Research ArticleImmunology

The purinergic receptor P2Y11 choreographs the polarization, mitochondrial metabolism, and migration of T lymphocytes

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Science Signaling  29 Sep 2020:
Vol. 13, Issue 651, eaba3300
DOI: 10.1126/scisignal.aba3300
  • Fig. 1 P2X4 and P2Y11 receptors promote T cell migration.

    (A) Human PBMCs were treated for 20 min with P2X4 antagonist (antag.) (5-BDBD, 10 μM), P2Y11 antagonist (NF340, 10 μM), or P2Y11 agonist (NF546, 1 μM). CD4 T cells were identified by CD4 labeling. CD4 T cell migration in response to SDF-1α (100 ng/ml) was monitored with video microscopy for 30 min, and migration speeds of individual cells (n ≥ 20 per experiment) were calculated. Box plots of the means of independent experiments (n = 3 to 6; indicated by circles) are shown. ***P < 0.001 compared to SDF-1α control (one-way ANOVA). (B) Jurkat T cells were treated with 5-BDBD, NF340, H89 (5 μM), NF546, or cell-permeable cAMP-AM (1 μM) or transfected with P2Y11-targeting, P2X4-targeting, or nontargeting control siRNA and cell migration in response to SDF-1α was analyzed. Mean migration speeds from at least three independent experiments (indicated by circles; n ≥ 20 cells per experiment) are shown as box plots. ***P < 0.001 and ###P < 0.001 compared to SDF-1α controls (one-way ANOVA). (C) Effect of 5-BDBD, NF340, H89, NF546, or cAMP-AM on the migration of CD4 effector T cells. Mean migration speeds from at least three independent experiments (indicated by circles; n ≥ 20 cells per experiment) are shown as box plots. ***P < 0.001 compared to control (one-way ANOVA).

  • Fig. 2 Autocrine P2Y11 receptor signaling regulates cell polarization and pseudopod formation.

    (A) Human effector CD4 T cells were obtained by stimulation for 3 days with anti-CD3/CD28 antibody–coated beads and then treated for 30 min with P2Y11 antagonist (NF340, 10 μM), PKA inhibitor (H89, 5 μM), P2Y11 agonist (NF546, 1 μM), or cell-permeable cAMP (cAMP-AM, 1 μM). Scale bar, 10 μm. (B to D) Cell polarization expressed as ratio between lengths (l) and widths (w) of individual cells (B), cell surface areas (C), and the number of pseudopods extended (D) were analyzed after treatment of effector T cells for 30 min with inhibitors and agonists as in (A). Box plots of n ≥ 65 cells from at least three independent experiments are shown. Circles indicate single cells. ***P < 0.001 compared to controls (Kruskal-Wallis test). (E to G) Freshly isolated naïve human CD4 T cells were treated with NF340 (antag.) or NF546 (agon.) for 10 min, stimulated with SDF-1α (100 ng/ml) or not (controls), and observed for 30 min by time-lapse video microscopy. Cell polarization (E), cell surface areas (F), and the number of pseudopods formed (G) were analyzed after 30 min. Box plots represent n ≥ 50 cells (indicated by circles) from independent experiments (n ≥ 3). ***P < 0.001 compared to SDF-1α controls (Kruskal-Wallis test).

  • Fig. 3 P2X4 receptor signaling defines the subcellular redistribution of P2Y11 receptors during T cell polarization.

    (A to C) P2Y11 and P2X4 receptor distribution patterns were analyzed in Jurkat cells expressing YFP-tagged P2Y11 or EGFP-tagged P2X4 receptors 10 min after the addition of SDF-1α (100 ng/ml) or vehicle control. In (C), cells were preincubated with the P2X4 receptor antagonist 5-BDBD (10 μM) for 10 min before stimulation. Arrows indicate the direction of cell migration. Images and corresponding receptor distribution profiles are representative of at least 20 (A and B) or 10 (C) cells from at least three separate experiments (scale bars, 10 μm); a.u., arbitrary units. (D) Correlation between P2Y11 receptor accumulation at the back of cells and the uropod retraction speed in migrating cells. Uropod retraction was tracked for 2 min (n = 12 cells from three independent experiments); r, Pearson’s correlation coefficient. (E) Jurkat cells expressing P2Y11-YFP were labeled with MitoTracker Red CM-H2Xros, and the distribution of P2Y11 receptors and active mitochondria after stimulation with SDF-1α was recorded over time. Representative images of 10 cells from different experiments and corresponding distribution profiles of P2Y11-YFP and active mitochondria summed over the whole cell area are shown. Arrows indicate the direction of migration. Scale bar, 10 μm.

  • Fig. 4 P2Y11 receptors restrict excitatory P2X4 receptor signaling to the front of migrating T cells.

    Jurkat cells expressing EGFP-tagged P2X4 receptors were labeled with MitoTracker Red CM-H2Xros and treated with P2Y11 antagonist (NF340, 10 μM), P2Y11 agonist (NF546, 1 μM), or vehicle control for 10 min. Then, cells were stimulated with SDF-1α (100 ng/ml), and redistribution of P2X4 receptors and active mitochondria was analyzed after 3 min. Representative images are shown in (A). Summarized results of 6 to 10 separate experiments are shown in (B). Dots indicate the distribution of single-cell fluorescence values, and lines represent averaged summarized line profiles of the whole cell areas. Scale bar, 10 μm.

  • Fig. 5 P2X4 and P2Y11 receptor signaling regulates mitochondrial activity.

