Targeting negative regulators downstream of the TCR represents a novel strategy to improve cancer immunotherapy. Two proteins that serve as critical inhibitory regulators downstream of the TCR are diacylglycerol kinase ζ (DGKζ), a regulator of Ras and PKC-θ signaling, and Casitas b-lineage proto-oncogene b (Cbl-b), an E3 ubiquitin ligase that predominantly regulates PI(3)K signaling. We sought to compare the signaling and functional effects that result from deletion of DGKζ, Cbl-b, or both (double knockout) in T cells and to evaluate tumor responses generated in a clinically relevant orthotopic pancreatic tumor model. We found that whereas deletion of Cbl-b primarily served to enhance NF-κB signaling, deletion of DGKζ enhanced TCR-mediated signal transduction downstream of Ras/Erk and NF-κB. Deletion of DGKζ or Cbl-b comparably enhanced CD8+ T cell functional responses, such as proliferation, production of IFN-γ, and generation of granzyme B when compared with wild type T cells. Double-knockout T cells demonstrated enhanced function above that observed with single-knockout T cells after weak, but not strong, stimulation. Deletion of DGKζ, but not Cbl-b, however, resulted in significant increases in numbers of activated (CD44hi) CD8+ T cells in both nontreated and tumor-bearing mice. DGKζ-deficient mice also had enhanced control of pancreatic tumor cell growth compared with Cbl-b–deficient mice. This represents a direct comparison between mice of these genotypes and suggests that T cell immunotherapies may be better improved by targeting TCR signaling molecules that are regulated by DGKζ as opposed to molecules regulated by Cbl-b.
Targeting inhibitory proteins within cytotoxic T cells as a means to improve T cell activity against cancer has led to impressive clinical successes, but only in a minority of treated patients (1). The primary current focus of cancer immunotherapy is to identify ways to increase the number of responding patients while limiting the induction of autoimmunity. This has resulted in accelerated research focused on understanding the mechanisms that tumors use to inactivate cytotoxic T cells and developing ways to overcome this inhibition (2, 3). Much of this work has centered on interfering with the activation of CTLA-4 and PD-1, two inhibitory cell surface receptors that antagonize TCR activation (4). Although mAbs that disrupt the interactions between inhibitory receptors and their cognate ligands have led to significant disease responses in a variety of cancers, only a minority of patients derive meaningful benefit from these therapies (5), and, in the case of PD-1, those who do benefit may develop tumor escape (6). Thus, current efforts have focused on finding novel targets to improve cytotoxic T cell activity that will augment existing therapies. These include therapies that target other immune checkpoint receptors such as Tim3 and Lag3 (2) or other inhibitory receptors including the adenosine 2A receptor, PGE2 receptor, and TGF-β receptor complex (7, 8). Recently, interest has developed regarding the possibility of targeting intracellular inhibitory proteins to improve T cell activity against cancer, including diacylglycerol kinase ζ (DGKζ) (9, 10) and Casitas b-lineage proto-oncogene b (Cbl-b) (11), which attenuate signal transduction events downstream of the TCR and CD28. Targeting these proteins may be superior to targeting cell surface proteins because their elimination confers simultaneous insensitivity to multiple cell-surface inhibitory receptors. For instance, we previously demonstrated that DGKζ-deficient T cells demonstrate partial insensitivity to several important mediators of immune inhibition within the tumor microenvironment, including PGE2 (12), adenosine (12), TGF-β (13), and PD-L1 (10).
DGKζ is one of two DGK isoforms in T cells that phosphorylate diacylglycerol (DAG). DAG is a second messenger of TCR signaling that is generated by the cleavage of phosphatidylinositol 4,5-bisphopshate by PLCγ1 and is required for activation of RasGRP1 and PKC-θ (14). DAG phosphorylation results in production of phosphatidic acid and termination of DAG-mediated signaling (15). Deletion of DGKζ from T cells results in prolonged TCR signal transduction downstream of DAG, resulting in enhanced activation of Ras, increased effector T cell proliferation, and amplified cytokine production, which leads to increased antitumor activity against s.c. implanted EL4 tumors or murine mesothelioma (12, 16, 17). DGKζ has been demonstrated to be the dominant isoform of DGK in T cells as assessed by functional changes in peripheral T cells and direct measurement of phosphatidic acid after TCR stimulation (18).
