Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca²⁺ mobilizing second messenger discovered to date, has been implicated in Ca²⁺ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca²⁺ signaling or the identity of the Ca²⁺ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca²⁺ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca²⁺ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca²⁺ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca²⁺ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca²⁺ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.

Introduction

Ca²⁺ signaling is critical in T lymphocyte activation upon stimulation through the T cell receptor (TCR).² It is essential for proliferation, differentiation, gene transcription, and effector functions (1, 2). In T cells, triggering through the TCR stimulates synthesis of second messengers that cause release of Ca²⁺ from internal stores. The resulting depletion of ER stores causes activation of store-operated Ca²⁺ entry (SOCE) via the STIM/Orai complex, which is the major factor allowing influx of external Ca²⁺ (3). Although much is known about the canonical Ca²⁺ release second messengers d-myo-inositol-1,4,5-triphosphate (IP₃) and cyclic ADP-ribose (cADPR), the mechanism of action of NAADP, the most potent Ca²⁺ releasing agent identified to date (4), is less clear in T-lymphocytes and in general. In Jurkat T cells, stimulation through the TCR causes a biphasic increase in cellular NAADP that is inhibited by tyrosine kinase inhibition. NAADP shows a sharp peak in the first 20 s after triggering followed by a low peak that is long lasting (5).

NAADP was found to be an essential regulator involved in the initiation and propagation of Ca²⁺ signals in Jurkat T lymphoma cells (6). Microinjection of NAADP into Jurkat cells caused Ca²⁺ release with a bell-shaped dose-response curve. Co-injection of an IP₃ receptor antagonist with NAADP reduced the overall Ca²⁺ release, suggesting that some Ca²⁺ release to NAADP may be due to synergistic Ca²⁺-induced Ca²⁺ release by other mediators. This amplification of other Ca²⁺ releasing signals by NAADP has been termed the “trigger hypothesis” (7). Microinjection of desensitizing high concentrations of NAADP into Jurkat cells reduced Ca²⁺ release to co-injected IP₃ and cADPR and reduced the overall Ca²⁺ increase to TCR triggering by OKT3 (6), which includes both internal Ca²⁺ release and influx of external Ca²⁺. Therefore, disruption of NAADP signaling could have a pronounced effect on T cell responses beyond the amount of Ca²⁺ that it directly releases. The molecular identity of the NAADP-binding protein is unknown and may differ among species (8–11). Whether it mobilizes Ca²⁺ from ER/SR stores or from lysosomal-like acidic stores or both is controversial (12, 13).

Although useful as a model to study NAADP-mediated Ca²⁺ signaling in T lymphocytes, Jurkat T lymphoma cells may not behave as primary conventional T cells. Specifically, it is unclear whether the target Ca²⁺ store of NAADP in conventional naive T cells involves lysosomal acidic stores, or whether NAADP evokes release of Ca²⁺ from the ER via the ryanodine receptor (RyR), as convincingly demonstrated by Guse and colleagues (14–17) in Jurkat T lymphoma cells, or both. In addition, the physiological importance of NAADP in regulating conventional naive T cell function has not yet been thoroughly investigated. These reasons prompted us to study NAADP-mediated Ca²⁺ signaling in conventional naive mouse CD4 and CD8 T cells and to determine the target Ca²⁺ stores sensitive to NAADP. Importantly, we also investigated the role of NAADP in regulating critical downstream physiological end points such as T cell proliferation and cytokine production.

Upon activation by antigen, naive CD4 T cells can differentiate under the influence of cytokines into several subsets of effector T cells including Th1, Th2, and Th17. How important NAADP-mediated Ca²⁺ release is for the function of these different subsets of effector T cells is not clear. For example, a recent study showed that NAADP mobilizes acidic Ca²⁺ stores in a primary human CD8 cytotoxic T lymphocyte clone (18), differing from the observations in Jurkat cells.

Thus, in this report, we also sought to answer the following questions: would NAADP exhibit the same mechanism in effector T cells as their counterpart naive T cells? Is NAADP-mediated Ca²⁺ signaling equally important for the effector function of different subsets of T cells? To this end, we addressed the involvement of intracellular Ca²⁺ stores in NAADP-mediated Ca²⁺ release in effector CD4 and CD8 T cells as well as the physiological importance of NAADP signaling for the functions of effector CD4 subsets (Th1, Th2, and Th17) and CD8 effector T cells. We also examined natural T regulatory cells (Tregs) for evidence of NAADP involvement in signaling.

Experimental Procedures

Mice

Six to 8-week-old female C57BL/6 (B6) and OT-II (B6.Cg-Tg (TcraTcrb) 425Cbn/J) mice were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were maintained in the animal facility at the University of Toledo Health Science Campus under a specific pathogen-free environment. All mouse experiments were performed according to NIH guidelines with approval of the Institutional Animal Care and Use Committee.

Medium

T cell medium was Roswell Park Memorial Institute medium (RPMI)-1640 with 116 μg/ml of arginine, 36 μg/ml of asparagine, 216 μg/ml of glutamine, 110 μg/ml of pyruvate, 6 μg/ml of folic acid, 10% fetal bovine serum (Atlanta Biological), 10 mm HEPES, 1000 units/ml of penicillin, 100 μg/ml of streptomycin, and 0.03 mm mercaptoethanol. In some experiments, HBSS (Sigma) with or without Ca²⁺ was used.

CD4 and CD8 Isolation

Single cell suspensions were prepared from mouse spleens, cleared of erythrocytes, and filtered through a 70-μm nylon mesh cell strainer (Fisher). A Dynabeads Flow-Comp Mouse CD4 or CD8 kit (Invitrogen) was used to positively isolate CD4 or CD8 T cells according to the protocol provided. FACS analysis showed that CD4 and CD8 cells were greater than 92% CD4⁺ or CD8⁺ cells, respectively. Natural T regulatory cells were isolated using a Dynabeads Flow-Comp Mouse CD4⁺CD25⁺ Treg kit.

Naive CD4 and CD8 Proliferation Assays

Ninety-six well plates were coated with anti-CD3 (2 μg/ml, eBioscience) and anti-CD28 (2.5 μg/ml, eBioscience) in a volume of 100 μl/well for 3 h at 37 °C and washed with phosphate-buffered saline (PBS) two times before using. Isolated CD4 or CD8 T cells (2 × 10⁶ cells/ml) in T cell medium were preincubated for 1 h at 37 °C and 5% CO₂ with increasing amounts of Ned-19 in DMSO or with DMSO alone and then added (4 × 10⁵ cells/well) to the prepared plates in triplicate wells. When BZ194 was used, cells were preincubated for 5 h with BZ194 prior to stimulation. BZ194 was synthesized according to the procedure described in Ref. 16. For antigen-specific proliferation, OT-II CD4 T cells were isolated and stimulated with ovalbumin (10 μg/ml) together with 10⁵ irradiated (1500 Rad) CD4-depleted spleen cells per well. As controls, PMA (100 ng/ml, Sigma) and ionomycin (200 ng/ml, Calbiochem) were used for stimulation. After 48 h of stimulation, [³H]deoxythymidine ([³H]dT) was added (1 μCi/well, Moravek Biochemicals, Inc.). After 24 h of [³H]dT incorporation, the cells were harvested onto a filter plate (PerkinElmer Uni-Filter-96 GF/B) and dried overnight. Proliferation was then evaluated by adding liquid scintillation mixture (MP Biomedicals) to the filter plate (40 μl/well) and scintillation counting in a PerkinElmer Top Count NXT.

Nonpolarized Effector CD4 and CD8 Generation and Proliferation Assays

Twelve-well plates (Fisher Scientific) were coated with anti-CD3 and anti-CD28 as above. Isolated naive CD4 or CD8 T cells (2 × 10⁶ cell/ml) in T cell medium supplemented with IL-2 (40 units/ml, PeproTech, Inc.) were added to the prepared plate in a volume of 2 ml/well and incubated for 48 h at 37 °C and 5% CO₂. The cells were then transferred to an uncoated 6-well plate with addition of 2 ml of fresh T cell medium and IL-2 (20 units/ml) and incubated for 3 more days. On day 5, CD4 or CD8 cells (1.5 × 10⁶ cells/ml) in fresh T cell medium were preincubated for 1 h at 37 °C and 5% CO₂ with increasing amounts of Ned-19 in DMSO or with DMSO alone and then re-stimulated in a 96-well plate pre-coated with anti-CD3 (2 μg/ml) in triplicate wells. For antigen-specific proliferation, CD4⁺ T cells were isolated from mice primed 2 weeks earlier with ovalbumin (100 μg/mouse) and the cells were stimulated with ovalbumin (10 μg/ml). The procedure was continued as described above for naive cells.

Th1, Th2, and Th17 Polarization

For the generation of functionally polarized Th1, Th2, and Th17 effector T cells, the isolated naive CD4 T cells were stimulated by anti-CD3/CD28 and cultured in the presence of polarizing conditions: IL-2 (40 units/ml), IL-12 (40 ng/ml, PeproTech, Inc.), and anti-IL-4 (20 μg/ml, Biolegend) for Th1 and IL-2 (40 units/ml), IL-4 (4 ng/ml, PeproTech, Inc.), anti-IFNγ (20 μg/ml, Biolegend), and anti-IL-12 (20 μg/ml, Biolegend) for Th2, and TGFβ (10 ng/ml, PeproTech, Inc.), IL-6 (100 ng/ml, Biolegend), anti-IL-4 (10 μg/ml), and anti-IFNγ (10 μg/ml) for Th17. On day 5, FACS analysis showed ≥90% pure Th1, Th2, or Th17 subsets were obtained. Cells were then re-stimulated with anti-CD3 in the presence of increasing concentrations of Ned-19 following the same proliferation assay described above.

