DGKζ serves as a critical negative regulator of DAG signaling downstream of the TCR and can modulate the strength of TCR signaling. Early experiments using the immortalized Jurkat T cell line showed that overexpression of DGKζ inhibits TCR signaling by reducing the levels of active GTP-bound Ras and, consequently, diminishing ERK activation (Zhong et al., 2002). Furthermore, the overexpression of DGKζ was associated with decreased AP-1 transcription factor activity and CD69 expression (an early T cell activation marker) following TCR stimulation, both of which are regulated by the Ras-ERK pathway. Importantly, Ca²⁺ flux was normal regardless of DGKζ overexpression, suggesting that DGKζ selectively regulated DAG-mediated signaling pathways downstream of the TCR. Further biochemical analysis through the use of a kinase dead DGKζ mutant revealed that the enzymatic activity of DGKζ was critical for its inhibitory effects on TCR signaling.

To test the physiological role of DGKζ in T cells, Zhong et al. generated DGKζ knockout mice (Zhong et al., 2003). Initial phenotypic analysis showed that DGKζ KO mice contained similar frequencies and numbers of CD4⁺ and CD8⁺ T cells in secondary lymphoid organs and displayed no obvious defects in lymphoid architecture or cellularity. Furthermore, thymic development as analyzed by the number and frequency of CD4 single-positive (SP), CD8 SP, double-positive (DP), and double-negative (DN) thymocytes in DGKζ-deficient mice was similar to wild-type (WT) mice. However, upon TCR stimulation, naïve DGKζ KO CD4⁺ and CD8⁺ T cells displayed enhanced upregulation of activation markers CD25 and CD69 and increased proliferation compared to WT T cells. The increased expression of activation markers was associated with enhanced phosphorylation of ERK but normal induction of non-DAG mediated pathways including Ca²⁺ flux and JNK activation. Importantly, bypassing TCR activation with a DAG analog, phorbol-12-myristate-13-acetate (PMA), abolished differences in activation between DGKζ KO and WT T cells, suggesting that the hyperactivation of DGKζ KO T cells was secondary to defective regulation of DAG.

In agreement with enhanced TCR signaling, DGKζ KO T cells also display improved anti-viral responses (Zhong et al., 2003). DGKζ KO mice infected with LCMV Armstrong showed enhanced viral-specific T cell responses as evidenced by decreased viral titers at day 7 following infection. This effect correlated with an increased number of total and CD44^hiCD62L^lo effector CD8⁺ T cells in the spleen. Furthermore, LCMV-infected DGKζ KO mice exhibited increased number of IFNγ-producing CD8⁺ and CD4⁺ T cells when restimulated with LCMV-specific peptides, suggesting that DGKζ KO T cells displayed enhanced effector function following LCMV infection.

Similarly, DGKζ-deficient mice also exhibit enhanced anti-tumor responses. DGKζ KO mice subcutaneously injected with OVA-expressing EL4 T cell lymphoma, had significantly reduced tumor mass compared to their WT counterparts (Riese et al., 2011). This effect was accompanied by an increased number of total and antigen-specific tumor-infiltrating CD44^hiCD8⁺ T cells. To show that T cells were responsible for the enhanced anti-tumor effect by DGKζ deficiency, DGKζ KO and WT OVA-specific OT-I T cells were adoptively transferred into naïve recipient WT mice. Mice receiving DGKζ KO compared to WT OVA-specific OT-I T cells also exhibited lower tumor mass upon challenge with OVA-expressing EL4 cells. Isolation of tumor-infiltrating OT-I cells revealed that the loss of DGKζ increased the frequency of CD44^hi and IL-2-producing OT-I cells in a cell-intrinsic manner. In addition to preventing tumor engraftment, DGKζ deficiency also improves tumor rejection of established tumors, as the adoptive transfer of DGKζ KO but not WT OT-I effector T cells into tumor-bearing mice significantly reduced tumor burden (Riese et al., 2013). Thus, DGKζ could represent a novel target for enhancing anti-tumor responses in adoptive immunotherapy. This could also be applied to engineered T cells that express chimeric antigen receptors (CAR) directed against the tumor, as DGKζ deficiency was also shown to promote CAR T cell-mediated anti-tumor responses (Riese et al., 2013). How DGKζ deficiency augments anti-tumor responses is unclear. Although DGKζ KO CD8⁺ T cells display increased cytokine production and increased proliferation, their cytotoxic function is comparable to WT CD8⁺ T cells (Riese et al., 2011). Nevertheless, these studies demonstrate that DGKζ serves to constrain T cell activation and anti-viral and anti-tumor T cell responses. Thus, inhibition of DGKζ might provide a therapeutic opportunity to enhance immune-mediated viral and tumor clearance.