Cbl-b is an E3 ubiquitin ligase that targets multiple components of the TCR signal transduction pathway for degradation through the proteasome to facilitate termination of TCR signaling (19, 20). Although several proteins have been demonstrated to be targets of Cbl-b, including the TCR itself, much of the inhibition attributable to Cbl-b results from ubiquitination of the p85α subunit of PI(3)K (21, 22). Similar to T cells deficient in DGKζ, Cbl-b–deficient T cells demonstrate enhanced proliferation, cytokine production, and antitumor activity in numerous tumor models (23, 24). Current phase I immunotherapy trials are underway to test the utility of inhibiting Cbl-b in PBMCs in the treatment of cancer patients (ClinicalTrials.gov: NCT02166255, NCT03087591).
Given the predominant role for DAG on regulating Ras/Erk and PKC-θ signaling and Cbl-b for regulating PI(3)K signaling, we hypothesized that T cells deficient in DGKζ or Cbl-b would alter TCR signal transduction through distinct mechanisms, such that DGKζ-deficient T cells would demonstrate strongly enhanced Ras/ERK activation, but only modestly enhanced NF-κB activation, and Cbl-b–deficient T cells would demonstrate modestly enhanced Ras/ERK activation and strongly enhanced NF-κB activation. We found that whereas DGKζ regulated Ras/ERK activation to a greater degree than Cbl-b, CD8+ T cells deficient in either DGKζ or Cbl-b exhibited similar enhancement of NF-κB signaling. We further determined that T cells deficient in DGKζ exhibit greater control of murine pancreatic tumor growth as compared with T cells deficient in Cbl-b. Moreover, we found that T cells from double-knockout (DKO) mice did not demonstrate enhanced tumor activity above that observed in DGKζ single-knockout (KO) mice. Additionally, we identified that combined deletion of DGKζ and Cbl-b resulted in similar impairment of antigen-specific memory CD8+ T cell generation and/or maintenance compared with KO. Our data suggests that targeting DGKζ could prove to be a more useful clinical approach to augment cytotoxic T cell activity against tumor than targeting Cbl-b, perhaps resulting from enhanced Ras/ERK signaling.
Materials and Methods
Mice deficient in DGKζ or Cbl-b, backcrossed to C57BL/6 mice, were described previously (16, 25). Cbl-b−/− mice were generously provided by Richard Hodes, National Institutes of Health (Washington, DC). DGKζ−/− and Cbl-b−/− mice were crossed to create DKO mice. C57BL/6 mice were purchased from The Jackson Laboratory. All experiments were performed in mice 6–13 wk old. Animal housing and experimentation were done in accordance with the Institutional Animal Care and Use Committee at the Medical College of Wisconsin.
Statistical analysis was performed using ANOVA and unpaired two-tailed t test with GraphPad Prism software (La Jolla, CA). A p value <0.05 was considered to indicate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).
T cell phenotyping
Phenotypic analyses of cell surface markers in spleen, lymph node, and thymus were performed on unchallenged mice at 6–8 wk of age. Spleens were processed into cell suspensions through a 70-μm strainer by a syringe plunger in RPMI 1640 with 10% FBS and then treated with ACK lysis buffer to remove RBCs. Lymph nodes were processed using glass sides and the thymi were minced. The resulting cell suspensions were centrifuged and stained with Abs specific for CD4 (RM4-5), CD8α (53-6.7), CD62L (MEL-14), and CD44 (IM7), all supplied by BD Pharmingen (San Jose, CA); CD25 (PC61.5) (eBioscience, San Diego CA); and a viability stain (Life Technologies, Carlsbad, CA). After staining for 25 min at 4°C, cells were fixed in PBS containing 2% paraformaldehyde. All flow cytometry data were acquired on an LSRII cytometer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Tree Star, Ashland, OR).
T cells from mouse splenocytes were purified with magnetic naive CD8+ T cell isolation kits using instructions provided by the manufacturer (Stemcell Technologies, Vancouver, Canada). A total of 4 × 105 purified CD8+ T cells in 100 μl of serum-free media were incubated with 5 μg/ml biotinylated anti-CD3 (2C11; BD Pharmingen) and anti-CD28 Abs (37.51; (BD Pharmingen) for 1 min at 37°C followed by the addition of 25 μg/ml streptavidin (Thermo Fisher Scientific, Waltham, MA) for 0, 5, and 15 min. Reactions were terminated with 1 ml of ice-cold PBS, and cells were centrifuged and resuspended in 50 μl of cell lysis buffer containing 1% NP-40 and protease inhibitors. Lysates were subjected to high-speed centrifugation to remove nuclei and cell debris, and total protein was quantified using the Pierce BCA protein assay kit (BD Pharmingen). Normalized protein lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted with primary Abs from Cell Signaling Technology (Danvers, MA) at concentrations recommended by the manufacturer.