Ca²⁺ Flux

Primary or effector CD4 and CD8 T cells were loaded with Fura-2 AM fluorescent dye (2 μm, ThermoFisher) for 30 min at 37 °C. Cells were then incubated at 37 °C with the reagent of interest, trans-Ned-19 (Tocris Biosciences) for 1 h, bafilomycin A1 (1 μm, LC Laboratories) for 30 min, thapsigargin (1 μm, Calbiochem) for 30 min, tetracaine (100 μm, Sigma) for 20 min, SKF96365 (25 μm, Cayman Chemical) for 10 min, 18β-glycyrrhetinic acid (100 μm, Santa Cruz Biotechnology) for 20 min, octanol (1 mm, Sigma) for 15 min, or dipyridamole (100 μm, Sigma) for 15 min. The cells were plated for 10–15 min on glass bottom dishes (Mattek P50G-1.5–30-F) that were pre-coated with poly-l-lysine in PBS (10–50 μg/ml) for 30–60 min prior to use. Determination of [Ca²⁺]_i was performed using digital fluorescence imaging microscopy, ×100 oil objective, with a monochronometer-based system and high speed CCD camera (T.I.L.L.-Photonics). Cells were alternately excited at 340/380 ± 15 nm and the fluorescence emission was collected through a 510 ± 25-nm band pass filter (Chroma). Ca²⁺ release was stimulated by exogenous NAADP or by the addition of biotinylated anti-CD3 monoclonal antibody (5 μg/ml, eBioscience) followed by streptavidin (50 μg/ml, Jackson ImmunoResearch). Unless noted otherwise, the increase in peak amplitude of the 340/380 ratio relative to baseline was measured for multiple fields per dish.

Imaging of Lysosomes in Intact T Cells

T cells were incubated with LysoTracker Red (30 nm, Molecular Probes) for 30 min and with or without Ned-19 for 1 h at 37 °C. Samples were acquired using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems, Bannockburn, IL) equipped with the LASAF software suite, conventional lasers (488, 561, and 633), and a tunable Ti-sapphire multi-photon laser (Coherent, Santa Clara, CA). Images were acquired in a 512 × 512 format at 800 Hz bi-directionally using a HCX PL APO CS 63.0X1.20 water UV objective in a sequential sequence to minimize any spectral overlap. The LysoTracker Red was imaged using excitation/emission of 577/590 nm. Ned-19 was imaged using the multi-photon laser excitation at 760 nm and collection in the range of 415–485 nm. In some experiments, the lysosomal pH gradient was disrupted by bafilomycin A1 (1 μm).

Flow Cytometry

An Accuri c6 (BD Biosciences) was used for immunophenotyping analysis. Anti-mouse antibodies used for surface staining were FITC anti-CD4 or CD8, APC anti-CD44, and APC anti-CD25 (50 μg/ml, all from eBioscience). For connexin-43 hemichannel expression, cells were fixed/permeabilized overnight and then incubated in 100 μl of PBS, 10% normal goat serum, and 0.3 m glycine to block nonspecific protein-protein interactions. The cells were then stained with anti-connexin-43 ab79010 antibody (1 μg/1 × 10⁵ cells, Abcam) for 30 min at room temperature and washed with 100 μl of 1× permeabilization buffer. The cells were then stained with the secondary antibody FITC-goat anti-mouse IgG (10 μg/ml, BD Pharmingen) for 30 min at room temperature followed by washing and re-suspension in 1× permeabilization buffer.

For immunophenotyping of polarized effector T cells, FITC anti-CD4 stained cells (1 × 10⁵) were fixed/permeabilized overnight using a fixation/permeabilization kit (eBioscience), and then stained for transcription factors with anti-T-bet (50 μg/ml, eBioscience), anti-GATA3 (50 μg/ml, eBioscience), or anti-RORγ (50 μg/ml, eBioscience) for 1 h. The cells were then washed with 1× permeabilization buffer and kept on ice in the dark until analyzed. FoxP3 expression in natural Tregs was determined using PE-anti-FoxP3 following fixation and permeabilization (eBioscience).

ELISA

CD4 or CD8 naive or effector T cells were stimulated in the presence or absence of Ned-19 (100 μm) for 48 h and the secreted IL-2, INFγ, IL-4, or IL-17 were quantitated using the relevant Mini ELISA Development Kit (PeproTech). All procedures were performed according to the kit protocol, except that 1× tetramethylbenzidine (Thermo Scientific) was used asthe substrate. The absorbance was measured at 620 nm on a SpectraMax^TM Plus spectrophotometer (Molecular Devices, Sunnyvale, CA).

Analysis of NFAT-1 and NF-κB Translocation

Naive CD4 T cells were stimulated with anti-CD3/CD28 in the presence or absence of Ned-19 (100 μm) and transferred onto glass coverslips pre-coated with poly-l-lysine (50 μg/ml). Samples were fixed in paraformaldehyde (4%) at 0, 1, 24, or 48 h after stimulation for 30 min and permeabilized with Triton X-100 (0.3%). Immunohistochemical staining was performed overnight at 4 °C using mouse monoclonal antibody against NFAT-1 (Abcam) or rabbit polyclonal antibody against NF-κB P65 (Santa Cruz). DyLight488-conjugated anti-mouse (Jackson ImmunoResearch) and Alexa Fluor 594-conjugated anti-rabbit (Jackson ImmunoResearch) were used as secondary antibodies. DAPI (Santa Cruz) was used for nuclear staining. Immunofluorescence images were taken with a Nikon Eclipse Ti microscope using a ×100 oil objective. Fluorescence was quantitated by NIS-Element BR. A region of interest was determined over the nucleus of each cell. The ratio of fluorescence in the NFAT or NF-κB channel to that of the DAPI channel was determined over that region of interest and averaged for 80–100 cells.

NAADP Treatment

NAADP was synthesized from NADP and nicotinic acid using ADP-ribose cyclase from Aplysia californica according to the procedure of Trabbic et al. (19) and dissolved in PBS for cell treatment.

NAADP Transport

[³²P]NAADP was synthesized according to the procedure outlined in Ref. 9. [³²P]NAD (6000Ci/mmol) was converted to [³²P]NADP using human NAD kinase. This was converted to [³²P]NAADP by base exchange with nicotinic acid via Aplysia ADP-ribosyl cyclase. The product was purified by AG MP-1 column chromatography and lyophilized. The specific activity of the [³²P]NAADP was adjusted with added unlabeled NAADP according to the final NAADP concentration desired. Cells (10⁶ per 0.1 ml in HBSS with 0.1 mg/ml of BSA) were layered over 0.1 ml of oil (silicone oil:paraffin oil 80:20), incubated for varying times with [³²P]NAADP, and rapidly centrifuged to pellet the cells, according to the procedure of Kim et al. (20).

Caged NAADP

1-(4,5-Dimethoxy-2-nitrophenyl)diazoethane-caged NAADP was synthesized according to Ali et al. (10). Cells in RPMI were loaded with 2 μm Fluo-4 AM (ThermoFisher) for 30 min at 37 °C and adhered as above. Cells were treated with 1 mm octanol or control for 15 min, followed by 30 μm caged NAADP for 10 min. Cells were washed with RPMI after treatment. Cells were excited at 488 ± 15 nm and monitored through a 510 ± 25 nm band pass filter. Free cytosolic Ca²⁺ was measured as in Ref. 10 after applying 8 flashes of UV light (360 ± 7.5 nm) to release active NAADP.

Statistical Analysis

Data are expressed as mean ± S.E. Statistical comparisons between three or more groups were performed with Tukey one-way analysis of variance. Significant differences between two groups were determined using the unpaired Student's t test. Statistical significance was set at p < 0.05.

Results

NAADP Antagonist Ned-19 Inhibits Receptor-mediated Ca²⁺ Signals in Naive T Cells

To test the role of NAADP in naive T cell Ca²⁺ signaling upon anti-CD3/TCR complex stimulation, we have used Ned-19, a molecule that has been previously shown to selectively antagonize NAADP-mediated Ca²⁺ release, but did not affect either IP₃ or cADPR-mediated Ca²⁺ release in sea urchin egg homogenates (21, 22). CD4 cells were isolated from spleens of untreated C57BL/6 mice. These were 85–90% CD44^lo naive cells (Fig. 1A, left panel). Purified T cells were first incubated with biotinylated anti-CD3, which was then cross-linked by addition of streptavidin to initiate signal transduction. The increase in cellular Ca²⁺ observed was dependent on both internal and external Ca²⁺, as absence of external Ca²⁺ or addition of the SOCE inhibitor SKF96365 greatly reduced but did not eliminate the increase (data not shown). As shown in Fig. 1, B and C, preincubation of T cells with Ned-19 altered Ca²⁺ signals in naive CD4 T cells evoked by anti-CD3 stimulation. Ned-19 delayed the response and significantly reduced both the initial Ca²⁺ peak and the sustained Ca²⁺ plateau. The inhibition by Ned-19 was concentration dependent with a maximal inhibition at 100 μm. A similar result was observed in naive CD8 T cells (Fig. 1, A, right panel, and D). As expected, the evoked Ca²⁺ signal was not totally eliminated, indicating the involvement of other Ca²⁺ signaling pathways. However, the Ca²⁺ signal was reduced by approximately two-thirds, suggesting that NAADP contributes prominently, either directly or indirectly, to overall Ca²⁺ increases during the activation of naive T cells. The effect of Ned-19 was observed at the level of intracellular Ca²⁺ stores. When Ned-19 was added to cells stimulated in the absence of extracellular Ca²⁺, the Ca²⁺ increase was still reduced (Fig. 1E).