It is possible that DGKζ is physiologically important for limiting over-activation and inducing anergy in inappropriately activated T cells. The expression level of DGKζ can be controlled depending on the type of stimulation the T cell receives. T cells that are stimulated through their TCR and co-stimulatory molecules downregulate DGKζ transcript levels, thereby allowing appropriately activated T cells to become fully activated (Macian et al., 2002; Olenchock et al., 2006a; Zha et al., 2006). In contrast, T cells that receive TCR stimulation alone in the absence of co-stimulation do not downregulate DGKζ levels, potentially leading to attenuated DAG-mediated signaling and decreased activation. Consistent with this notion, DGKζ KO T cells resist anergy induction when activated by TCR alone in the absence of co-stimulatory signals (Olenchock et al., 2006a). In addition to TCR-mediated regulation, DGKζ might also be regulated by environmental cytokines. In particular, IL-33 has been shown to up-regulate DGKζ in cardiomyocytes following stimulation (Rui et al., 2012). Although it is unknown if IL-33 can upregulate DGKζ in immune cells, it is tantalizing to speculate that cytokine signaling can affect the TCR responsiveness of T cells by regulating DGKζ levels.

NK cells are cytotoxic members of the innate lymphoid cell (ILC) family and play an important role in protection against viral infection and clearance of tumors (Artis and Spits, 2015). Unlike their adaptive counterparts (CD8⁺ T cells), they do not possess a somatically-rearranged antigen receptor but rather express a variety of activating receptors specific for ligands displayed on virally-infected, stressed, or transformed cells (Lanier, 2008). NK cell activating receptors can be categorized into three main families based on the signaling adaptors used to relay downstream activation signals. These families include SAP-dependent (e.g., 2B4), ITAM-dependent (e.g., CD16), or DAP10-dependent (e.g., NKG2D) receptors (McVicar et al., 1998; Wu et al., 1999; Chen et al., 2006; Lanier, 2008). The activation of any of these three families of receptors relies on proximal signaling complexes involving SLP-76, which subsequently leads to the activation of PLCγ and the production of DAG (Wu and Koretzky, 2004; Tassi et al., 2005; May et al., 2013). In addition to these activating receptors, NK cells express an assortment of inhibitory receptors, many of which bind to MHC class I alleles and negatively regulate activating receptor signaling by the recruitment of phosphatases such as SHP-1 and SHIP (Binstadt et al., 1996; Lanier, 2008).

NK cell activation is determined by the net balance of the activating and inhibitory inputs that the NK cell receives through its receptors. For example, NK cells are activated when neoplastic cells upregulate ligands such as RAE-1 or MICA, which are recognized by the activating receptor NKG2D (Jung et al., 2012). Likewise, NK cells are activated through disinhibition when tumor cells lose MHC class I, a process known as missing self recognition (Kärre et al., 1986). Since SHP-1 and SHIP negatively regulate activating receptor signaling, one might predict that the loss of these molecules would boost the effector function of NK cells. Surprisingly, however, SHP-1 and SHIP deficiency in NK cells renders them less functional than their WT counterparts (Lowin-Kropf et al., 2000; Viant et al., 2014; Gumbleton et al., 2015). One explanation of this seemingly paradoxical finding is that NK cells continuously adjust their responsiveness to activating stimuli in their local environment, a phenomenon known as tuning (Höglund and Brodin, 2010). Thus, NK cells that chronically lack inhibitory signals, such as in SHP-1 or SHIP deficiency, require more stimulation to achieve their threshold of activation (Lowin-Kropf et al., 2000; Viant et al., 2014; Gumbleton et al., 2015). While NK cell tuning may protect the host from NK cell-mediated immunopathology, this process can hamper important effector responses against chronic viral infections or tumors.