Assessment of proliferation and cytokine production
Purified T cells from the spleen were labeled with CellTrace CFSE (Invitrogen, Carlsbad, CA) using protocols and reagents supplied by the manufacturer. A total of 1 × 105 cells were placed in individual wells of a 96-well plate, and cells were precoated with 100 μl of varying concentrations of plate-bound anti-CD3 (2C11; BD Pharmingen) and 5 μg/ml anti-CD28 Abs in PBS. Three days later, cells were surface-stained with Abs against CD4 (RM4-5) and CD8α (53–6.7) (Life Technologies) and subjected to flow cytometry for evaluation of CFSE dilution. IFN-γ and IL-2 production from supernatants of individual wells was determined using an ELISA (BioLegend, San Diego, CA). To assess granzyme B production, CFSE-labeled cells were stimulated for 24 or 72 h, surface-stained as described above, permeabilized using a Cytofix/Cytoperm kit (BD Pharmingen), and then stained with flurochrome-labeled granzyme B Ab (GB11; Life Technologies) at 4°C for 30 min prior to flow cytometric analyses. Naive CD8+ T cell experiments were performed with 5 × 104 cells per stimulation. Cells were stimulated in the presence or absence of 100 U/ml IL-2 (PeproTech, Rocky Hill, NJ) or 5 μg/ml anti–IL-2 (eBioscience).
Orthotopic injection of KPC1242 tumors
The KPC1242 cell line, derived from a spontaneous pancreatic tumor that arose in a KPC mouse, was a generous gift of David Tuveson (Cold Spring Harbor Laboratories, Cold Spring, NY). A total of 1 × 106 KPC1242 tumor cells were injected orthotopically into the pancreas as previously described (26). Fifteen days later, mice were euthanized, tumor weight was measured, and T cells in the spleen, ascites, and tumor were analyzed. Spleens and tumors were mashed through a 70-μm strainer by a syringe plunger in RPMI 1640 with 10% FBS. Spleens were then treated with ACK lysis buffer to remove RBCs. Splenocytes, ascites, and tumor cells were stained with a viability dye and Abs against CD4, CD8α, CD62L (MEL-14), CD44 (IM7), and CD25 (PC61.5; eBioscience) and were analyzed by flow cytometry. Additionally, an aliquot of 4 × 106 splenocytes was stained for Foxp3 expression using methods and reagents, provided by the manufacturer (eBioscience), prior to flow cytometric analyses.
Lymphocytic choriomeningitis virus infection
Mice were infected i.p. with 2 × 106 PFU of lymphocytic choriomeningitis virus (LCMV) (strain Armstrong) in PBS. Blood was collected on weeks 1, 2, 4, and 6 to assess virus-specific T cell expansion and contraction. Blood samples were underlaid with histopaque (Sigma-Aldrich, St. Louis, MO) and centrifuged at 2000 rpm for 15 min. Lymphocytes were harvested from the gradient interface and washed before further analysis. Cells were surface-stained with Abs against CD8α, CD62L, CD44, CD27 (LG.7F9; eBioscience), KLRG-1 (2F1), and CD127 (A7R34; BioLegend), along with viral epitope–specific GP33 tetramer for 25 min at 4°C. Cells were then analyzed by flow cytometry. Six weeks postinfection, mice were euthanized, and spleens and inguinal lymph nodes were isolated. Cells were stained with the same panel as described for blood samples, with the addition of fluorochrome-labeled tetramers capable of binding to TCR clones GP276 or NP369, which were generated as previously described (27). For intracellular cytokine staining, lymphocytes were cultured in 96-well flat-bottom plates at 1 × 106 cells/well in 200 μl RPMI 1640 supplemented with 10% FBS in the presence or absence of GP33, GP276, and NP369 peptides. Stimulations were performed for 5 h at 37°C in the presence of GolgiPlug (1:1000; BD Pharmingen), surface-stained as described above, permeabilized, and stained with Abs against IFN-γ (XMG1.2) (BD Pharmingen). Flow cytometry was performed, and cells were analyzed as described above.
