For pharmacological stimulation, we used phorbol 12-myristate 13-acetate (PMA; Sigma) and ionomycin (Calbiochem). For antibody (Ab)-based stimulation, we used mouse antihuman or hamster antimouse monoclonal anti-CD3 and -CD28 Ab (555336, 555725, 553058, 553295; all from BD Pharmingen). For flow cytometry analyzes, we used LIVE/DEAD fixable red cell death staining kit (L23102, Invitrogen) and antimouse CD45-APC (L23102, eBioscience) or Percp-Cy5.5, CD8-BV610 and CD25-PCy7 (103132, 100744, and 102016, respectively; all from Biolegend), TCRβ-FITC (732247, Beckman Coulter) and PD-1-APC780 (47998582, eBioscience), antihuman PD-1-APC (17-9969-42, eBioscience) and CD25-PE (A07774, Beckman Coulter) and paraformaldehyde (TedPella). For western blot analysis, we used anti-DGKα (11 547–1-AP, ProteinTech), DGKζ (105195, Abcam), α tubulin (T5168, Sigma), GAPDH (G-9, Santa Cruz), horseradish peroxidase-conjugated antimouse and rabbit IgG (P0447, P0260, Dako), antirabbit IgG Dylight 800 (SA5-35571, Invitrogen) and mouse IgG AlexaFluor 680 (175775, AbcamLife). IKKβ inhibitor PS-1145 was from Sigma-Aldrich, MEK inhibitor PD98059 was from Calbiochem, and calcineurin (CaN) inhibitor FK506 was from Merck Millipore. Anti-PD-1 (nivolumab) humanized Ab was from BioVision. For TIL isolation, we used collagenase type I (Worthington), dispase II and DNase I (both from Roche). Trypsin was from Biowest.

Human Jurkat JE6.1-derived TPR cells, transduced to express NFAT-GFP, NFκB-CFP (cyan fluorescent protein) and AP-1-Cherry, were generated and selected as reported.33 TPR cells retrovirally transduced with vectors encoding PD-1 were described.33 Human leukemic Jurkat cells were from ATCC. T cell stimulator (TCS) cells, a murine thymoma cell line (Bw5147) engineered to stably express an antihuman CD3 single-chain fragment anchored to the cell membrane via a human CD14 stem, have been described.34 TCS cells expressing no human costimulatory molecule were used as control TCS (TCS-control) and compared with cells retrovirally transduced to express human CD86 (TCS-CD86), either alone or in the presence of PD-L1 (TCS-CD86/PD-L1). Murine MC38 adenocarcinoma cell line was a kind gift from Prof. Santos Mañes (CNB-CSIC). All cells were maintained in complete RPMI medium (cRPMI) consisting of RPMI (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 1% HEPES (Sigma), 2 mM L-glutamine and penicillin and streptomycin (100 U/mL; Biowest) (37°C, 5% CO₂).

C57BL/6J-DGKζ-deficient mice were reported previously.35 Lymph nodes were isolated from 6 to 12 weeks mice. Cells were maintained in cRPMI supplemented with 50 mM β-mercaptoethanol (Merck) (37°C, 5% CO₂).

For pharmacological/Ab-based stimulation, TPR cells (5×10⁴) in 50 µL were stimulated in cRPMI medium with PMA (50 µM) and ionomycin (1 µM; Sigma-Aldrich) for indicated times, or with anti-CD3/CD28 mAb (1 µg/mL each) for 24 hours.

For experiments with mouse cells, T lymphocytes were isolated from lymph nodes according to standard protocols and maintained in complete medium throughout the assay. Cells (2.5×10⁶) were cultured on anti-CD3 mAb-coated plates with medium containing anti-CD28 mAb (72 hours, 37°C).

For stimulation with TCS cells, TPR or Jurkat cells (5×10⁴) were cocultured with TCS cells (2×10⁴) in 100 µL medium in a 96-well round-bottom plate for the indicated times (37°C, 5% CO₂). Where indicated, 30 µM PS-1145 (IKKβ inhibitor), 50 µM PD98059 (MEK inhibitor), 20 nM FK506 (CaN inhibitor) or 0.5 µg/mL anti-PD-1 (nivolumab) was added.