Although the molecular mechanism of NK cell tuning is unknown, stimulation of NK cells with PMA and a calcium ionophore, ionomycin, can bypass the hyporesponsiveness of SHP-1 and SHIP KO NK cells (Viant et al., 2014; Gumbleton et al., 2015). These data suggest that the tuning process is proximal to PLCγ-mediated production of DAG. Thus, we speculated that NK cells may not be able to tune their responsiveness in response to enhanced DAG-mediated signaling by DGKζ deficiency. Indeed, we recently demonstrated that DGKζ KO NK cells are hyperfunctional compared to WT NK cells (Yang et al., 2016). DGKζ KO NK cells displayed increased cytokine production and cytotoxicity following stimulation through ITAM, SAP, and DAP10-dependent activating receptors. In contrast, IFNγ production by DGKζ KO and WT NK cells was similar following stimulation with IL-12 and IL-18, which utilize a DAG-independent signaling pathway, suggesting that the loss of DGKζ selectively augmented NK cell responsiveness to DAG-dependent stimuli. Like T cells, the hyperfunctionality of DGKζ KO NK cells was dependent on enhanced ERK signaling. Importantly, DGKζ KO mice cleared the NK cell-sensitive RMA-S tumor more efficiently than WT mice. Thus, the inactivation of negative regulators distal to PLCγ such as DGKζ might prove therapeutically useful in enhancing NK cell function.

B cells comprise the second arm of the adaptive immune system and are critical for the generation of protective antibody responses during infection. The induction of antibody production results from the stimulation of the somatically rearranged B cell receptor (BCR) by cognate antigen (McHeyzer-Williams and McHeyzer-Williams, 2005; Kurosaki et al., 2010). Similar to the TCR, activation of the BCR leads to downstream biochemical cascades that ultimately result in the generation of DAG through PLCγ and, subsequently, the activation of ERK (Hashimoto et al., 2000; Saijo et al., 2003). ERK has been shown to play multiple roles during B cell responses, including the promotion of B cell survival, proliferation, and differentiation into antibody-secreting plasma cells (Richards et al., 2001; Coughlin et al., 2005; Yasuda et al., 2011). Furthermore, attenuation of ERK activation has been shown to important during B cell development, since ERK signals decrease as B cells progress from the immature transitional stage to mature follicular B cells (Yasuda et al., 2008; Gross et al., 2009; Rowland et al., 2010).

Given the role of ERK in these B cell processes, controlling the levels of BCR-induced DAG through DGKζ might be important in regulating B cell development, activation, and antibody secretion capabilities. For example, mRNA transcripts of DGKζ are upregulated as B cells progress from early transitional to the mature follicular stage, which is associated with decreased ERK activation (Wheeler et al., 2013). Accordingly, the loss of DGKζ only affected ERK activation and IκBα degradation in the follicular but not early immature transitional B cell pool in response to BCR stimulation. Importantly, the augmentation of BCR-induced activation in DGKζ KO follicular B cells was seen even under less optimal BCR activation conditions, suggesting that DGKζ might control the BCR activation threshold in these cells.

The effects of DGKζ on B cell signaling threshold translate to functional consequences on B cell effector responses. BCR stimulation of purified DGKζ KO splenic B cells in vitro led to increased expression of CD69 and enhanced proliferation compared to WT B cells. DGKζ KO mice displayed enhanced antibody responses to T-independent and T-dependent antigens (Wheeler et al., 2013). The heightened antibody response by DGKζ-deficiency was accompanied by increased antigen-specific expansion of both germinal center (GC) B cells and plasma cells. These results demonstrate that regulation of DAG-dependent ERK activation by DGKζ is critical for selectively controlling the activation threshold of mature B cells to limit their activation.

Regulatory T cells (Tregs) are a key subset of T cells that display suppressive function and are important for the regulation of adaptive immune responses. Tregs are governed by the master transcription factor, forkhead box P3 (Foxp3), and exert their immunosuppressive function via the production of immunoregulatory cytokines and through cell contact dependent mechanisms (Josefowicz et al., 2012). Loss of function mutations in the Foxp3 gene, as seen in Scurfy mice and humans with immune dysregulation, polyendocrinopathy, and X-linked lymphoproliferative disease (IPEX), leads to lethal systemic autoimmunity early in life, highlighting the importance of Tregs in inducing immunotolerance against self antigens (Chatila et al., 2000; Bennett et al., 2001; Brunkow et al., 2001; Wildin et al., 2001).

