GrassiScienceSignal2008Suppl

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Unformatted text preview: www.sciencesignaling.org/cgi/content/full/1/39/ra6/DC1 Supplementary Materials for Purinergic Control of T Cell Activation by ATP Released Through Pannexin-1 Hemichannels Ursula Schenk, Astrid M. Westendorf, Enrico Radaelli, Anna Casati, Micol Ferro, Marta Fumagalli, Claudia Verderio, Jan Buer, Eugenio Scanziani, Fabio Grassi* *To whom correspondence should be addressed. E-mail: [email protected] Published 30 September 2008, Sci. Signal. 1, ra6 (2008) DOI: 10.1126/scisignal.1160583 This PDF file includes: Fig. S1. Decreased CRAC inactivation and unaltered mitochondrial membrane potential in crt−/− T cells. Fig. S2. Analysis of ATP release from activated T cells. Fig. S3. Reduced Ca2+ entry, ATP synthesis, and ATP release in Tregs. Fig. S4. Analyses of purinergic receptor expression and function in T cells. Fig. S5. Influence of oATP and pannexin hemichannel inhibitors on productive T cell activation. A TG 1.8 1.6 ΔF 340/380 nm 1.4 1.2 1 0.8 0.6 0.4 0 200 crt+/+ Iono 1.8 1.6 ΔF 340/380 nm 1.4 1.2 1 0.8 0.6 400 600 Time (s) 800 1000 0.4 0 200 TG crt-/Iono 400 Time (s) 600 800 B Naïve CD4+ T cells Effector/memory CD4+ T cells Cell number crt-/- Cell number crt+/+ TMRM crt-/- crt+/+ TMRM Fig. S1. Decreased CRAC inactivation and unaltered mitochondrial membrane potential in crt-/- T cells. (A) To score CRAC inactivation, T cells were loaded with Fura-2 and plated on poly-L-lysine–coated coverslips. ER stores of Ca2+ were depleted by the addition of thapsigargin (TG) to medium devoid of Ca2+. Following complete ER store depletion, Ca2+ was added twice for 100s (green filled bars). Both additions of Ca2+ resulted in Ca2+ influx through CRACs. Because these channels are inactivated by Ca2+, the second rise in Ca2+ occurred with a slower rate and lower amplitude than did the first. Crt-/- cells showed diminished CRAC inactivation. Differently colored traces represent Ca2+ fluxes in individual cells, whereas the black line represents the average response. Addition of ionomycin at the end of the assay (third green bar) identifies viable cells. (B) Tetramethylrhodamine methyl ester (TMRM) staining of sorted naïve (CD44-CD62L+) and effector/memory (CD44+CD62L-) CD4+ T cells from crt-/- and crt+/+ FLC mice showed that deletion of crt did not modify mitochondrial membrane potential. A 5 min 15 min 20 min B +CBX 3.0 2.5 ROI 1 ROI 2 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 800 900 1000 1100 1200 1300 1400 -1.0 300 400 500 600 700 800 900 ROI 1 ROI 2 [ATP] (μM) Naïve T cell Anti-CD3 antibody-coated microbead 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 Time (s) Time (s) C 1000 D Fluorescence (AU) *** 800 600 400 200 E Relative IL-2 secretion (%) 200 1.5 150 100 50 *** *** [ATP] (μM) * 1.0 0.5 0 ctrl oATP 10 0 panx1 GAP26 ctrl oATP ctrl αCD3avidin αCD3avidin CBX αCD3avidin 18αGA αCD3avidin oATP Fig. S2. Analysis of ATP release from activated T cells. (A) ATP release from T cells stimulated with anti-CD3 antibody-coated microbeads was measured as the increase in NADPH fluorescence generated by a two-enzyme assay (see Methods). Pseudocolor images show the increased fluorescence in the pericellular region of a naïve CD4+ T cell after synapse formation. (B) Representative traces of ATP concentrations in a region of interest (ROI) placed above the T cell (black) or in proximity to the same cell (red). Note that when a T cell is stimulated in the presence of 5 μM carbenoxolone (+CBX, right panel), which inhibits pannexin hemichannels, the fluorescence increase is limited to the cell body (black line), indicative of Ca2+-induced mitochondrial synthesis of NADPH, but no fluorescence is detected in the pericellular region of the same cell (red line). NADPH fluorescence was converted to [ATP] by means of a standard curve, prepared with varying concentrations of ATP as described in the Methods. (C) Histograms and pseudocolor images of carboxyfluorescein uptake in CD4+ naïve T cells either untreated or stimulated with cross-linked anti-CD3 antibodies in the absence or presence of the indicated drugs. Three independent experiments were performed (ctrl, n = 141; anti-CD3, n = 142; CBX, n = 224; 18αGA, n = 156; oATP, n = 58). (D) Naïve CD4+ T cells were stimulated for 48 h with plate-bound anti-CD3 and anti-CD28 antibodies