TPR/TCS or Jurkat/TCS cocultures were stained with anti-mouse CD45-APC or Percp-Cy5.5 to exclude TCS cells from analysis. Live cells were gated using forward and side scatter parameters within the CD45^- subset. Expression of NFAT-GFP, NFκB-CFP and AP-1-Cherry, was determined by flow cytometry analysis of the live cell population using a CytoFLEX S flow cytometer (Beckman Coulter). Mean and SD of the geometric mean fluorescence intensity (gMFI) of the whole population and the percentage of positive cells were determined from triplicate wells. Transcription factor activity was calculated by normalizing data to the maximal activation for each experiment.

Jurkat and LN mouse T cells were washed after stimulation and stained with LIVE/DEAD fixable red cell death staining kit, and stained with a mixture of Ab. Cells isolated from tumors were subjected to the same procedure, in this case CD45 staining was used to exclude MC38 cells from analysis. CD8⁺ T cells were identified among total T cells (CD45⁺ TCR⁺) and the expression of CD25 and PD-1 evaluated with the corresponding Ab.

Data were analyzed using FlowJo software (V.10.2, FlowJo LLC, Ashland, Oregon, USA).

Cells were lysed and processed as in27 and western blots probed with specific antibodies. Protein bands were visualized by enhanced chemiluminescence (ECL detection, Amersham Bioscience) or with an Odyssey scanner (LI-COR). Densitometric analysis of proteins was performed using ImageJ.

TPR or Jurkat cells in logarithmic growth (4–5 x 10⁵ cells/mL) were transfected with 15–20 µg plasmid DNA by electroporation using Gene Pulser II (BioRad) (270 mV, 975 µF). For DGKζ silencing experiments, a validated sequence for the DGKζ isoform was selected to generate the shRNAi pSUPER construct.27 Equivalent murine sequences were cloned and used as negative controls.36 Expression was evaluated from days 1 to 4 post-transfection by western blot. Plasmids encoding the interleukin 2 (IL-2) promoter region were reported.23 The IL-2 promoter was obtained from Addgene. Renilla luciferase vector was from Promega.

TPR cells were transfected with 15 µg of the IL-2 promoter construct, with 200 ng renilla as internal control. After 24 hours, cells were washed, allowed to recover (6 hours) and stimulated using TCS cells for 24 hours (37°C, 5% CO₂). Cells were harvested and assayed for luciferase activity using the Dual-Luciferase Reporter assay (Promega). Luciferase activity was reported relative to renilla luciferase activity.

Supernatants from TPR/TCS cocultures were collected and IL-2 levels measured using the ELISA MAX Deluxe Set Human IL-2 kit (Biolegend).

MC38 cells were trypsinized from subconfluent monolayers, washed, and injected subcutaneously in 100 µL (5×10⁵) into one flank of female 6- to 8-week-old C57BL/6J wild type (wt) or DGKζ-deficient mice. Tumor growth was monitored in a blind manner with calipers, and area and volume were estimated according to the formulas: area=a x b/volume = (a² x b)/2, where a is tumor width and b is tumor length in mm. Mice were sacrificed when wt tumors reached 1 cm³, at ~19 days postinjection, and tumors were excised, measured and weighed.

For TIL isolation, tumors were fragmented into 1 mm³ pieces using a scalpel. Fragments were suspended in DMEM culture medium (Invitrogen) supplemented with 20 mM HEPES, with 2 mg/mL collagenase type I, 2.5 mg/mL dispase II and 0.1 mg/mL DNase I, and incubated with gentle shaking (15 min, 37°C). The resulting suspension was filtered with a 70 µm filter, washed with PBS+5% FBS and centrifuged (5 min, 300 X g, 4°C). Resulting pellets were processed for flow cytometry analysis.