T cells that strongly recognize self antigens are deleted during thymic development in a process known as negative selection. Specifically, T cells that receive strong TCR signals in the thymus, implying overt self reactivity, undergo apoptosis. As an alternative fate, strong TCR stimulation in developing thymocytes can also lead to Treg differentiation (Josefowicz et al., 2012). Thus, we hypothesized that enhancement of TCR-mediated DAG signaling by DGKζ deficiency in developing thymocytes may increase Treg generation. Indeed, the loss of DGKζ resulted in a significant increase in Treg development in the thymus in a cell-intrinsic manner (Schmidt et al., 2013). DAG-mediated signaling leads to the activation of the NF-κB (through activation of PKC) and ERK pathways. One NF-κB family member, c-Rel, was previously shown to be important for inducing Foxp3 expression in thymocytes (Long et al., 2009; Ruan et al., 2009). Although Treg generation in DGKζ KO mice was reduced in the absence of c-Rel, there was still residual Tregs in the thymus, suggesting that c-Rel was only partially responsible for the increased generation of Tregs in DGKζ KO mice (Schmidt et al., 2013). In fact, ERK activation appeared to be more important in the enhancement of Treg generation in DGKζ KO mice. Using an in vitro Treg development assay, we found that the inhibition of ERK phosphorylation by a MEK inhibitor led to decreased Treg generation in a dose-dependent manner, whereby the level of phosphorylated ERK (pERK) directly correlated to the magnitude of Treg generation. Importantly, Treg generation was also increased in sevenmaker mice (Sharp et al., 1997), which express a gain of function ERK mutation that leads to increased resistance to dephosphorylation of active pERK, suggesting that the selective enhancement of the ERK pathway alone is sufficient to increase Treg generation.

In addition to Treg generation in the thymus, TCR signaling plays an important role in the function of Tregs. Although some Treg function may be preserved in the absence of TCR signaling, we demonstrated that Tregs lacking SLP-76 cannot suppress TCR-driven proliferation of conventional T cells (Schmidt et al., 2015). Furthermore, Tregs with a Y → F mutation at tyrosine 145 (Y145F) of SLP-76, which leads to defective PLCγ activation, also display attenuated suppressive function, suggesting that PLCγ is important for Treg function. Consistent with this notion, Tregs lacking DGKζ display significantly increased suppression of TCR-driven conventional T cell proliferation compared to WT Tregs. Together, these data demonstrate that DGKζ limit Treg generation and function. Thus, DGKζ deficiency may indirectly lead to the suppression of immune responses through Tregs.

Mast cells are critical mediators in type 2 immune responses involved in protection against helminthes and in pathologic responses in asthma and allergy (Locksley, 2010; Voehringer, 2013). A key feature of mast cell function is the immediate release of pre-formed inflammatory mediators such as histamine, cytokines, and proteases in a process called degranulation. In addition, mast cells produce arachidonic acid metabolites and cytokines in a protracted manner (Voehringer, 2013). One major stimulus for the release of these inflammatory mediators is crosslinking of FcεRI, the high affinity receptor for the Fc region of immunoglobulin E (Kinet, 1999).

The interaction of allergens with IgE-FcεRI complexes results in formation of signaling complexes that converge on the activation of PLCγ (Atkinson et al., 1992; Schneider et al., 1992). PLCγ and subsequent PKC activation have been shown to be critical in controlling mast cell degranulation, suggesting that controlling the levels of DAG might be important for regulating this process (Nechushtan et al., 2000; Wang et al., 2000; Leitges et al., 2002; Wen et al., 2002). Indeed, the loss of DGKζ in FcεRI-stimulated mast cells leads to increased DAG levels, along with enhancement of downstream DAG-dependent signals, including RasGTP and ERK (Olenchock et al., 2006b). Accordingly, DGKζ deficiency leads to enhanced mast cell production of IL-6 following FcεRI stimulation.