FIGURE 1.

The NAADP antagonist Ned-19 inhibits Ca²⁺ signaling in naive T cells. A, CD4 or CD8 cells were stained with anti-CD4 or CD8-FITC, respectively, and anti-CD44-APC. Eighty to 90% were CD44^lo naive cells. B, representative ratiometric traces (340/380) of Ca²⁺ concentration in single naive CD4 T cells induced by anti-CD3 stimulation. Fura-2-labeled cells were incubated with medium (Control) or Ned-19 for 1 h and then stimulated biotinylated anti-CD3 (αCD3) followed by streptavidin (X) or PBS. C, concentration-response curve of anti-CD3-induced Ca²⁺ signaling in naive CD4 T cells following treatment with increasing concentrations of Ned-19; mean maximal increases in peak amplitude from 3 experiments; *, p < 0.001. D, same as C for naive CD8 T cells; mean maximal increases were from 3 experiments; *, p < 0.01; **, p < 0.001. E, anti-CD3-induced Ca²⁺ signaling in naive CD4 T cells in the absence of external Ca²⁺ with or without Ned-19 treatment. F, store depletion was induced via thapsigargin (TG) following biotinylated anti-CD3 (αCD3) and streptavidin (X) stimulation of Ned-19-treated naive CD4 T cells (red) or in cells that received neither Ned-19 nor anti-CD3 stimulation (untreated, blue). G, naive CD4 T cells were preincubated for 1 h with Ned-19 (100 μm) or PBS (control). Cyclopiazonic acid (CPA, 30 μm, arrow) was added in the absence of external Ca²⁺ (EGTA) followed by addition of 2 mm Ca²⁺ containing medium to induce SOCE; mean maximal increases from 3 experiments after subtracting background are summarized in H.

The strong inhibition of Ca²⁺ increases in the presence of extracellular Ca²⁺ is likely due to a combination of inhibition of internal Ca²⁺ release and reduced influx of extracellular Ca²⁺ due to incomplete store depletion. To test this hypothesis, we induced store depletion using thapsigargin in Ned-19-treated naive CD4 T cells after they were stimulated with anti-CD3. The Ca²⁺ signal was similar to that induced in untreated cells, which received neither anti-CD3 stimulation nor Ned-19 treatment (Fig. 1F). This indicated that anti-CD3 stimulation does not deplete stores in Ned-19-treated cells. In addition, Ned-19 did not directly inhibit SOCE. As shown in Fig. 1, G and H, Ca²⁺ signaling in naive CD4 T cells induced by cyclopiazonic acid, a specific inhibitor of the reticular Ca²⁺-ATPase that causes store depletion and resultant SOCE via STIM/Orai activation, was not affected. Our data suggest a critical role for NAADP in triggering Ca²⁺ signals during the early events of the receptor-mediated response in naive T cells.

Acidic Ca²⁺ Stores Contribute to Receptor-mediated Ca²⁺ Signals in Naive T Cells

As a possible target for NAADP, we investigated the involvement of lysosomal like Ca²⁺ stores in Ca²⁺ signaling. We depleted the endolysosomal Ca²⁺ using the vacuolar proton pump (H⁺-ATPase) inhibitor bafilomycin A1 and monitored cytosolic Ca²⁺ evoked by anti-CD3 stimulation. In this set of experiments, the punctate LysoTracker Red labeling of acidic compartments was abolished with bafilomycin A1 treatment compared with control (Fig. 2A), indicating the dissipation of the pH gradient and leakage of dye into the cytosol. As shown in Fig. 2, B and C, bafilomycin A1 treatment reduced the receptor-mediated Ca²⁺ signal observed in naive CD4 T cells by two-thirds. The effects of Ned-19 and bafilomycin A1 were quite similar and the question arose whether Ned-19 acts on the same Ca²⁺ store as bafilomycin A1. To answer this question, we repeated this experiment using cells pretreated with both Ned-19 and bafilomycin A1 to determine whether the effects were additive. Interestingly, the resulting Ca²⁺ signals from combined treatment with Ned-19 and bafilomycin A1 were similar to those observed after treatment with either Ned-19 or bafilomycin A1 alone (Fig. 2, B and C). Furthermore, we determined that Ned-19 fluorescence localized to acidic stores in naive CD4 T cells. As shown in Fig. 2D, Ned-19 and LysoTracker Red labeled the same acidic compartments in naive CD4 T cells after combined staining with both fluorescent molecules. The possibility of autofluorescence was ruled out because no fluorescence was observed in non-treated naive CD4 T cells compared with Ned-19-treated cells (data not shown). These data suggest that lysosomal-like acidic Ca²⁺ stores are involved in the generation of receptor-mediated Ca²⁺ signals in naive T cells. This involvement is perhaps dependent on NAADP because IP₃ and cADPR are known to mediate Ca²⁺ release via the ER and would not be directly affected by bafilomycin treatment.

FIGURE 2.

Effect of bafilomycin A1 on receptor-mediated Ca²⁺ signaling in naive CD4 T cells reveals lysosomal sensitivity to NAADP. A, LysoTracker Red labeling of naive CD4 T cells in the absence (Control) or presence of bafilomycin A1 (Baf). Expanded view is boxed. B, similar inhibition of Ca²⁺ signaling in naive CD4 T cells after treatment with Baf (1 μm) for 30 min, Ned-19 (100 μm) for 1 h, or both. Signaling was induced with biotinylated anti-CD3 (anti-CD3) and streptavidin (X). C, summary of mean maximal increases from 3 experiments after subtracting background; *, p < 0.001. D, overlay of fluorescent labeling of CD4 T cells with 100 μm Ned-19 (green) together with 30 nm LysoTracker Red (red); overlay (yellow). The expanded view is boxed. E, representative traces showing significant reduction in receptor-mediated Ca²⁺ signals following pretreatment with thapsigargin (Tg) (1 μm) for 30 min or a mixture of 100 μm Ned-19, 1 μm Baf, and 1 μm Tg for 30 min; F, summary of mean maximal increases from 3 experiments after subtracting background; *, p < 0.001.

Although we showed that the Ca²⁺ signal was reduced by about two-thirds following treatment with the NAADP antagonist Ned-19 or upon the inhibition of lysosomal Ca²⁺ uptake, our data does not exclude the possibility of the co-involvement of ER stores. To examine the involvement of the ER, we monitored the Ca²⁺ signals evoked by anti-CD3 stimulation after the depletion of Ca²⁺ from the ER stores using a known inhibitor of the SERCA-ATPase pump, thapsigargin. As shown in Fig. 2, E and F, the resulting Ca²⁺ signal was significantly reduced as expected, because Ca²⁺ increase after TCR stimulation depends on both ER store release and store-operated Ca²⁺ entry. Combined treatment with Ned-19, bafilomycin A1, and thapsigargin led to a complete loss of Ca²⁺ signaling, indicating that the Ca²⁺ source inhibited by Ned-19 and bafilomycin A1 is different from the source affected by thapsigargin.

Exogenous NAADP-mediated Ca²⁺ Signaling in Naive T Cells

Although many mammalian cells are impermeable to NAADP, as is expected for an anionic dinucleotide, previous studies have shown that extracellular application of NAADP could induce Ca²⁺ release in astrocytes (23) and in a rat basophilic cell line (24). Recent reports further showed that exogenous NAADP was transported into human lymphokine-activated killer cells and induced Ca²⁺ release from lysosomal-like acidic stores in those cells (25) and in mouse pancreatic β cells and adipocytes (26). These observations prompted us to test this approach to investigate the NAADP-mediated Ca²⁺ release mechanism in a more specific manner through direct activation of Ca²⁺ release in naive T cells by NAADP.

To determine the concentration-response relationship, we administered a known concentration of NAADP into plated naive CD4 cells and monitored the [Ca²⁺]_i fluorometrically as before. As shown in Fig. 3A, exogenous NAADP as low as 0.03 μm elicited a Ca²⁺ response, whereas PBS had no effect. The concentration-response relationship showed a bell-shaped curve for the Ca²⁺ peak with an optimal NAADP concentration at 3 μm. A bell-shaped dose response to NAADP is observed in other mammalian cells (6, 7). To test the possibility of purinergic receptor contribution, we added equimolar concentrations of the similar nucleotide NADP under the same conditions. As shown in Fig. 3, B and C, the signal induced by NADP was comparable with that induced by PBS, but not significant compared with the signal induced by NAADP. Although NAADP was reported to be an agonist of the human P2Y11 purinergic receptor, the optimal stimulation of this purinergic receptor was observed at 100 μm NAADP with no effect at 1 μm (27). The maximum Ca²⁺ signal induced by NAADP approximately equals two-thirds of the signal induced by anti-CD3 in the absence of Ned-19 (compare Figs. 1C with 3C), supporting the idea that NAADP can initiate some but not all of the Ca²⁺ signals generated by receptor stimulation.