T cells deficient in DGKζ and Cbl-b demonstrate differential enhancement of Erk and NF-κB signaling
Stimulation of the TCR results in cleavage of phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate, which induces intracellular calcium flux, and DAG, which binds to RasGRP1 and PKC-θ to contribute to activation of these proteins (28). DGKζ phosphorylates DAG, terminating DAG-mediated protein activation. Cbl-b targets the p85α subunit of PI(3)K for degradation, resulting in termination of substrate activation downstream of phosphatidylinositol 3,4,5-triphosphate (21). Previous work demonstrated that loss of either DGKζ or Cbl-b results in enhanced activation of both Erk and NF-κB pathways in T cells after stimulation of the TCR (29); however, the relative intensity of signaling changes downstream of the TCR in T cells after loss of each protein has not been directly compared. To define the signaling changes resulting from each genotype, we isolated naive CD8+ T cells from mice deficient in DGKζ, Cbl-b, or both in DKO; stimulated the cells through their TCRs; and evaluated phosphorylation of Erk and phosphorylation and degradation of IκBα. We observed that Erk phosphorylation was modestly enhanced in Cbl-b−/− CD8+ T cells when compared with wild type (WT) T cells but was greatly enhanced in DGKζ−/− and DKO CD8+ T cells when compared either with Cbl-b−/− or WT CD8+ T cells (Fig. 1A). This indicates that DGKζ plays a greater role in limiting Erk activation in CD8+ T cells than Cbl-b. We also observed that IκBα phosphorylation and degradation were enhanced in DGKζ−/−, Cbl-b−/−, and DKO CD8+ T cells, indicating that DGKζ and Cbl-b are important regulators of NF-κB activation (Fig. 1B). These data indicate that DGKζ and Cbl-b differentially regulate TCR signal transduction.
DGKζ, Cbl-b, and DKO mice exhibit normal T cell development and peripheral T cell numbers but decreased naive CD8+ T cells
Previous studies on DGKζ−/− and Cbl-b−/− transgenic mice have shown phenotypically normal thymic development but decreased presence of naive T cells (e.g., CD44loCD62Lhi) in peripheral immune organs (17). The mechanism underlying a decrease in naive T cells is thought to result from several factors, including increased non–TCR-mediated (e.g., homeostatic) T cell proliferation and increased TCR-mediated activation (12, 17). To compare T cell phenotypes between DGKζ−/− and Cbl-b−/− mice, and to evaluate DKO mice, we isolated thymi, lymph nodes, and spleens from mice of each genotype and determined expression of T cell surface markers. In the thymus, expression of CD4 and CD8 varies during T cell maturation, such that cells initially do not express CD4 or CD8 (double negative [DN]) after arriving to the thymus, then express both CD4 and CD8 (double positive [DP]) after TCR rearrangement, and finally downregulate either CD4 or CD8 to generate mature single-positive cells that exit into the periphery. We observed no significant differences between percentages of DN and DP populations in the thymi from mice of each genotype (Fig. 2A); however, some changes in absolute number of single-positive populations were decreased in DKO and DGKζ−/− mice, which is of unclear significance. Overall, the results indicate that T cell development is grossly normal in mice that lack DGKζ, Cbl-b, or both.
We next evaluated T cell numbers in peripheral immune organs and found comparable numbers of CD8+ and CD4+ T cells in spleens of WT mice compared with KO or DKO mice with some modest differences in CD8+ and CD4+ T cell percentages between genotypes (Fig. 2B). To test the activation state of peripheral T cells, we evaluated for the presence of two surface markers, CD44 and CD62L. Whereas naive T cells express low levels of CD44 and high levels of CD62L, acutely activated CD8+ T cell express high levels of CD44 and low levels of CD62L, and memory CD8+ T cells express high levels of both CD44 and CD62L. Importantly, evaluation of activation markers on peripheral CD8+ T cells showed decreased numbers of naive (CD44loCD62Lhi) T cells in splenocytes of KO mice (Fig. 2C), as has been previously observed (17), which was enhanced in DKO mice. A similar trend was observed in activated CD4+ T cells (Supplemental Fig. 1). Together, these data indicate that peripheral T cell numbers are relatively preserved in KO or DKO mice compared with WT mice but that fewer naive CD8+ T cells are present in all genotypes when compared with WT.