Intriguingly, however, FcεRI-stimulated DGKζ KO mast cells display impaired degranulation and are resistant to local skin anaphylaxis (Olenchock et al., 2006b). The differential effect of DGKζ deficiency on mast function (the hypersecretion of IL-6 vs. decreased degranulation) may be explained by the negative feedback of DAG on PLCγ activation in mast cells. The elevation of DAG by DGKζ deficiency appears to negatively regulate the phosphorylation and subsequent activity of PLCγ. Thus, although DAG accumulates, the production of IP₃, and hence Ca²⁺ flux is attenuated in DGKζ KO mast cells. As degranulation responses are highly dependent on elevation of intracellular Ca²⁺ levels, this may cause a differential effect on degranulation and cytokine production by mast cells (Ozawa et al., 1993; Olenchock et al., 2006b). Thus, as opposed to T cells and NK cells, DGKζ exerts both activating and inhibitory effects on mast cell functional responses.

Macrophages and dendritic cells (DC) play a key role in bridging the adaptive and innate immune responses (Medzhitov, 2001; Janeway and Medzhitov, 2002; Akira and Takeda, 2004). Toll-like receptors (TLR) serve as an important mechanism for equipping macrophages and DCs with the ability to recognize the presence of pathogenic infection and, subsequently, instruct adaptive immune cells on the type of response needed to effectively clear the infection. TLRs can signal through either MyD88 and/or TRIF to induce activation of the NF-κB and ERK pathways (Akira and Takeda, 2004). While TLR activation does not generally lead to PLCγ activation, DAG has been shown to be induced in macrophages following stimulation with LPS (TLR4 agonist) and lipopeptide (TLR2 agonist) (Monick et al., 1999; Zhang et al., 2001a,b). Furthermore, inhibition of PLC or PLD reduced cytokine production and nitric oxide formation by macrophages following TLR stimulation, suggesting that control of DAG levels through DGK might be important in regulating TLR-mediated responses.

Interestingly, modulation of DAG levels by the loss of DGKζ resulted in impairment rather than enhancement of cytokine production by macrophages and DCs in response to TLR stimulation. Specifically, in a developmentally independent manner, bone marrow derived macrophages (BMMΦ) and splenic DCs produced substantially less IL-12p40 and TNFα following in vitro stimulation through a variety of TLR agonists (Liu et al., 2007). This paradoxical finding may be explained by the role of DGK in converting DAG into PA. Biochemical analysis revealed that the loss of DGKζ resulted in selective elevation of the PI3K-Akt pathway but no difference in activation of the ERK or NK-κB pathways following TLR stimulation. Activation of the PI3K pathway has been shown to negatively regulate TLR stimulation (Fukao et al., 2002; Guha and Mackman, 2002; Martin et al., 2005) and chemical inhibition of the PI3K restored LPS-induced IL-12p40 production from DGKζ KO BMMΦs (Liu et al., 2007). Intriguingly, the addition of PA also restored LPS-induced IL-12p40 production, suggesting that the cytokine production defect in DGKζ KO DCs and macrophages may be due to reduced PA rather than elevated DAG levels. Exactly how PA rescues TLR-induced cytokine production is unknown, but one possible mechanism is through the recruitment of SHP-1 to negatively regulate PI3K activation (Cuevas et al., 1999; Frank et al., 1999; Zhang et al., 2003).

Defective cytokine production was also observed in vivo following intraperitoneal injection of TLR agonists, which correlated with enhanced survival of DGKζ KO mice after LPS-induced septic shock (Liu et al., 2007). However, while DGKζ KO mice were protected from TLR-mediated pathology, the loss of DGKζ conferred susceptibility to Toxoplasma gondii. DGKζ KO mice infected with T. gondii displayed decreased serum IL-12p40 and IFNγ levels compared to WT mice. Furthermore, IFNγ production by DGKζ KO splenocytes isolated at day 15 and 30 post-infection was significantly attenuated following restimulation with T. gondii antigen STAg. Intriguingly, total CD4⁺ and CD8⁺ T cell numbers were similar between WT and DGKζ KO mice following infection with the frequency of CD44⁺CD62^lo effector T cells higher in infected DGKζ KO mice. As TLR-induced IL-12p40 production and the subsequent induction of a Th1 response are critical for protection against T. gondii infection, the impairment of immune responses against T. gondii by DGKζ KO mice could be secondary to a defect in macrophage and DC-derived cytokines that drive Th1 responses.

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.