FIGURE 3.

Exogenous NAADP-mediated Ca²⁺ release in naive CD4 T cells. A, concentration-response curve for exogenously added NAADP; mean maximal increases were from 3 experiments; *, p < 0.01. B, representative ratiometric traces of Ca²⁺ concentration in naive CD4 T cells induced by exogenously added NAADP (3 μm), NADP (3 μm), or PBS; mean maximal increases from 3 experiments are summarized in C; *, p < 0.001. D, Ca²⁺ release response to exogenous NAADP (3 μm) in the presence or absence of the SOCE inhibitor SKF96365 (25 μm) in HBSS or in Ca²⁺-free HBSS (0 Ca²⁺), mean maximal increases from 3 experiments after subtracting background. E, sensitivity of acidic Ca²⁺ stores to NAADP. Ca²⁺ signals evoked by exogenous NAADP (3 μm) following pretreatment with Ned-19 (100 μm) for 1 h, Baf (1 μm) for 30 min, or both; mean maximal increases were from 3 experiments after subtracting background; *, p < 0.001. F, ER involvement in NAADP-mediated Ca²⁺ signaling. Significant reduction of Ca²⁺ signals evoked by exogenous NAADP (3 μm) following pretreatment with the RyR antagonist tetracaine (Tet, 100 μm) for 20 min, thapsigargin (Tg) (1 μm) for 30 min, or a mixture of 100 μm Ned-19, 1 μm Baf, and 1 μm Tg for 30 min; mean maximal increases were from 3 experiments after subtracting background; *, p < 0.001. G, expression of connexin-43 hemichannels in naive CD4 T cells. Cells were stained with (gray peak) or without (black peak) anti-connexin-43 ab79010 followed by FITC anti-mouse IgG; mean data (n = 3) are summarized as mean fluorescence intensity. H, effect of connexin-43 hemichannel blockers or nucleoside transporter inhibitor on Ca²⁺ release evoked by exogenous NAADP (3 μm). Cells were pretreated with 18β-glycyrrhetinic acid (100 μm), octanol (1 mm), dipyridamole (100 μm), or octanol and dipyridamole combined for 15 min; mean maximal increases were from 3 experiments after subtracting background; *, p < 0.05; **, p < 0.01.

It was clear that exogenous NAADP elicited a Ca²⁺ response, but what was not clear was whether this Ca²⁺ signal was due to NAADP gating a plasma membrane channel, causing Ca²⁺ influx, or due to NAADP internalization and induced Ca²⁺ release from internal stores. In fact, the response to exogenous NAADP was slow, as shown in Fig. 3B, compared with the Ca²⁺ signals mediated upon TCR/CD3 ligation as in Fig. 1B, which started within a few seconds. This might indicate the time needed for NAADP to be imported into the cells before inducing Ca²⁺ release internally, but it does not exclude the possibility of external Ca²⁺ influx. Previous studies have shown that NAADP regulates transient receptor potential (TRPM2) channels in T lymphocytes (28), and that NAADP-mediated Ca²⁺ influx is triggered from TRPM2 channels in human neutrophils (29). To determine whether Ca²⁺ influx contributes to NAADP-induced Ca²⁺ signals in naive CD4 T cells, we monitored the change in [Ca²⁺]_i evoked by exogenous NAADP in Ca²⁺-free medium. As shown in Fig. 3D, the Ca²⁺ signal in Ca²⁺-free medium was reduced compared with that observed in Ca²⁺-containing medium. Similarly, pretreatment with the SOCE inhibitor SKF96365 reduced the Ca²⁺ increase caused by external NAADP. This data indicated that Ca²⁺ release caused by externally added NAADP is partially from internal stores and partially from influx through activation of store-operated Ca²⁺ entry.

Consistent with this idea, we determined which intracellular Ca²⁺ stores were affected by exogenous NAADP. First, we depleted the endolysosomal Ca²⁺ using bafilomycin A1 and monitored cytosolic Ca²⁺ evoked by exogenous NAADP. As shown in Fig. 3E, the Ca²⁺ signal was reduced by 80% in bafilomycin A1-treated cells compared with control cells. This was consistent with the observation that Ned-19 reduced the Ca²⁺ response by 80% compared with control (Fig. 3E). Moreover, treatment with both Ned-19 and bafilomycin A1 did not cause significant inhibition beyond that caused by either one alone (Fig. 3E). These data are similar to the bafilomycin A1-mediated reduction of Ca²⁺ signaling after TCR/CD3 ligation shown earlier in Fig. 2C. These data support our conclusion that Ned-19 inhibits NAADP-stimulated Ca²⁺ release from lysosomal compartments in naive T cells and that Ned-19 is selective for NAADP.

Because the Ca²⁺ signal was not completely abolished, the residual signal may have come from the ER, and therefore we investigated ER store involvement. In fact, several reports using human Jurkat T cells demonstrated direct involvement of the RyR in NAADP-mediated Ca²⁺ release (14–16). Thus, we blocked the RyR with the RyR antagonist tetracaine or depleted the Ca²⁺ from the ER store using thapsigargin and monitored cytosolic Ca²⁺ evoked by exogenous NAADP. The Ca²⁺ signal observed upon blocking RyR or depleting the ER was also significantly reduced compared with control as shown in Fig. 3F. When Ca²⁺ was depleted from both acidic and ER stores, the signal was diminished to background (Fig. 3F). Collectively, these data demonstrate that acidic stores are a primary source of Ca²⁺ for NAADP-mediated Ca²⁺ release in conventional naive T cells, but do not exclude the involvement of ER stores and the possibility that the RyR is directly targeted by NAADP, inducing additional signals to those of acidic stores. NAADP-initiated Ca²⁺ release can thus stimulate store-operated Ca²⁺ entry.

Transport of External NAADP or Caged NAADP

Although we showed that exogenous NAADP induced Ca²⁺ release from internal acidic stores, how NAADP was taken up into the cells was not clear. Studies in cultured cells (30, 31) have demonstrated that connexin-43 hemichannels mediate the export of NAD and import of cADP-ribose back into the cell after its synthesis by CD38. To test if such import is possible for NAADP, we first determined the expression of these channels in naive T cells. Consistent with the level of mRNA expression of connexin-43 hemichannels reported previously (32), we observed low expression of these channels in naive CD4 T cells (Fig. 3G). To determine the role of connexin-43 hemichannels in importing exogenous NAADP into naive CD4 T cells, we monitored the change in [Ca²⁺]_i evoked by exogenous NAADP in the presence of saturating concentrations of the connexin-43 hemichannel inhibitors 18β-glycyrrhetinic acid or octanol. As shown in Fig. 3H, the Ca²⁺ signal was reduced to half after blocking these channels with either inhibitor, which indicates a positive role of these channels in importing NAADP into the cell, but also suggests the existence of another transporter. In fact, it has been suggested that NAADP has specific nucleoside transporters (24, 26). To test the role of nucleoside transporters, we used a saturating concentration of dipyridamole, a nucleoside transport inhibitor, to ensure blockage of these transporters. The Ca²⁺ signal evoked by external NAADP was also reduced by half upon pretreatment with 100 μm dipyridamole (Fig. 3H), which is consistent with a previous report in which the same concentration of this inhibitor partially blocked the transport of NAADP into other cells (24). However, combined treatment with octanol and dipyridamole to block both connexin-43 hemichannels and nucleoside transporters further diminished the signal (Fig. 3H).

We examined uptake of external NAADP into the cells by a [³²P]NAADP transport experiment similar to that used to demonstrate uptake by pancreatic β cells (26) and LAK cells (25). CD4 T cells showed a concentration and time-dependent increase in [³²P]NAADP (Fig. 4A). The concentration and time ranges for uptake were similar to those observed for the Ca²⁺ release response.

FIGURE 4.

NAADP uptake by naive CD4 cells. A, approximately one million cells were incubated with [³²P]NAADP at the indicated concentrations for 1 (♦), 5 (□), or 10 (▴) min at room temperature. Cells were pelleted through an oil cushion and radioactivity in the pellet and aqueous layer was determined. Average of two separate experiments. B, representative fluorometric traces of Ca²⁺ release in single naive CD4 T cells induced by the photolysis of caged NAADP. CD4 T cells in RPMI medium were loaded with Fluo-4 (2 μm) and adhered to the plate. Some were treated with octanol (1 mm) for 15 min followed by treatment with caged NAADP (30 μm) for 10 min, washing was done fresh RPMI after each treatment. Free cytosolic Ca²⁺ was measured after applying 8 flashes of UV light to release active NAADP at 30 and 200 s. C, the cumulative data from B plotted as % increase of the Ca²⁺ peak relative to the baseline; mean data of 3 experiments; p < 0.05.

To further confirm uptake of external NAADP, we incubated Fluo-4-labeled naive CD4 T cells with 30 μm 1-(4,5-dimethoxy-2-nitrophenyl)diazoethane-caged NAADP, a photoactivatable precursor of NAADP, for 10 min, washed the cells, and photoreleased active NAADP inside the cells with flashes of UV light. The Ca²⁺ release in response to uncaging (arrow) is shown in Fig. 4, B and C. A second set of UV flashes caused additional Ca²⁺ release. No release was seen without caged NAADP. Reduced release was observed if the cells were pretreated with octanol prior to incubation with caged-NAADP. This supports the proposal that NAADP and caged NAADP can enter cells through connexin-43 hemichannels.