T cell effector functions are enhanced in DKO mice compared with DGKζ−/− or Cbl-b−/− mice under suboptimal stimulatory conditions
Deletion of DGKζ or Cbl-b is known to enhance the effector functions of CD8+ T cells (16, 17, 19, 25). To directly compare functional changes in DGKζ−/− and Cbl-b−/− mice and to determine if DKO CD8+ T cell demonstrated an enhanced functional phenotype relative to KOs, we stimulated purified T cells obtained from spleens of each genotype with limiting dilutions of anti-CD3 and/or anti-CD28 and measured CD8+ T functions such as proliferation, production of effector cytokine IFN-γ, and generation of granzyme B. We found that, relative to WT T cells, KO T cells and DKO T cells demonstrated comparable enhancement of proliferation with high amounts (0.3 μg/ml) of anti-CD3 stimulation as assessed by dilution of CFSE (Fig. 3A, bottom panels), but proliferation occurred in DKO T cells only, after stimulation with low amounts (0.1 μg/ml) of anti-CD3 (Fig. 3A, middle panels). Further, although we could detect enhancement of granzyme B production in DKO T cells relative to WT, DGKζ−/−, or Cbl-b−/− T cells at early time points (Fig. 3B, upper panels), we did not observe enhanced granzyme B production in DKO relative to DGKζ−/− or Cbl-b−/− T cells at later time points (Fig. 3B, lower panels), at higher or lower concentrations of anti-CD3 Ab, or with addition of anti-CD28 Ab (Supplemental Fig. 2B). Lastly, we observed that production of IFN-γ was generally greater in DKO and Cbl-b−/− T cells relative to DGKζ−/− T cells, which was increased above the level observed in WT T cells (Fig. 3C). Previous work with DGKζ−/− CD8+ T cells showed that increased TCR-mediated proliferation relative to WT T cells can be largely attributed to increased IL-2 production (17, 25). To determine if this was also true for Cbl-b−/− and DKO T cells, we isolated naive CD8+ T cells from mice deficient in DGKζ, Cbl-b, or both, prior to stimulation with anti-CD3 (Fig. 3D–F). We found that, similar to the responses observed after stimulation of mixed T cell populations, stimulated naive CD8+ T cells from DGKζ−/−, Cbl-b−/−, and DKO mice demonstrated enhanced proliferation compared with WT T cells (Fig. 3D). However, DGKζ−/− CD8+ T cells were less responsive to low amounts (0.3 μg/ml) of anti-CD3 stimulation than similarly stimulated Cbl-b−/− and DKO CD8+ T cells (Fig. 3D) or to DGKζ−/− CD8+ T cells present in a mixed T cell population (Fig. 3A). The addition of IL-2, as expected, enhanced the proliferation of WT T cells, but not completely to levels observed with DGKζ−/− CD8+ T cells + IL-2 or to Cbl-b−/− and DKO CD8+ T cells (Fig. 3D). Furthermore, an increased amount of IL-2 was present in the supernatants of anti-CD3–stimulated DKO and Cbl-b−/−–stimulated CD8+ T cells relative to DGKζ−/− CD8+ T cells, which was increased relative to WT CD8+ T cells (Fig. 3E). Lastly, we determined the amount of IFN-γ produced after administration of exogenous IL-2 or anti–IL-2 to naive CD8+ T cells. As seen with stimulated T cells (Fig. 3C), Cbl-b−/− and DKO CD8+ T cells produced higher levels of IFN-γ compared with DGKζ−/− or WT CD8+ T cells (Fig. 3F) in a manner that was enhanced by the presence of additional IL-2 (Fig. 3F). In contrast, the addition of anti–IL-2 inhibited the production of IFN-γ in all genotypes tested (Fig. 3F). Together, these data indicate that the deletion of DGKζ or Cbl-b confer different effects on CD8+ T cell functions, and that combined deletion of the proteins decrease the threshold for inducing CD8+ T cell functions after TCR activation.