Flow cytometry data were analyzed with GraphPad Prism V.6 software. Data are shown as mean±SEM Samples were assumed to fit normality. When more than two conditions were analyzed, we applied analysis of variance and Bonferroni post-test analysis. If not applicable, parametric unpaired t tests were performed. In all cases, differences were considered statistically not significant (ns) for p>0.05, and significant for p values *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

The TPR cell model allows the concurrent flow cytometry analysis of NFAT, NFκB and AP-1 transcriptional activation.33 These three transcription factors classically represent the end-point activation of Ca²⁺-dependent CaN activation, as well as of Ras/extracellular signal-regulated kinase (ERK)- and protein kinase C (PKC) θ/ΙκΒ kinase (IKK) β-regulated pathways. Flow cytometry analysis of fluorescent proteins coupled to transcription factors enables simultaneous quantification of the signal intensity as determined by the reporter gene induction on a per cell basis (gMFI). The percentage of responding cells reflects the digital characteristics of TCR-delivered signals that ensures scaled T cell responses according to dose and affinity for the antigens encountered.37 Stimulation of TPR cells with phorbol 12-myristate 13-acetate (PMA) and the Ca²⁺ ionophore ionomycin evidenced a strong, uniform cell response with distinct kinetics for the different reporters (figure 1A). The early, robust NFAT induction correlated with its direct nuclear entry as the result of its CaN-dependent dephosphorylation.38 The induction of NFκB or AP-1, which require successive activation of small GTPases and kinases, accumulated over time (figure 1A).

At difference from the uniform response induced by pharmacological stimulation, 24 hours treatment of TPR cells with agonistic mAb to the TCR-CD3 complex resulted in a small percentage of NFAT-positive and AP-1-positive cells but a higher proportion of NFκB activation (figure 1B). Addition of anti-CD28 mAb increased the activity of the three reporters (figure 1B), especially that of NFκB and AP-1 (figure 1B, middle and right), in accordance with the specific contribution of DAG-regulated pathways downstream of CD28 costimulation.23

As described,33 TPR coculture with control TCS cells (TCS control) promoted strong induction of NFAT and NFκB, with lower AP-1 induction (figure 1C). CD28 stimulation with CD86-expressing TCS cells (TCS-CD86), as shown for the CD80 ligand,33 further increased expression of the three reporters. Individual transcription factor analysis revealed maximal increase in the frequency of AP-1-expressing cells after costimulation (figure 1D). The effect of CD28 stimulation on a per cell basis was also observed for the three transcription factors, with maximal effect for NFκB expression (figure 1E). Bi-parametric analyzes confirmed a marked increase in the percentage of NFAT/AP-1-expressing cells following incubation with TCS-CD86 cells (online supplemental figure 1). These results add new information to existing data on this model, showing that it accurately reproduces the strict requirement for CD28 engagement to reach the AP-1 transcription threshold.

The activation of CaN in response to Ca²⁺ not only triggers NFAT nuclear translocation but also cooperates with PKCθ in NFκB and AP-1 regulation.39 In agreement, CaN inhibitor FK506 treatment prevented induction of the three transcription factors (figure 1F). NFAT transcription was independent on the PKCθ/IKKβ signals, as demonstrated by the minimal effect of the IKKβ inhibitor PS-1145. Pharmacological IKKβ blockade nonetheless markedly diminished NFκB and AP-1 activation, in accordance with results from primary human T cells40 (figure 1F). MEK inhibition by PD98059 limited AP-1 transcription but also partially decreased NFAT and NFκB induction (figure 1F), which confirmed the reported role for MEK, an intermediate kinase of the Ras/ERK axis, in the activation of these two transcription factors.41 42 Overall, these results confirm the critical requirement for the Ca²⁺/CaN axis, and reveal AP-1 transcriptional activation as the sensor most sensitive to signal limitation.

Biochemical studies combined with luciferase-based assays demonstrate that DGKζ silencing in Jurkat T cells leads to enhanced Ras/ERK and PKCθ/AKT pathway activation, which translates into enhanced NFκB transcription.27 The TPR model allowed us to examine the result of DGKζ silencing on the simultaneous activation of NFAT, NFκB and AP-1. We used previously validated shRNAi sequences to attenuate DGKζ expression in TPR cells. DGKζ targeting achieved similar extent of silencing than that reported in Jurkat T cells,27 without affecting that of DGKα (figure 2A and online supplemental figure 2). Flow cytometry analysis of DGKζ-silenced TPR cells at different times after coculture with TCS-control or TCS-CD86 cells showed an enhanced percentage of NFAT-, NFκB- and AP-1-positive cells that was detected as early as 4–6 hours poststimulation (figure 2B). The effect of DGKζ silencing varied for each transcription factor, with increased NFAT and AP-1 induction at early times that contrasted with the sustained, enhanced activation of NFκB throughout the kinetic course. Analysis on a per cell basis showed no major effect of DGKζ depletion for NFAT induction (figure 2C, left), but exhibited increased NFκB and AP-1 expression (figure 2C, middle and right). As also reported for mAb-stimulated Jurkat cells,27 43 DGKζ silencing did not replace the CD28 requirement for AP-1 induction (figure 2B–C, right). Analysis of the percentage of cells that coexpressed the three reporters confirmed a marked elevation in DGKζ-silenced cells in response to TCR alone or to TCR/CD28 stimulation (figure 2D). Our results illustrate the utility of fluorescent protein-based TPR cells to demonstrate that, as shown in mice, DGKζ potently limits the threshold of responding T cells.