Ned-19 Suppresses Naive T Cell Proliferation

Having demonstrated the effect of NAADP on the initiation and propagation of Ca²⁺ signaling in naive T lymphocytes, we next studied the role of NAADP on the activation and proliferation of these cells. As shown in Fig. 5, A and B, Ned-19 significantly suppressed proliferation of naive CD4 and CD8 T cells to anti-CD3/CD28 stimulation, respectively, in a concentration-dependent manner. At 50 μm or higher concentration of Ned-19, 90% inhibition was observed. Additionally, Ned-19 inhibited ovalbumin-induced antigen-specific proliferation of naive T cells (Fig. 5C). One might suggest that Ned-19 exerts cytotoxicity at higher micromolar concentrations and that the inhibition effect seen is a result of toxicity. To test this hypothesis, naive T cells were stimulated with anti-CD3 and anti-CD28 antibodies first, followed by the addition of 100 μm Ned-19 at different time points after stimulation. Ned-19 addition after 24 h did not impair naive T cell proliferation (Fig. 5D). Furthermore, addition of Ned-19 to CD4 cells stimulated by phorbol myristate acetate (PMA) and ionomycin had only a slight inhibitory effect at high concentrations (Fig. 5E). These results provide evidence that the inhibition of naive T lymphocyte proliferation occurred as a result of blocking the NAADP signaling pathway at an early stage of the response.

FIGURE 5.

NAADP antagonists inhibit naive T cell proliferation. Naive T cells were stimulated by anti-CD3/CD28 for C57BL/6 mice or by ovalbumin-pulsed APCs (10 μg/ml) for OT-II mice with increasing concentrations of Ned-19 or BZ194. Ned-19 was added 1 h prior to stimulation and BZ194 was added 5 h prior. Cell proliferation was determined by [³H]dT incorporation 3 days later. A, effect of Ned-19 on naive CD4 T cell proliferation; untreated stimulation index (S.I.) = 38.5; mean data (n = 3); *, p < 0.001. B, effect of Ned-19 on naive CD8 T cells proliferation; untreated S.I. = 35.5; mean data (n = 3); *, p < 0.05; **, p < 0.001. C, effect of Ned-19 on ovalbumin-induced proliferation of naive OT-II CD4 T cells; untreated S.I. = 7.8; mean data (n = 3); *, p < 0.01; **, p < 0.001. D, effect of Ned-19 is strongest at the time of stimulation. Ned-19 (100 μm) was added to stimulated naive CD4 cells at different times after cell activation by anti-CD3/CD28. Proliferation was determined by [³H]dT incorporation 3 days later and indicated as percent of control (no Ned-19); untreated S.I. = 52.3; mean data (n = 3); *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with t = 0. E, Ca²⁺ ionophore overcomes Ned-19 inhibition of proliferation. Cells were incubated with the indicated amount of Ned-19 and stimulated with anti-CD3/CD28 (black bars) or PMA/ionomycin (white bars). Cell proliferation was determined by [³H]dT incorporation 2 days later; mean data (n = 3); *, p < 0.001. F, effect of BZ194 on naive T cell proliferation to anti-CD3/CD28 indicated as percent of control; untreated S.I. = 47.8.

Minimal Effect of BZ194 on Naive T Cell Proliferation

The derivatized nicotinic acid BZ194 was reported to antagonize NAADP-mediated Ca²⁺ signaling via the type 1 RyR in Jurkat T lymphocytes and to inhibit rat effector T cells (16). We investigated its effect on naive T cell proliferation following 5 h preincubation. BZ194 had little effect on naive T cell proliferation even at higher micromolar concentrations (Fig. 5F). The maximal inhibition induced by 800 μm BZ194 was only 10%, whereas Ned-19 induced almost 50% inhibition at only 10 μm. These data support our previous observation of the different behavior in response to NAADP between Jurkat lymphoma T cells and conventional naive T cells.

Ned-19 Suppresses Cytokine Production by Naive T Cells

Next, we investigated whether NAADP signaling has any role in downstream events following T cell activation such as cytokine production and cytokine receptor expression. In stimulated naive CD4 T cells, as shown in Fig. 6, A and B, high-affinity IL-2 receptor (CD25) expression was only slightly affected by Ned-19; however, the secretion of IL-2 was suppressed almost completely compared with control (Fig. 6C). Similarly, as shown in Fig. 6, D and E, the amount of IL-2 and INFγ secreted, respectively, by naive CD8 T cells was significantly reduced by Ned-19 compared with control cells. These data demonstrate the importance of NAADP-mediated Ca²⁺ signaling for the secretion of critical cytokines after naive T cell activation.

FIGURE 6.

NAADP signaling is essential for cytokine production by naive T cells. Naive T cells were stimulated by anti-CD3/CD28 for 48 h with or without Ned-19 (100 μm) 1 h prior to stimulation. A, expression of the high-affinity IL-2 receptor (CD25). Representative histogram for APC anti-CD25 (gray) and APC isotype control (black). B, mean fluorescence intensity of staining as in A. No significant difference between ±Ned-19. C, inhibition of IL-2 secretion by naive CD4 T cells treated with Ned-19 (100 μm). Supernatants were assayed for IL-2 by ELISA; mean data (n = 3); *, p < 0.001. D, inhibition of IL-2 secretion by naive CD8 T cells treated with Ned-19 (100 μm) as in C; mean data (n = 3); *, p < 0.007. E, inhibition of IFNγ secretion by naive CD8 T cells treated with Ned-19 (100 μm). Supernatants were collected and assayed for IFNγ by ELISA; mean data (n = 3); *, p < 0.0001. F, addition of IL-2 did not rescue the proliferation of naive CD4 T cells. IL-2 (40 units/ml) was added at the initiation of culture on anti-CD3/CD28-coated wells. Cells were pretreated for 1 h with the indicated concentration of Ned-19. Mean data (n = 3); no significant difference between ±IL-2. G, addition of IL-2 did not rescue the proliferation of naive CD8 T cells as in F; mean data (n = 3); no significant difference between ±IL-2.

IL-2 is a very important cytokine for T cell proliferation and differentiation. Because both events of IL-2 secretion and proliferation in naive T cells were inhibited by Ned-19, we tested the possibility of a direct connection between lack of IL-2 secretion and inhibition of proliferation. To determine this, we measured the effect of Ned-19 on naive T cell proliferation in the presence of exogenous IL-2. As shown in Fig. 6, F and G, IL-2 added at the initiation of culture did not rescue the proliferation of naive CD4 and CD8 T cells, respectively.

NAADP has been implicated in secretion in several systems, including externalization of granzymes by cytotoxic T cells (18). To confirm that the effect of Ned-19 was not just an inhibition of secretion, we measured intracellular cytokines in Ned-19-treated cells. Ned-19 reduced intracellular IL-2 staining in CD4 cells (Fig. 7, A–C), whether or not the inhibitor of secretion BFA was present. It also reduced intracellular staining of IFNγ in CD8 cells (Fig. 7, D–F). In untreated cells, BFA-mediated increase in intracellular cytokines was measurable. Because no accumulation was observed, this supports an effect of Ned-19 on cytokine synthesis, although we would not be able to distinguish an effect of Ned-19 on both synthesis and secretion.

FIGURE 7.

Ned-19 inhibits the synthesis of cytokines by naive T cells. Naive CD4 or CD8 T cells were stimulated by anti-CD3/CD28 for 24 h with or without Ned-19 (100 μm) 1 h prior to stimulation. Cells were further cultured for 5 h in the presence or absence of BFA (3 μg/ml). Cells were tested for intracellular cytokine by FACS. A, an overlay of representative dot plot for APC anti-IL-2 of BFA-treated CD4 T cells in the absence (red) or presence (green) of Ned-19. B, mean fluorescence intensity of staining as in A after background (APC isotype control) subtraction; mean data (n = 3); *, p < 0.05. C, percent of CD4 T cells positive for IL-2 after background subtraction; mean data (n = 3); *, p < 0.05. D, an overlay of representative dot plot for APC anti-IFNγ of BFA-treated CD8 T cells in the absence (red) or presence (green) of Ned-19. E, mean fluorescence intensity of staining as in D after background (APC isotype control) subtraction; mean data (n = 3); *, p < 0.05. F, percent of CD4 T cells positive for IL-2 after background subtraction; mean data (n = 3); *, p < 0.05.

NAADP-mediated Ca²⁺ Signaling Is Necessary for Stimulating NFAT and NFκB Nuclear Translocation

Ca²⁺ signals in T cells result in the activation and nuclear translocation of several transcription factors. The most critical of these factors are NFAT and NF-κB. To determine whether NAADP-mediated Ca²⁺ signals are necessary to stimulate NFAT translocation into the nucleus in naive T cells, we stimulated naive CD4 T cells with anti-CD3/CD28 in the presence or absence of Ned-19 followed by immunohistochemistry. Naive T cells have little cytoplasm and very low levels of NFAT and NF-κB prior to initial stimulation; therefore, time is required to synthesize more of these transcription factors before significant amounts can be detected in the nucleus at 24 and 48 h. As shown in Fig. 8, A and B, Ned-19 significantly reduced NFAT-1 nuclear translocation at both 24 and 48 h after stimulation. Ned-19 prevented blast formation after stimulation, which is seen as the control cells have enlarged at 48 h. Similarly, the translocation of NF-κB was also significantly reduced after 48 h (Fig. 8, A and C).