Enhanced control of KPC1242 pancreatic tumor in DGKζ−/− and DKO mice relative to Cbl-b−/− or WT mice
DGKζ−/− mice and T cells deficient in DGKζ are known to demonstrate enhanced clearance of tumors in s.c. models (12, 17, 30, 31). Similarly, Cbl-b−/− mice and T cells lacking Cbl-b demonstrate improved control of s.c.-implanted tumors (23) and disseminated leukemia (32), along with decreased spontaneous tumor formation in ATM−/− mice (23) and UV B-treated mice (24). However, tumor phenotypes in DGKζ−/− or Cbl-b−/− mice have not been directly compared. To evaluate tumor growth in mice of each genotype, we used an orthotopic implant model derived from a spontaneous pancreatic tumor arising in a C57BL/6 KPC mouse (33), which expresses mutant forms of K-Ras and p53 selectively in pancreatic tissue. We chose this model because 1) it more closely recapitulates features of human pancreatic cancer than do s.c.- implanted tumors, 2) it can be used in immunocompetent mice to permit assessment of immune responses, and 3) the cells grow in vivo with predictable kinetics (34). Fifteen days after orthotopic implantation, we detected a trend toward smaller tumors in Cbl-b−/− mice when compared with WT mice, and significantly smaller tumors in DGKζ−/− and DKO mice, including a complete absence of tumors in several DGKζ−/− and DKO mice (Fig. 4A). To assess whether changes in T cell numbers within the tumors could be responsible for the observed differences in tumor size, we processed the spleen and tumors from mice and calculated percentages of CD4+ and CD8+ T cells. We found that the percentages of T cells were similar in tumors among all genotypes; however, DKO mice were incidentally noted to have decreased amounts of splenic total CD8+ T cells (Fig. 4B). Note that tumor could not be evaluated in all DGKζ−/−or DKO mice, as tumor was not present in ∼50% of animals. We then assessed activation of status of T cells. We observed an increased percentage of activated (CD44hi) CD8+ T cells in tumors of DGKζ-deficient mice relative to WT mice (Fig. 4C). Further, consistent with T cell phenotypes in nontumor-bearing mice (Fig. 2), we observed an increased percentage of spleen-derived CD8+ T cells expressing high levels of the activation marker CD44 in DGKζ−/− or DKO mice that had been inoculated with tumor when compared with Cbl-b−/− or WT mice (Fig. 4C). In a reciprocal manner, tumor-inoculated DGKζ−/− or DKO mice demonstrated a decrease in percentages of naive (CD44hiCD62Llo) CD8+ T cells within the spleen when compared with WT mice and, in the case of DGKζ−/− mice, when compared with Cbl-b−/− mice (Fig. 4C). We also evaluated the presence of CD4+ regulatory T cells (Tregs) within the spleen because Tregs are known to play an important role in limiting antitumor immunity and because an increase in natural Tregs has been reported in DGKζ−/− mice (29). Consistent with prior reports, an increase in percentages of splenic Tregs was observed in DGKζ−/− and DKO mice in comparison with WT or Cbl-b−/− mice (Fig. 4D). Collectively, these data indicate that DGKζ−/− mice exert improved control of orthotopically implanted KPC1242 tumors, compared with WT mice, in a manner that may result from changes in the number of intratumoral activated CD8+ T cells in DGKζ−/− mice.
Inefficient CD8+ T cell memory development in mice deficient in DGKζ or Cbl-b
Whereas acute CD8+ T cell effector responses are enhanced in DGKζ-deficient mice postinfection with intracellular pathogens, persistent memory formation is impaired, most dramatically when both DGKζ and DGKα, the other isoform of DGK that metabolizes DAG downstream of the TCR in T cells, are deleted (35, 36). To determine the impact of Cbl-b deletion on memory formation in conjunction with DGKζ deficiency, we inoculated WT, DGKζ−/−, Cbl-b−/−, and DKO mice with LCMV (strain Armstrong). Consistent with prior reports on effector cells (35), we observed that, among CD8+ T cells specific for the dominant epitope of LCMV (GP33), the percentages of short-lived effector cells (KLRG1+CD127−) were increased, and the percentages of memory precursor cells (KLRG1+CD127+) were decreased in the peripheral blood of DGKζ−/−, Cbl-b−/−, and DKO mice relative to WT mice after acute infection (Fig. 5A). Although similar numbers of total splenic gp33-LCMV–specific T cells were observed between genotypes (Fig. 5B), the distribution of gp33-LCMV–specific CD8+ T cell effector and memory subsets were altered, such that there was a significantly lower percentage of short-term effector T cells and reciprocal changes in memory precursor cells among gp33-specifc CD8+ T cells in WT relative to DKO mice (Fig. 5C), with a trend in changes in absolute cell numbers, consistent with temporal data from peripheral blood (Fig. 5A). As has been noted by others (35), T cells from infected DGKζ−/− or DKO mice demonstrated enhanced IFN-γ production after stimulation with LCMV-specific peptides gp33 and np396 (Fig. 5D), and T cells from infected DGKζ−/− mice demonstrated enhanced IFN-γ production after stimulation with LCMV-specific peptide gp276 relative to T cells from WT mice (Fig. 5D). Peptide stimulation of T cells from Cbl-b−/− infected mice were similar in their ability to produce IFN-γ relative to WT controls (Fig. 5D), as previously seen in mice injected with a low dose of LCMV Docile (37). These data indicate that enhanced TCR signaling resulting either from deletion of DGKζ or Cbl-b results in skewed effector versus memory development postinfection with LCMV. Deletion of DGKζ or Cbl-b also results in differential effects on memory CD8+ T cell expansion and effector cytokine production.
