Nicotinic acid and its derivative BZ194 inhibit NAADP-mediated Ca²⁺ signaling in T cells. (A) Structures of NAADP and NADP. (B) NAADP-induced Ca²⁺ signaling in T cells is inhibited by nicotinic acid. Jurkat T cells were loaded with Fura-2/AM and were microinjected with 100 nM NAADP, 100 nM NAADP plus 1 mM nicotinamide, or 100 nM NAADP plus 1 mM nicotinic acid, or intracellular buffer. Free cytosolic Ca²⁺ ([Ca²⁺]_i) was measured. Mean data of 4–12 experiments are shown (Upper). The bar chart (Lower) summarizes the data (Ca²⁺ peak and Ca²⁺ plateau; means ± SEM., n = 4 – 12; ∗, P ≤ 0.05; ns, not significant). (C) Inhibition of NAADP-induced Ca²⁺ signaling upon co-injection with BZ194. Jurkat T cells were co-injected with 100 nM NAADP and increasing concentrations of BZ194. Mean data of 5–13 experiments are shown. (D) Concentration-response curve of NAADP-induced Ca²⁺ signaling upon microinjection with BZ194 (mean ± SEM, n = 5–13; ∗, P ≤ 0.05). (E) BZ194 does not directly interfere with InsP₃- or cADPR-mediated Ca²⁺ release. Jurkat T cells were loaded with Fura2/AM. Thereafter, single cells were co-injected with 1 mM BZ194 and either NAADP (100 nM), InsP₃ (4 μM), or cADPR (100 μM), and Ca²⁺ signaling was recorded. Relative values are shown as percentages of NAADP-induced [Ca²⁺]_i. Data represent means ± SEM, n = 5–13; ∗, P ≤ 0.05; ns, not significant.

RyR expression and antagonism of Ca²⁺ signaling in rat MBP-specific effector T cells. (A) Expression of RyR subtypes in effector T cells. Immunofluorescence for RyR1 to 3 (red) and the endoplasmatic reticulum marker protein disulphide isomerase (PDI, green). (Scale bar, 10 μm.) (B) Suppression of Ca²⁺ signaling in Fura2-loaded T_MBP cells stimulated in the presence of MBP-pulsed astrocytes. Rat F10 astrocytes with up-regulated MHC II expression were pulsed with MBP. T_MBP cells, either preincubated with BZ194 (2 mM, MBP + BZ194) or left untreated (MBP), were loaded with Fura-2/AM and added to the astrocytes. In control experiments, astrocytes were not pulsed with MBP (control). Time point 0 was defined as first visible contact between a T cell and an astrocyte. Ca²⁺ imaging was performed as detailed in SI Methods. (B) shows a representative experiment with T cells highlighted by white lines in the brightfield images. (Scale bar, 5 μm.) (C) Mean global [Ca²⁺]_i data from 14 (control: no MBP), 30 (MBP pulsed astrocytes), or 31 (MBP pulsed astrocytes plus T cells preincubated with BZ194) T cells are displayed. (D) Resting rat T_MBP cells were loaded with Fura2/AM and cell suspension measurements were carried out as described in Methods. Arrows indicate the addition of αCD3 or crosslinking (X) antibodies. Cells were preincubated with 500 μM BZ194 overnight. (E) Concentration-response curves summarizing data from (D) (transient Ca²⁺ peak and Ca²⁺ plateau; mean± SEM, n = 8–9). (F) Effect of BZ194 preincubation time on Ca²⁺ signaling (mean ± SEM, n = 8–9). (G) Cells were preincubated with 1 mM BZ194 overnight and thapsigargin (1 mM, arrow) was added to induce capacitative Ca²⁺ signaling; mean data ± SEM (n = 9) are summarized in H. ∗, P ≤ 0.05 according to Student's t test; ns, not significant.

Targeting of NAADP RyR1 interaction by BZ194. (A) Silver-stained gel demonstrating the purity of RyR1 preparation. (B) Kinetics of NAADP-induced association of [³H] ryanodine to the purified RyR1 at 30 °C. Specific [³H]ryanodine binding in the absence (control) or presence of 300 nM NAADP; BZ194 (100 μM) was combined with both control and NAADP experiments. Asterisk (*) denotes statistical significance of NAADP over control, cross (#) that of BZ194 plus NAADP over NAADP alone. Data are mean ± SD. (n = 4). (C) After 2 h of association, dissociation of bound [³H] ryanodine (20 nM) was triggered by 20 μM cold ryanodine (ry) in the absence or presence of 300 nM NAADP or 100 μM BZ194 (BZ) at 30 °C. Data are mean ± SD. (n = 4). (D) Inhibition of NAADP-stimulated [³H]ryanodine binding to RyR1 by increasing concentrations of BZ194 after 2 h at 30 °C. Data are mean ± SEM (n = 2–14).

NAADP signaling is essential for efficient activation of antigen-specific effector T cells. (A) BZ194 reduces NFAT-1 nuclear translocation. Immunocytochemistry of BZ194-treated or control (DMSO- treated) T_MBP cells 0, 30, or 60 min after stimulation with αCD3/CD28 antibodies. Red, DAPI staining; green, NFAT-1 or isotype control staining. (Scale bars, 10 μm.) Representative data of 3 independent experiments are shown. (B) Quantification of NFAT-1 nuclear translocation, expressed as percent of cells. Values respresent means ± SD, data from 3 independent experiments including at least 150 cells per counting. P values: ∗, P ≤ 0.05; ∗∗, P ≤ 0.01. (C–E) BZ194 suppresses activation-induced IL-2 expression in rat and human T_MBP cells. (C) Quantitative PCR of rat T_MBP cells challenged with control antigen- (OVA, white) or MBP- (black) pulsed APCs in the presence of vehicle (control), 0.5 or 1 mM BZ194. mRNA was extracted 48 h after stimulation. Values are normalized to β actin mRNA. Representative data of 3 independent experiments are shown. All differences are statistically significant (P value, P at least ≤ 0.05). (D) IL-2 protein release of T_MBP cells 48 h after stimulation, determined by ELISA. Values represent means ± SD of triplicate measurements. Representative data of at least 2 independent experiments are shown. ∗, P ≤ 0.0001. (E) BZ194 blocks antigen-induced T cell proliferation, but does not interfere with T-cell activation cascades beyond Ca²⁺ signaling. T_MBP cells incubated with the indicated amounts of BZ194 were stimulated with αCD3/CD28 antibodies (black bars), PMA/ionomycin (white bars) or αCD3/CD28 antibodies + ionomycin (gray bars). T_MBP cells reactivity was determined by [³H]dT incorporation 2 d later. Proliferation is indicated as percent of control (no BZ194 treatment; CD3/CD28 stimulation: 6445 ± 475 cpm; CD3/CD28 + ionomycin stimulation: 6624 ± 320 cpm; PMA/ionomycin stimulation: 5264 ± 521cpm). Data represent means ± SD of measurements from 5 independent experiments. ∗, P ≤ 0.01.