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 Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) 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 Ca(2+) 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.

Keywords: Ned-19; T cell; calcium imaging; calcium intracellular release; cellular immune response; nicotinic acid adenine dinucleotide phosphate (NAADP).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.