Recently, there has been progress in the treatment of patients with malignancy through targeting receptors on the surface of CD8+ T cells that inhibit their activity within the highly suppressive tumor microenvironment. Clinically, these efforts have been most successful when targeting the inhibitory receptor PD-1, or its ligand PD-L1, with overall response rates of 20–35% for malignancies with favorable characteristics, including high expression of PD-L1 on tumor cells, high mutational burden within tumor cells, or robust CD8+ T cell infiltration within a tumor prior to treatment. A current focus of immuno-oncology research is to identify additional T cell targets that are able to improve antitumor efficacy while maintaining a side effect profile suitable for clinical development. Whereas the vast majority of investigations in the field have focused on inhibitory cell surface proteins such as CTLA4, Tim3, Lag-3, TGF-β–R, and others, much less is known about intracellular regulators that could be targeted to enhance T cell–mediated immune responses. The studies described in this article directly compared the functional characteristics of T cells deficient in two intracellular negative regulators of TCR signal transduction, DGKζ and Cbl-b. We chose these two proteins because they regulate TCR signaling through distinct mechanisms. DGKζ, a lipid kinase, phosphorylates the second messenger DAG, terminating DAG-mediated activation of RasGRP1 and PKC-θ, whereas Cbl-b, an E3 ubiquitin ligase, facilitates ubiquitination and subsequent degradation of the p85 subunit of PI(3)K. Previous studies have evaluated numerous aspects of T cell biology in the two models (16–18, 25, 38), but the signaling and functional effects of DKGζ deficiency and Cbl-b deficiency in T cells had not been directly compared.
In the studies presented in this article, we sought to directly compare signaling downstream of the TCR resulting from deletion of DGKζ or Cbl-b. Although other reports suggest that T cells deficient in either DGKζ or Cbl-b demonstrate enhanced signal transduction downstream of the TCR (14, 16, 17, 25, 39), we show that DGKζ is more important than Cbl-b in limiting Erk activation in T cells. However, DGKζ and Cbl-b both play a role in the negative regulation of NF-κB. We also show that deletion of both DGKζ or Cbl-b results in enhanced signaling events reflective of each individual deletion but that little synergy is apparent among downstream pathways. To evaluate physiological alterations resulting from deletion of DGKζ or Cbl-b, we first sought to evaluate T cell development among WT mice and each of the three genetic deletion models. As previously reported, we observed no changes in T cell development in DGKζ−/− and Cbl-b−/− mice (16, 25) and found that DKO mice also displayed grossly normal T cell development. These characterizations were requisite because work by others demonstrated that expression of a constitutively active form of another DGK allele important in regulating DAG levels in T cells, DGKα, resulted in developmental blockade of T cells between progression from DN to DP (40). Furthermore, although T cell development in the thymus appeared normal, the distribution of T cells expressing markers consistent with a naive versus activated state was altered, resulting in decreased numbers of naive T cells in peripheral lymphoid tissues of DGKζ−/−, Cbl-b−/−, and DKO mice compared with WT mice. This alteration likely can be attributed to a combination of decreased persistence of memory T cells (41) and enhanced homeostatic proliferation resulting from increased strength of signal downstream of the TCR (16).