Previous studies used the TPR system to demonstrate that PD-1 ligation results in strong NFAT/NFκB downregulation with a minimal effect on AP-1.33 We used the same PD-1-expressing TPR (TPR-PD-1) cells, but here we incubated them with TCS cells expressing CD86 alone or in combination with PD-L1 (TCS-CD86/PD-L1) to study the effect of PD-1 ligation in the presence of costimulatory signals. At difference from the effect reported for TCR single stimulation, PD-1 triggering with CD28 costimulation reduced the percentage (figure 3A) and the expression (figure 3B) of NFAT and AP-1, but had no effect on NFκB activation. These results coincide with the inhibition of TCR-driven early signals by PD-1 engagement as well as with studies showing that, in human T lymphocytes, CD28 promotes NFκB activation in the absence of TCR triggering.44

Correct and timely coordinated activation of NFAT, NFκB and AP-1 is indispensable for transcription of IL-2, which orchestrates the ensuing clonal expansion of effector populations.45 Analysis of a luciferase-coupled IL-2 promoter in TPR-PD-1 cells demonstrated the need for CD28 stimulation, and confirmed the PD-1-driven limitation of IL-2 transcription (figure 3C). Determination of IL-2 secretion by stimulated cells fully reflected the observations in the promoter experiments; only those cells stimulated by TCS-CD86 cells showed robust secretion of this cytokine, which was greatly restricted by PD-1/PD-L1 ligation (figure 3D). These results confirm a strict requirement for CD28 triggering to reach the correct AP-1 threshold, which acts in concert with NFAT and NFκB,46 47 and the limitation on IL-2 production imposed by PD-1 engagement, even in the presence of costimulatory signals.48 TPR-PD-1 cells stimulated with TCS-CD86/PD-L1 cells restored IL-2 production after incubation with an anti-PD-1 humanized Ab (nivolumab, nivo) (figure 3D). Our analyzes confirm the utility of the TPR cell model to reproduce the specific PD-1 contribution to T cell hyporesponsive states, as well as the effect of blocking antibodies used in the clinic.

We investigated the effect of DGKζ silencing in the context of PD-1 inhibitory function. As observed in TPR cells, DGKζ silencing in TPR-PD-1 cells increased the frequencies of NFκB- and AP-1-expressing cells, but diminished that of NFAT, compared with control cells (figure 4A–C). PD-1 ligation decreased NFAT transcription both in control and DGKζ-silenced cells (figure 4A), and had no obvious effect on NFκB induction (figure 4B). Nivolumab-mediated blockade of PD-1/PD-L1 interaction restored NFAT in control and DGKζ-silenced cells (figure 4A). Compared with control cells, DGKζ-silenced cells showed less sensitivity to PD-1-dependent AP-1 inhibition and displayed further induction of AP-1 following nivolumab treatment (figure 4C). Although the effects of DGKζ silencing were small in the analysis of each independent transcription factor, the evaluation of percentage of TPR-PD-1 cells that coexpressed the three reporters confirmed a significantly higher proportion of responding cells following DGKζ silencing, which nonetheless decreased after PD-1 triggering and was fully restored by nivolumab treatment (figure 4D). The biological significance of these changes was reflected by the analysis of IL-2 secretion, which confirmed enhanced IL-2 production by DGKζ-silenced TPR-PD-1 cells (figure 4E). DGKζ silencing did not confer resistance to PD-1 engagement, resulting in severe impairment of IL-2 secretion, with partial but significant recovery following nivolumab addition (figure 4E).