after which, the amount of IL-2 secreted by these cells into the culture supernatant was determined by ELISA. The pannexin-inhibitory peptide 10panx1, but not the connexin-inhibitory peptide GAP26, inhibited IL-2 secretion comparably to that of oATP. (E) Two-enzyme assay of ATP release by naïve CD4+ T cells stimulated with anti-CD3 antibody-coated microbeads. Histograms representing ATP released by cells that were untreated or preincubated with oATP are displayed. *, P<0.05; ***, P<0.0001. A avidin Ionomycin B 2.00 1.75 Naïve T cell Regulatory T cell 3.5 Fold-increase in intracellular [ATP] Naïve T cell 3.0 2.5 Effector/memory T cell ΔF 340/380 nm 1.50 1.25 1.00 0.75 0.50 0 100 200 300 400 500 600 2.0 1.5 Regulatory T cell 1.0 0.5 0 50 100 150 200 Time (s) Time (min) C 1.75 Extracellular [ATP] (μM) 1.5 1.25 ** 1 0.75 0.5 0.25 0 CD4+ Naive CD4+CD25high Regulatory T cells Fig. S3. Reduced Ca2+ entry, ATP synthesis, and ATP release in Tregs. (A) Cytosolic Ca2+ profiles after CD3 stimulation of naïve CD4+ T cells and Tregs. Note that CCE in Treg cells is more transient and of a lower amplitude than that of naïve CD4+ T cells. (B) Changes in the concentrations of cytosolic ATP in sorted naïve and effector/memory CD4+ T cells and Tregs at different time points after T cell activation with anti-CD3 antibodies. (C) ATP release measured by two-enzyme assay in either naïve CD4+ T cells or Tregs stimulated with anti-CD3 antibody-coated beads. **, P <0.01. A P2X1 P2X1 P2X4 P2X7 P2Y1 P2Y12 P2Y13 P2Y14 B ATP + Ca2+ 1000 3 000 ATP - Ca2+ ΔF 340/380 0 50 1 00 150 200 250 30 0 ΔF 340/380 900 2 500 2 000 800 1 500 700 1 000 600 500 500 0 50 100 150 200 250 300 Time (s) Time (s) C αβ methyl ATP 3000 MeSATP 3000 3000 Bz-ATP ΔF 340/380 ΔF 340/380 ΔF 340/380 2500 2500 2500 2000 2000 2000 1500 1500 1500 1000 1000 1000 500 0 50 100 150 200 500 250 300 0 50 100 150 200 250 300 500 0 50 100 150 200 250 300 Time (s) Time (s) Time (s) D MeSADP 3000 3000 UDP-Gluc ΔF 340/380 2500 ΔF 340/380 0 50 100 150 200 250 300 2500 2000 2000 1500 1500 1000 1000 500 500 0 50 100 150 200 250 300 Time (s) Time (s) Fig. S4. Analyses of purinergic receptor expression and function in T cells. (A) RT-PCR analysis showed that mRNAs for the P2X1, P2X4, and P2X7 receptors and for the P2Y1 and P2Y12 to P2Y14 purinergic receptors are all expressed in T cell clones. (B) Ca2+ imaging experiments in normal medium (left panel) or medium devoid of Ca2+ (right panel) confirm the presence of both ionotropic (P2X) and metabotropic (P2Y) receptors on CD4+ T cell clones. (C) Preferential agonists for P2X receptors (αβ methyl ATP for P2X1, MeSATP for all P2X receptors, and Bz-ATP for P2X7) confirm the functional competence of the P2X receptors whose expression was detected in A. (D) Functional P2Y receptors are also present on T cell clones, as shown by the response to preferential agonists (MeSADP for P2Y1, P2Y12, and P2Y13, UDP-glucose for P2Y14). Differently colored traces represent individual cells monitored in the same field. A Control 100 80 60 40 20 CD3/28 +PMA +PMA B Cell number 600 33568 400 200 0 100 101 102 103 104 600 400 200 0 100 101 102 103 104 6830 600 400 200 3443 C Cell number 500 27388 300 100 ctrl 0 300 200 100 10 Cell number ctrl 0 100 100 80 60 40 20 0 100 101 102 101 102 103 104 CD3/28 CD3/28 +PMA +oATP Cell number 600 24001 400 200 Cell number 0 100 101 102 103 104 oATP 6442 0 panx1 Cbx +oATP 103 104 Cell number 0 100 101 102 103 104 18αGA 1200 52285 900 600 300 10 CD69 CFSE 0 100 101 102 103 104 panx1+PMA CFSE Fig. S5. Influence of oATP and pannexin hemichannel inhibitors on productive T cell activation. (A) Antibody labeling of surface CD69 after 6h of stimulation of naïve CD4+ T cells with plate-bound anti-CD3 and anti-CD28 antibodies either in the absence (top panel) or presence (bottom panel) of oATP. The addition of PMA fully restored TCRstimulated surface CD69 in the presence of oATP. (B) T cell proliferation of naïve CD4+ T cells labeled with CFSE and stimulated for 16 h with plate-bound anti-CD3 and antiCD28 antibodies. Dilution of CFSE (and indicator of cell proliferation) was strongly affected by carbenoxolone (cbx) and oATP, but not by α18-glycyrrhetinic acid. (C) 10 Panx1 peptide mediated inhibition of T cell proliferation (middle panel) was reversed by the addition of PMA (lower panel). ...
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