FIGURE 8.

NAADP signaling is necessary to stimulate NFAT and NF-κB nuclear translocation in naive T cells. A, Ned-19 reduces NFAT-1 and NF-κB nuclear translocation. Immunohistochemistry of naive CD4 T cells stimulated with anti-CD3/CD28 for 0, 1, 24, or 48 h in the presence or absence of Ned-19 (100 μm). Blue for DAPI staining, green for NFAT-1 staining, and red for NF-κB staining. All images are at the same scale and the scale bar in the right top panel, 10 μm, applies to all panels. B and C, quantification of NFAT-1 and NF-κB nuclear translocation, respectively, expressed as the ratio of the mean fluorescence intensity of NFAT-1 or NF-κB staining to the mean fluorescence intensity (MFI) of DAPI. Mean data represent a total of between 80 and 100 cells from 2 experiments; *, p < 0.001.

NAADP Contributes to Receptor-mediated Ca²⁺ Signals in Effector T Cells

Because effector T cells are known to differ from naive T cells in their requirements for activation, we asked whether the NAADP signaling pathway is as important for TCR-mediated Ca²⁺ signals in effector T cells. As in naive T cells, the increase in cellular Ca²⁺ observed is dependent on both internal and external Ca²⁺, as the absence of external Ca²⁺ or the inhibition of SOCE with SKF96365 greatly reduced the increase in cellular Ca²⁺ (data not shown). As shown in Fig. 9, A and B, Ned-19 reduced the amplitude and plateau levels of Ca²⁺ signals in nonpolarized effector CD4 T cells in a concentration-dependent manner, with a maximal inhibition at 100 μm. Similarly, inhibition was also observed in effector CD8 T cells (Fig. 9C). The Ca²⁺ signal was not completely abolished and the residual Ca²⁺ signal is likely generated by other Ca²⁺ signaling pathways. However, the Ca²⁺ signal was reduced by approximately two-thirds, which indicates how significantly NAADP contributes, either directly or indirectly via Ca²⁺-induced Ca²⁺ release to overall Ca²⁺ increases during the receptor-mediated activation of effector T cells. The strong inhibition of Ca²⁺ increase by NAADP antagonist Ned-19 is likely due to reduction in both internal Ca²⁺ release and external Ca²⁺ influx as a result of reduced store depletion. This data were consistent with our observations in naive T cells except that the magnitude of the Ca²⁺ signal in effector T cells was generally lower than that observed in naive T cells.

FIGURE 9.

NAADP antagonist Ned-19 inhibits Ca²⁺ signaling in effector T cells. A, representative ratiometric (340/380) traces of Ca²⁺ concentration in single effector CD4 T cells induced by TCR/CD3 stimulation. Cells loaded with Fura-2/AM were incubated without Ned-19 (Control) or with the indicated concentration of Ned-19 for 1 h. Ca²⁺ release was stimulated by biotinylated anti-CD3 (αCD3) followed by streptavidin (X) or PBS as vehicle. B, concentration-response curve of TCR/CD3-induced Ca²⁺ signaling in effector CD4 T cells following treatment with increasing concentrations of Ned-19; mean maximal increases from 3 experiments; *, p < 0.001. C, same as B for effector CD8 T cells; mean maximal increases from 3 experiments; *, p < 0.05; **, p < 0.001.

External NAADP Stimulates Ca²⁺ Flux in Effector T Cells in a Similar Manner

We confirmed that connexin-43 hemichannels are expressed at low levels by effector CD4 T cells (Fig. 10A). We administered 3 μm NAADP, which was found to be the optimal concentration to induce Ca²⁺ release in naive T cells, onto plated effector T cells and monitored the evoked changes in cytosolic Ca²⁺. As shown in Fig. 10B, exogenous NAADP, but not NADP or PBS induced Ca²⁺ signals in effector CD4 T cells. As shown in Fig. 10, B and C, Ned-19 inhibited Ca²⁺ signals evoked by external NAADP in effector CD4 T cells, which is inconsistent with the results obtained above from the receptor-mediated response. Interestingly, the amplitude of the Ca²⁺ signal evoked by exogenous NAADP in effector T cells seemed to be comparable with that evoked by TCR/CD3 stimulation. Collectively, these data demonstrate a critical role for NAADP for the initiation and propagation of Ca²⁺ signals during the effector response of T cells. To determine whether Ca²⁺ influx contributes to NAADP-induced Ca²⁺ signals in effector CD4 T cells, we monitored the change in [Ca²⁺]_i evoked by exogenous NAADP in Ca²⁺-free medium or upon pretreatment with the SOCE inhibitor SKF96365. As shown in Fig. 10, D and E, the Ca²⁺ signal was significantly reduced compared with control. This data indicated that the Ca²⁺ increase evoked by externally added NAADP is partially from Ca²⁺ release from internal stores and partially from Ca²⁺ influx due to the activation of SOCE. This data support the strong inhibitory effect of Ned-19 on intracellular Ca²⁺ increase during the receptor-mediated response as observed earlier.

FIGURE 10.

Effect of exogenous NAADP on effector T cells. A, expression of connexin-43 hemichannels in effector CD4 T cells. Cells were stained with (red peak) or without (black peak) anti-connexin-43 ab79010 followed by FITC anti-mouse IgG. B, representative ratiometric traces of Ca²⁺ concentration in single effector CD4 T cells induced by exogenously added NAADP, in the presence or absence of Ned-19, NADP, or PBS at the indicated time (X); mean maximal increases from 3 experiments are summarized in C; *, p < 0.001. D, representative ratiometric traces of Ca²⁺ release in single effector CD4 T cells induced by exogenously added NAADP in the presence (Control) or absence (0 Ca²⁺) of external Ca²⁺ or SKF96365 (25 μm, SKF); mean maximal increases from 3 experiments after subtracting background are summarized in E; *, p < 0.001.

To test whether NAADP mediates Ca²⁺ release from lysosomal Ca²⁺ stores, we depleted the endolysosomal Ca²⁺ using bafilomycin A1 and monitored cytosolic Ca²⁺ evoked by external application of NAADP. The punctate LysoTracker Red labeling of acidic compartments was abolished with bafilomycin A1 treatment compared with control as shown in Fig. 11A. Similar to the inhibitory effect of Ned-19, the Ca²⁺ signal evoked by external application of NAADP following bafilomycin A1 treatment was significantly reduced (Fig. 11B). Moreover, we repeated this experiment using cells pretreated with both Ned-19 and bafilomycin A1 and the resulting Ca²⁺ signals from combined treatment were similar to those observed after treatment with Ned-19 or bafilomycin A1 alone (Fig. 11B). These results suggest that Ned-19 and bafilomycin A1 act on the same Ca²⁺ store to mediate the inhibition of NAADP-mediated Ca²⁺ release. Furthermore, we determined that Ned-19 fluorescence also localized to acidic stores in effector CD4 T cells. As shown in Fig. 11C, Ned-19 and LysoTracker Red labeled the same acidic compartments after combined staining with both fluorescent molecules.

FIGURE 11.

Effect of bafilomycin A1 on NAADP-mediated Ca²⁺ signaling in effector CD4 T cells reveals lysosomal sensitivity to NAADP. A, LysoTracker Red labeling of effector CD4 T cells in the absence (Control) or presence of bafilomycin A1 (Baf). B, similar inhibition of NAADP-mediated Ca²⁺ signaling in effector CD4 T cells after treatment with Baf (1 μm) for 30 min, Ned-19 (100 μm) for 1 h, or both; mean maximal increases from 3 experiments after subtracting background; *, p < 0.001. C, overlay of fluorescent labeling of effector CD4 T cells with 100 μm Ned-19 (green) together with 30 nm LysoTracker Red (red); overlay (yellow). Expanded view is boxed. D, reduction of NAADP-mediated Ca²⁺ signals following pretreatment with a mixture of 100 μm Ned-19 and 1 μm Baf, tetracaine (Tet) (100 μm) for 20 min and thapsigargin (Tg) (1 μm) for 30 min; mean maximal increases were from 3 experiments after subtracting background; *, p < 0.001.

To investigate the involvement of the ER store, we monitored the Ca²⁺ signals evoked by exogenous NAADP in the presence of the RyR antagonist tetracaine and after the depletion of Ca²⁺ from the ER store using thapsigargin. As shown in Fig. 11D, the Ca²⁺ response to external NAADP was reduced after tetracaine and thapsigargin treatments, but the inhibition was not as significant as that observed following pretreatment with Ned-19 or bafilomycin A1. The Ca²⁺ signal was reduced by 44 and 50% after tetracaine and thapsigargin treatments, respectively, whereas it was reduced by 63% after Ned-19/bafilomycin A1 treatment.

