKζ in regulating innate immune responses, further studies are required to gain a better understanding of the underlying molecular mechanisms. In addition, the involvement of other DGK isoforms in the regulation of innate immune responses remains to be investigated. The varied biological functions of DGK activity in T cells and other immune cells are summarized in Table 1.

Over the past few years, a remarkable number of elegant studies that have furthered our understanding of the roles of DAG-mediated and PA-mediated signaling pathways, and their regulation by enzymes of the DGK family, in T cell development and function. A role for DGK activity has been identified in a variety of critical processes including conventional αβ T cell and iNKT cell development, T cell activation and anergy, directional secretion, and suppression of CD8 responses against viruses and tumors. While a multitude of interesting and fundamental questions in the field have been addressed by these recent studies, it is important to note that perhaps just as many others await answers. The roles of DGK-derived PA and PLD-derived PA in T cell development and function have proved challenging to dissect, as have differences between DGK isoforms in terms of substrate specificity and subcellular localization. Key elements such as transcription factors, microRNAs, and post-translational modifications that control the dynamic expression and function of DGKs during a T cell’s lifetime also remain relatively unexplored. Applying the scientific method to answer these intriguing questions is likely to yield a better understanding of how DAG and PA signals and DGK activity regulate immune responses, enhancing our ability to modulate such responses to quell self-reactivity or generate protective immunity.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.