The previous experiments demonstrate the utility of the TPR model for studying the contribution of DGKζ-regulated pathways in the activation of positive and negative receptors in human T cells, and suggest that this platform could be used to profile isoform-specific DGK inhibitors. TPR cells are nonetheless an artificial system in which receptors can be analyzed at a fixed level of expression. Naïve T cells do not constitutively express PD-1, which is induced following activation.49 PD-1-mediated inhibition depends on TCR signal strength, with greater inhibition at low levels of TCR stimulation.50 In line with this fact, PD-1-delivered inhibition can be overcome by CD2851 or IL-2.48 IL-2 production in response to CD28 engagement is central to CD28 ability to rescue from PD-1-driven negative regulation.48 IL-2 leads to cell expansion by ensuring expression of CD25, which constitutes the high affinity receptor for this cytokine. We studied the kinetics of CD25 and PD-1 induction in Jurkat T cells stimulated with PMA and ionomycin or TCS-CD86 cells. As shown, both proteins were rapidly induced as early as 6 hours in response to both stimuli (figure 5A–C). In agreement with IL-2-dependent regulation of both proteins,52 by 24 hours CD25 expression was further increased, whereas PD-1 expression was reduced (figure 5A–C).

Concurring with its function as a negative regulator of CD28 signals, DGKζ silencing further reduced the percentage of PD-1⁺ Jurkat cells (figure 5D). The decrease in PD-1 expression was particularly marked in the CD25⁺ T cell population (figure 5E). Consistent with observations in the Jurkat model, stimulation of mouse T cells from DGKζ-deficient mice also led to a notable reduction in the percentage of PD-1⁺ CD8⁺ T cells compared with wt mice (figure 5F). These results indicate conservation in human and mouse T lymphocytes of a positive feedback by which DGKζ promotes PD-1expression and negative function.

Antigen recognition facilitates PD-1 upregulation in TIL, whereas PD-L1 expression by tumor or stromal cells increases the probability of PD-1 ligation.10 This leads to a scenario in which IL-2 production is reduced and the expansion of CD8⁺ CTL populations is limited.13 Our previous observations showing a correlation between enhanced IL-2 production and reduced PD-1 expression as a result of DGKζ depletion might be a plausible mechanism to explain the enhanced control of engrafted tumors observed in DGKζ-deficient mice.28–30 Although the Jurkat cell line is a good platform in which to study cytokine production, these cells lack cytotoxic abilities. We, thus, moved to mouse models that facilitate the examination of the DGKζ modulation of antitumor T cell functions.

At difference from findings in human colorectal tumors, which are in most cases PD-1-resistant, the induction of orthotopic tumors in mice by subcutaneous injection of MC38 adenocarcinoma cells is a classical model of sensitivity to PD-1/PD-L1 immune checkpoint blockade.53 We selected this tumor model to test whether the observed modulation of the CD25/PD-1 axis by DGKζ could affect tumor development. Analysis of growth kinetics of MC38 engrafted cells showed a notable growth lag in DGKζ-deficient mice, which developed considerably smaller tumors than those in wt animals by the time at which they had to be sacrificed (figure 6A–B). We explored existing differences in CD25⁺ and PD-1⁺ CD8⁺ T cell populations in TIL isolated from tumors implanted in wt and DGKζ-deficient mice. TIL analysis showed similar percentage of live (figure 6C) and CD45⁺ cells (figure 6D) with a tendency, not statistically significative, of higher CD8⁺ T cell percentage in TIL isolated from tumors in DGKζ-deficient mice (figure 6E). TIL isolated from tumors grown in DGKζ-deficient mice showed a significant increase in CD25⁺ CD8⁺ T cell frequency (figure 6F) and in CD25 expression per cell (figure 6G) compared with those from wt mice. TIL analysis showed no marked differences in the percentage of PD-1⁺ CD8⁺ T cells (figure 6H) but, in agreement with findings in Jurkat and mouse T cells, PD-1 expression specifically in the CD25⁺ CD8⁺ T cell population was lower in TIL from DGKζ-deficient than in those from wt mice (figure 6I). These results confirm a role for DGKζ in limiting effector functions, and suggest DGKζ targeting as a potential means to avoid exhausted T cell phenotypes, even in the context of immunosuppressive milieus.