Ned-19 Suppresses Proliferation of Nonpolarized Effector T Cells

Nonpolarized effector T cells that were previously stimulated with anti-CD3 and anti-CD28 antibodies were re-stimulated with anti-CD3 antibody in the presence or absence of varying concentrations of NAADP antagonist Ned-19. As shown in Fig. 12, A and B, Ned-19 significantly suppressed proliferation of effector CD4 and CD8 T cells, respectively, in a concentration-dependent manner. Moreover, we determined the effect of Ned-19 on antigen-specific proliferation of effector T cells. We used ovalbumin to immunize B6 mice and re-stimulated CD4 T cells 2 weeks after immunization. Similar to the proliferation results induced by anti-CD3 above, the ovalbumin-induced specific proliferation of effector CD4 T cells was also inhibited up to 70% by Ned-19 (Fig. 12C). To test if Ned-19 caused nonspecific toxic effects, we measured effector CD4 T cell proliferation after stimulation with PMA and ionomycin in the presence or absence of Ned-19. The addition of Ned-19 to effector CD4 T cells stimulated by PMA and ionomycin had no significant effect (Fig. 12D). This indicates that the inhibition by Ned-19 of effector T cell proliferation stimulated by anti-CD3 occurred as a result of the inhibition of Ca²⁺ flux, as stimulating the cells in excess Ca²⁺ conditions (ionomycin) rendered the cells non-susceptible to Ned-19 inhibition. These results suggest that NAADP signaling is essential for the expansion of effector cells during an immune response.

FIGURE 12.

NAADP signaling is essential for effector T cell proliferation. Effector T cells were re-stimulated by anti-CD3 in the presence or absence of increasing concentrations of Ned-19. Cell proliferation was determined by [³H]dT incorporation 3 days later. A, effect of Ned-19 on effector CD4 T cell proliferation; mean data (n = 4); *, p < 0.001. B, effect of Ned-19 on effector CD8 T cell proliferation; mean data (n = 4); *, p < 0.001. C, effect of Ned-19 on ovalbumin-induced proliferation of effector CD4 T cells. Mice were immunized with ovalbumin (100 μg) 2 weeks prior the isolation and stimulation of CD4 T cells by ovalbumin-pulsed APCs (10 μg/ml); mean data (n = 3); *, p < 0.001. D, Ned-19 effect on effector CD4 proliferation is reversed with excess Ca²⁺. Cells were incubated with or without Ned-19 and re-stimulated with anti-CD3 (black bars) or PMA/ionomycin (white bars). [³H]dT incorporation was measured 2 days later; mean data (n = 3); *, p < 0.001 compared with DMSO control. E, addition of IL-2 did not rescue the proliferation of effector CD4 T cells. IL-2 (40 units/ml) was added at the initiation of culture on anti-CD3-coated wells. Cells were pretreated for 1 h with the indicated concentration of Ned-19. Mean data (n = 3); no significant difference between ±IL-2. F, addition of IL-2 did not rescue the proliferation of effector CD8 T cells as in E; mean data (n = 3); no significant difference between ± IL-2.

Because IL-2 is required for effector T cell proliferation, we also asked whether lack of IL-2 was solely responsible for the lack of proliferation. Supplementation of the cultures with IL-2 did not restore proliferation of CD4 (Fig. 12E) or CD8 (Fig. 12F) effector cells.

Ned-19 Suppresses Proliferation of Different Subsets of Effector T Cells

During an immune response, CD4 T cells can differentiate, giving rise to several subsets of effector T cells, among which are Th1, Th2, and Th17 cells, which secrete different cytokines that lead to different immune responses. This differentiation depends on the nature of the pathogen encountered and the type of immune response required to clear it. It is not known whether NAADP-mediated Ca²⁺ signaling is necessary for the activation of Th1, Th2, and Th17 cells or varies from one subset to another and therefore contributes to specific subset-mediated immune responses. Therefore, we investigated how critical NAADP-mediated Ca²⁺ release was for the proliferation of these subsets of effector CD4 T cells. To determine that, we first polarized CD4 T cells into Th1, Th2, or Th17 cells and obtained up to 90% pure populations of each cell subset. As shown in Fig. 13, A–C, the majority of the cells have polarized and shifted to Th1, Th2, or Th17 as about 90% of the cells expressed characteristic transcription factors T-bet, Gata3, or RORγ, respectively. As shown in Fig. 13, D–F, proliferation of Th1, Th2, and Th17 cells was inhibited up to 80% by Ned-19.

FIGURE 13.

NAADP signaling contributes to the proliferation of different subsets of effector CD4 T cells. Polarized cells were stained with anti-CD4-FITC and anti-T-bet, anti-GATA3, or anti-RORγ to confirm expression of characteristic transcription factors of Th1, Th2, and Th17 cells. A, T-bet expression in the polarized Th1 cells (gray) versus isotype control (black). B, GATA3 expression in the polarized Th2 (gray) versus isotype control (black). C, RORγ expression in the polarized Th17 (gray) versus isotype control (black). Th1, Th2, and Th17 T cells were re-stimulated by anti-CD3 in the presence or absence of increasing concentrations of Ned-19. Cell proliferation was determined by [³H]dT incorporation 3 days later. D, effect of Ned-19 on Th1 effector T cell proliferation; mean data (n = 4); *, p < 0.001. E, effect of Ned-19 on Th2 effector T cell proliferation; mean data (n = 4); *, p < 0.001. F, effect of Ned-19 on Th17 effector T cell proliferation; mean data (n = 3); *, p < 0.001.

Ned-19 Suppresses Effector Cytokine Production by Different Subsets of Effector T Cells

A critical role for NAADP in downstream events of CD4 T cell clones has been reported (16). However, different effector T cells have different and specialized functions that are carried out by cytokines of different types and different combinations or by cytotoxic products of cytotoxic CD8 T cells. We determined the role of NAADP signaling for the production of the major effector molecules, IFN-γ, IL-4, and IL-17 by Th1, Th2, and Th17 cells, respectively. We blocked NAADP-mediated Ca²⁺ signaling using the concentration of Ned-19 (100 μm) that showed maximum inhibition of Ca²⁺ signals and proliferation and measured the production of these cytokines via ELISA. As shown in Fig. 14, A–C, Ned-19 significantly inhibited the production of IFN-γ, IL-4, and IL-17 by Th1, Th2, and Th17 cells, respectively. The suppression was not complete, particularly with the Th17 cells. Likewise, we tested the effect of Ned-19 on IFN-γ production by effector CD8 T cells. As shown in Fig. 14D, IFN-γ production was suppressed in Ned-19-treated effector CD8 T cells as compared with control. We previously showed that inhibition of T cell cytokine production by Ned-19 in naive T cells was not simply inhibition of secretion, as IL-2 or IFNγ did not accumulate within treated cells. These data demonstrate the importance of NAADP-mediated Ca²⁺ signaling for the production of such crucial effector cytokines and thus for the overall immune response.

FIGURE 14.

NAADP signaling is critical for cytokine production by different subsets of effector T cells. Effector T cells were re-stimulated by anti-CD3 for 48 h in the presence or absence of Ned-19 (100 μm) and supernatants were collected and assayed for secreted cytokines by ELISA. A, Ned-19 inhibited IFNγ production by Th1 effector T cells; mean data (n = 3); *, p < 0.05. B, Ned-19 inhibited IL-4 production by Th2 T cells; mean data (n = 3); *, p < 0.01. C, Ned-19 inhibited IL-17 production by Th17 T cells; mean data (n = 3); *, p < 0.05. C, Ned-19 inhibited IFNγ secretion by effector CD8 T cells; mean data (n = 3); *, p < 0.05.

NAADP Is Less Significant for Natural Regulatory T Cells

Natural Tregs are a very critical but low abundance subset of T cells that play a role in the maintenance of self-tolerance, down-regulation of the immune response, and prevention of the development of autoimmunity. We investigated if NAADP plays a role in natural Treg Ca²⁺ signaling. Tregs express the transcription factor FoxP3 that is used to identify T cells with regulatory functions. We first isolated the small number of CD4⁺CD25⁺ T cells from mouse spleen cells and confirmed that they were FoxP3 positive as shown in Fig. 15A. Interestingly, Ned-19 did not have any effect on natural Treg Ca²⁺ signaling upon anti-CD3 stimulation compared with control (Fig. 15B). The overall Ca²⁺ flux was about one-third of that observed with CD4 naive T cells and also less than that of CD4 effector cells. Moreover, exogenously added NAADP induced only a weak Ca²⁺ signal that was not affected by either bafilomycin A1 or Ned-19 (Fig. 15C). We confirmed that connexin-43 hemichannels were expressed by Treg cells (Fig. 15D). Finally, we tested if inhibiting NAADP played any role in natural Treg proliferation. As shown in Fig. 15E, Ned-19 did not inhibit Treg proliferation. It is worth noting that these cells exhibited very low proliferation, with a stimulation index of ∼1.5 compared with the unstimulated cells. This indicates that NAADP participation in natural Treg Ca²⁺ signaling and proliferation differs from conventional naive and effector cells. These findings regarding the sensitivity of naive T cells to NAADP antagonist Ned-19 versus the non-sensitivity of natural Tregs might be of immunological importance in differentially regulating the response of certain immune T cells without affecting other cells with regulatory functions.

FIGURE 15.