A major goal of these studies was to compare the functional consequences of enhanced signaling through Ras-Erk/AP-1 versus NF-κB downstream of TCR stimulation. As previously seen (16, 19, 42), CD8+ T cells deficient in DGKζ or Cbl-b demonstrated comparable increases in proliferation compared with WT T cells. More specifically, we found that T cells from DGKζ−/− and Cbl-b−/− mice exhibit similar levels of enhanced proliferation at low levels of TCR stimulation. However, stimulated naive DGKζ−/− CD8+ T cells are less responsive to low levels of anti-CD3 stimulation than Cbl-b−/− CD8+ T cells. This is likely due to enhanced production of IL-2 and possibly related to a decreased dependence on CD28 engagement for full stimulation in Cbl-b deficient CD8+ T cells (25). In contrast, proliferation differences seen between DGKζ−/− or Cbl-b−/− CD8+ T cells mostly dissipate upon administration of exogenous IL-2, which indicates that the enhanced production of IL-2 in stimulated Cbl-b−/− CD8+ T cells is the primary factor responsible for the observed differences between the two genotypes. Consistent with previously reported analyses, we also observed enhanced IFN-γ and granzyme B production in DGKζ and Cbl-b–deficient T cells. Further, DKO T cells demonstrated enhanced production of IFN-γ compared with WT T cells, in a manner quantitatively similar to Cbl-b–deficient T cells. When we isolated naive CD8+ T cells and stimulated with low level of anti-CD3, we also observed enhanced IFN-γ production in Cbl-b−/− and DKO T cells relative to DGKζ−/− or WT T cells. This is likely due to differences in cell number resulting from dissimilar proliferation among genotypes (Fig. 3D–F). After addition of exogenous IL-2, the levels of IFN-γ production in culture media of stimulated CD8+ T cells increased in all genotypes, again likely attributable to cell number (Fig. 3F). However, the absolute amount of IFN-γ did not completely normalize between DGKζ−/− and Cbl-b−/− CD8+ T cells. Because cell number was similar between the two genotypes (Fig. 3D), this indicates that at least some intrinsic difference in cytokine production is present between DGKζ−/− and Cbl-b−/− CD8+ T cells. In contrast to addition of exogenous IL-2, addition of anti–IL-2 resulted in relatively little proliferation and thus little production of IFN-γ in any of the genotypes (Fig. 3F). As an additional finding, we observed little stimulation of purified naive DGKζ-deficient CD8+ T cells, relative to Cbl-b–deficient or DKO cells (compare Fig. 3A with Fig. 3D), at limiting dilutions of anti-CD3 (0.3 μg/ml), when compared with DGKζ-deficient T cells in a mixed population. This suggests that DGKζ-deficient CD8+ T cells are more dependent on help from CD4+ T cells at limiting dilutions of anti-CD3 stimulation. Together, these data indicate that DGKζ and Cbl-b may differentially regulate the threshold of cytokine production in CD8+ T cells.
To evaluate the functional impact of deleting of DGKζ, Cbl-b, or both molecules (DKO) on T cell immune responses, we evaluated two model systems: tumor growth in a model of pancreatic cancer and memory T cell responses in LCMV infection. First, we tested T cell responses to tumor in the setting of DGKζ deficiency, Cbl-b deficiency, or both. We observed enhanced tumor rejection in DGKζ-deficient mice and DKO mice relative to WT or Cbl-b–deficient mice. The decreased tumor size and enhanced tumor rejection in DGKζ-deficient mice indirectly suggests that enhanced Erk signaling may be superior to enhanced NF-κΒ activation in facilitating T cell activity against tumor, especially because DKO mice did not exhibit improved tumor control relative to DGKζ−/− mice. This provides evidence that targeting DGKζ may prove superior to targeting Cbl-b in cancer immunotherapies and that combined targeting may not be beneficial. In our second model, we tested CD8+ T cell responses to LCMV infection. Results demonstrated that mice deficient in DGKζ, Cbl-b, or both molecules generate impaired memory T cell responses relative to WT mice, confirming previous reports with mice deficient in DGKζ (41) or Cbl-b (19). This implies that TCR signal strength is titrated to balance generation of effector responses with sustained memory (43, 44), such that decreased TCR signaling results in poor acute effector memory expansion and enhanced TCR signaling results in diminished establishment of memory. The mechanistic basis of decreased memory establishment secondary to enhanced TCR signaling is an area of ongoing investigation. DKO mice, however, have a decreased percentage of memory precursor cells and an increase in short-lived effector cells compared with WT mice. These data are consistent with previous work in DGKζ−/− mice, in which others identified decreased percentages and total numbers of splenic CD8TetG+ (tetramer of H-2Dbgp33–41) cells 4 mo postinfection (35).
Together our data support the notion that targeting DGKζ, a negative regulator of T cell activation, or other regulators of Erk activation, could be useful in the treatment of human malignancy and that additional preclinical studies of intracellular “brakes” on T cell activation are warranted.
We thank Joseph Barbieri, Thomas Zahrt, and Carol Williams for their suggestions and Sandra Holzhauer for technical assistance.
This work was supported by National Institutes of Health Grant K08-CA151893 (to M.J.R.) and an American Cancer Society Institutional Award (to M.J.R.).
Abbreviations used in this article:
Casitas b-lineage proto-oncogene b
diacylglycerol kinase ζ
lymphocytic choriomeningitis virus
regulatory T cell
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.