NAADP signaling in natural regulatory T cells. A, FoxP3 expression in natural CD4⁺CD25⁺ T cells. CD4⁺CD25⁺ T cells were isolated and stained with anti-CD4-FITC, anti-CD25-APC, and anti-FoxP3-PE. Left panel, CD4 FITC (gray) or unstained (black); center panel, CD4⁺CD25⁺ (gray) or CD4⁺ alone (black); right panel, cells gated as CD4⁺CD25⁺ were analyzed for FoxP3⁺ (gray) versus isotype control (black). B, no effect of Ned-19 on receptor-mediated Ca²⁺ signaling in natural Tregs. CD4⁺CD25⁺ cells were loaded with Fura-2/AM (2 μm) and treated with or without Ned-19 (100 μm) for 1 h. Ca²⁺ signaling was induced by anti-CD3 ligation or control PBS and the change in [Ca²⁺]_i was measured; mean maximal increases from 3 experiments. C, NAADP was added directly to Fura-2-loaded natural Treg, either untreated (control) or pretreated with Baf or Ned-19, and the maximal change in fluorescence was measured; mean maximal increases from 3 experiments after subtracting background. D, expression of connexin-43 hemichannels in regulatory T cells. Natural Tregs were stained with (gray peak) or without (black peak) anti-connexin-43 ab79010 followed by FITC-anti-mouse IgG. E, no effect of Ned-19 on natural Treg proliferation. CD4⁺CD25⁺ cell proliferation was determined by [³H]dT incorporation 3 days after stimulation with anti-CD3/CD28 and gave a low stimulation index of 1.5 (³H = 3044 mean cpm compared with ³H = 1928 mean cpm for unstimulated cells). Proliferation is indicated as percent of control, untreated stimulated cells, mean of 3 experiments.

Discussion

In this study, we report the first description of NAADP-mediated Ca²⁺ signaling in conventional naive T lymphocytes that is tied to functional effects. We demonstrated that Ned-19: (i) effectively suppressed the release of intracellular Ca²⁺ in naive T cells evoked by ligation of CD3 in a concentration-dependent manner, (ii) blocked the signal triggered directly by exogenous NAADP, (iii) totally blocked the proliferation of naive T cells, (iv) inhibited the production of IL-2 by naive CD4 and of IL-2 and INFγ by naive CD8 T cells, and (v) significantly reduced NFAT and NF-κB nuclear translocation. Effector CD4 and CD8 T cells showed a similar profile of NAADP or NAADP antagonist effects.

Our proliferation data shows that Ned-19 can exhibit its antagonistic effect at concentrations as low as 10 μm with only a 30 min to 1 h preincubation period. The lack of effect of Ned-19 addition at 24 h after naive T cell activation and on PMA/ionophore-activated proliferation strongly suggests that Ned-19 did not have cytotoxic effects on naive T cells. These data, along with the fact that Ned-19 did not directly affect store-operated Ca²⁺ entry in the absence of TCR signaling, support its selectivity for NAADP-mediated Ca²⁺ signaling. A different NAADP antagonist, BZ194, has been shown to inhibit Ca²⁺ flux, proliferation, IL-2 production, and NFAT nuclear translocation in rat effector T cell lines; however, it requires a long preincubation time, up to 24 h, for maximal effect (16), which might not be feasible for naive T cells. We found that BZ194 had no effect on naive T cell proliferation when preincubated with the cells up to 5 h. This is in agreement with the observation in which naive T cells were less susceptible to NAADP antagonism by BZ194 (33). Differences have been observed in BZ194 effects depending on the cell type analyzed (10). BZ194 did not inhibit NAADP-mediated Ca²⁺ release nor show any binding affinity for the NAADP receptor in sea urchin egg homogenates.

In our study, we report a clear distinction between Jurkat lymphoma T cells and conventional naive T cells in the sensitivity of their lysosomal Ca²⁺ pools to NAADP. We demonstrated: (i) that depleting the endolysosomal Ca²⁺ by bafilomycin A1 antagonized Ca²⁺ signaling evoked by stimulation via anti-CD3 and by exogenous NAADP, (ii) that Ned-19 fluorescence localized to the acidic compartments consistent with a study of pancreatic acinar cells (21), (iii) that a combination of Ned-19 and bafilomycin A1 did not exhibit any further inhibition than that exerted by either one alone, and (iv) that inhibition of the RyR by tetracaine or depletion of the ER store by thapsigargin did not cause as much of a decrease in Ca²⁺ flux as did Ned-19 or bafilomycin A1. A similar pharmacological profile was observed in effector T cells stimulated with exogenous NAADP. This evidence that NAADP targets acidic organelles is consistent with previous observations in a human cytotoxic T lymphocyte clone and Jurkat 1G4-transfected cells (18), but contrasts with results obtained using the Jurkat cell line in which the ER Ca²⁺ stores rather than lysosomes appeared to be sensitive to NAADP (34). However, a portion of the Ca²⁺ release in naive and effector T cells appears to involve the ER stores and that RyR-mediated release may be more dependent on other stores in primary T cells. Perhaps Ca²⁺ release in Jurkat cells is less dependent on this trigger. Recent work by Wolf et al. (17) confirms a role for the RyR in both Jurkat and primary mouse T cells. However, other sources such as external Ca²⁺ were found to participate in primary cells. Our data support the trigger hypothesis that, in response to NAADP, the initial Ca²⁺ signals are derived from acidic stores and amplified by the RyR through the process of Ca²⁺-induced Ca²⁺ release (7). The lysosomal sensitivity of NAADP in conventional naive T cells versus the non-sensitivity in Jurkat T cells might reflect the diversity of the Ca²⁺ stores involved and the target receptor for NAADP. This is compatible with our previous findings in which we demonstrated a difference in specificity between human and sea urchin NAADP receptor (10).

We demonstrated that externally applied NAADP, but not NADP, induced Ca²⁺ release from lysosomal-like acidic stores in naive and effector T cells. Although it was reported that NAADP-mediated Ca²⁺ influx is triggered from TRPM2 channels (29), the study using lymphokine-activated killer cells (25) demonstrated that blocking TRPM2 channels prevented a NAADP-mediated sustained Ca²⁺ increase, but not NAADP-mediated initial Ca²⁺ increase. Wolf et al. (17) also showed normal early Ca²⁺ release in TRPM2 knock-out T cells. Importantly, we report a role for nucleoside transporters and connexin-43 channels in transporting NAADP into these cells.

Effects of Ned-19 on NFAT and NF-κB translocation are expected because NFAT activation is highly regulated by Ca²⁺/calcineurin (35) and NFAT-dependent gene transcription requires strong and sustained Ca²⁺ signals (36). However, in contrast to NFAT, NF-κB requires no prolonged elevation of the Ca²⁺ level. Only a transient increase in Ca²⁺ is sufficient for NF-κB activation and subsequent gene expression (36). These data suggest that NAADP-mediated Ca²⁺ signaling is essential for efficient activation of naive T cells.

IL-2 and INFγ are synthesized and secreted after T cell activation through a pathway requiring an increase in intracellular Ca²⁺ concentration. It is known that IL-2 production is dependent on the Ca²⁺-dependent phosphatase calcineurin (37), which activates NFAT (35) that transcriptionally regulates the induction of IL-2. Similarly, it has been shown that the INFγ promoter has an element that behaves like an NFAT binding site on the IL-2 promoter and is Ca²⁺-inducible and cyclosporine sensitive (38). The inhibition of NAADP-mediated Ca²⁺ release by Ned-19 reduced the nuclear translocation of NFAT, and consequently expression of NFAT-dependent IL-2 and INFγ genes. NFAT is also involved in the induction of the IL-2 receptor promoter (39). After stimulation, expression of the IL-2 receptor was slightly reduced; however, the effect was not as significant as that on IL-2 production. Although analysis of intracellular cytokines supported the effect of Ned-19 on synthesis of cytokines, the treatment could also affect secretion.

The addition of exogenous IL-2 to Ned-19-treated naive or effector cells did not overcome inhibition of proliferation. Proliferation induced in Ca²⁺ sufficient conditions such as by anti-CD3/PMA in the presence of CsA/FK-506 can be restored by IL-2 addition (40). Our data suggest that the lack of IL-2 is not the only reason that Ned-19-treated cultured cells failed to proliferate. It suggests that NAADP signals are involved in the activation of not only the calcineurin pathway, but also other Ca²⁺-dependent pathways essential for full activation of T cells (41).

Natural Tregs proliferate poorly in response to anti-CD3/CD28 when compared with the response of naive T cells. Our observations of this hypoproliferative response to anti-CD3/CD28 as well as low Ca²⁺ signals might be explained by previous findings (42) in which Tregs have a defect in phospholipase Cγ and its downstream signals that enable Tregs to suppress the initiation of TCR signals and thus limit the accumulation of second messengers essential for IL-2 production and proliferation. In addition, the action of FoxP3 upon the transcriptional activity of NFAT and other transcription factors (43) may provide further limitation on IL-2 induction and proliferation by Tregs. However, Tregs did not respond to exogenously added NAADP and were not sensitive to the NAADP antagonist Ned-19, which may suggest down-regulation of the NAADP receptor in this subset of T cells. This would allow another level of control of Treg responsiveness.

In conclusion, our results revealed that NAADP plays a central role in the activation, proliferation, and cytokine production of naive and effector CD4 and CD8 T cells. This provides an additional target for manipulation of T cell responses. Our results indicated that lysosome-like acidic stores are a primary source of NAADP-evoked Ca²⁺ release in naive and effector T lymphocytes although ER stores also participate. The role of NAADP in Th1, Th2, and Th17 cells is similar to other types of T cells, but apparently different from natural Tregs.

Author Contributions

R. A. A. planned and conducted almost all of the experiments, analyzed data, prepared figures, and wrote most of the paper. C. C. and K. W. assisted with optimizing and conducting experiments. T. F. W. prepared [³²P]NAADP and assisted with uptake experiments. S. B. and D. R. G. assisted with calcium flux assays. J. T. S., T. F. W., D. R. G., and K. A. W. conceived the project, performed experiments, and assisted with writing the paper. All authors reviewed the results and approved the final version of the manuscript.