    (A and B) Human CD4 T cells were labeled with Rhod-2 and treated for 20 min with P2X4 antagonist (5-BDBD, 10 μM), P2Y11 antagonist (NF340, 10 μM), PKA inhibitor (H89, 5 μM), P2Y11 agonist (NF546, 1 μM), or cell-permeable cAMP (cAMP-AM, 1 μM). Mitochondrial Ca2+ levels after SDF-1α (100 ng/ml) stimulation were analyzed by fluorescence microscopy. Results show fold changes of mean Rhod-2 fluorescence values ± SEM (A) or peak fluorescence values (B) from 30 to 85 cells (indicated by circles) derived from separate experiments (n ≥ 3); ***P < 0.001 compared to SDF-1α–stimulated control (Kruskal-Wallis test). (C) Jurkat T cells were treated for 30 min with 5-BDBD, NF340, H89, NF546, or cAMP-AM—stimulated or not (unstim.) with SDF-1α (100 ng/ml) or vehicle (handling control)—and ATP levels in the supernatants were determined after 5 min. Data are means ± SD, circles indicate independent experiments (n = 3 to 5); ***P < 0.001 compared to SDF-1α–stimulated control (one-way ANOVA).

  • Fig. 6 Interference with P2Y11 receptor signaling impairs cytosolic Ca2+ signaling.

    (A and B) Human CD4 T cells were labeled with Fluo-4 and treated for 20 min with P2X4 antagonist (5-BDBD, 10 μM), P2Y11 antagonist (NF340, 10 μM), PKA inhibitor (H89, 5 μM), P2Y11 agonist (NF546, 1 μM), or global cAMP stimulation (cAMP-AM, 1 μM). Cytosolic Ca2+ levels after stimulation with SDF-1α (100 ng/ml) were analyzed by fluorescence microscopy. Results show fold changes of mean Fluo-4 fluorescence values ± SEM (A) or box plots of averaged peak fluorescence values (B) from 5 to 12 separate experiments (indicated by circles; n ≥ 15 cells per experiment). **P < 0.01 and ***P < 0.001 compared to SDF-1α–stimulated control (one-way ANOVA).

  • Fig. 7 Paracrine interference with P2Y11 receptor signaling impairs T cell functions.

    (A and B) Proposed model by which autocrine purinergic signaling regulates the polarization and migration of T cells. (A) Stimulation of CXCR4 by SDF-1α induces the release of ATP through pannexin-1 (Panx1) channels and P2X4 receptor–induced Ca2+ influx that initiates cell polarization, including the translocation of P2Y11 receptors to the back of cells. (B) Autocrine P2Y11 receptor signaling at the back shuts down the activation of nearby mitochondria and confines mitochondrial activity to the front of cells where mitochondrial ATP production amplifies P2X4 receptor signaling and energizes the cytoskeletal rearrangements needed for pseudopod protrusion and T cell migration. (C) Effect of apyrase on cell migration speed of CD4 effector T cells. Data are means ± SD of three to six independent experiments (n = 20 to 40 cells per experiment). *P < 0.05 compared to control (one-way ANOVA). (D) Human PBMCs were treated with P2Y11 agonist (NF546, 1 μM), exogenous ATP (10 μM), nonhydrolyzable ATP analog (ATPγS, 10 μM), or vehicle control and stimulated with SDF-1α (100 ng/ml; migration) or anti-CD3 antibodies (0.25 μg/ml; CD69, IL-2, and proliferation). Migration speed of CD4 T cells (n ≥ 20 cells per experiment) was analyzed by time-lapse video microscopy. CD69 expression in CD4 T cells was analyzed by flow cytometry after 4 hours. IL-2 in the supernatant was measured after 6 hours with ELISA. Proliferation of CD4 T cells was assessed 72 hours after cell stimulation by CFSE dye dilution. Data are from at least three independent experiments (indicated by circles) with cells from different donors. ***P < 0.001 compared to controls (one-way ANOVA).

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/13/651/eaba3300/DC1

    Fig. S1. P2Y11 receptors regulate the polarization and migration of Jurkat T cells.

    Fig. S2. Redistribution of P2Y11 receptors defines the subcellular distribution of active mitochondria and cell protrusions during T cell migration.

    Fig. S3. P2Y11 receptor stimulation does not increase intracellular Ca2+ levels in human CD4 T cells.

    Fig. S4. Adenosine does not affect human T cell migration.

    Movie S1. P2Y11 receptor silencing impairs Jurkat T cell migration.

    Movie S2. P2X4 and P2Y11 receptors regulate T cell migration.

    Movie S3. P2Y11 receptors direct cell protrusions during T cell migration.

    Movie S4. P2Y11 receptors restrict P2X4 receptor signaling and mitochondrial activation to the front of migrating T cells.

    Movie S5. Proposed model of how P2X4 and P2Y11 receptors synergize to regulate T cell migration.

    Movie S6. Extracellular ATP impairs T cell migration.

  • The PDF file includes:

    • Fig. S1. P2Y11 receptors regulate the polarization and migration of Jurkat T cells.
    • Fig. S2. Redistribution of P2Y11 receptors defines the subcellular distribution of active mitochondria and cell protrusions during T cell migration.
    • Fig. S3. P2Y11 receptor stimulation does not increase intracellular Ca2+ levels in human CD4 T cells.
    • Fig. S4. Adenosine does not affect human T cell migration.
    • Legends for movies S1 to S6

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). P2Y11 receptor silencing impairs Jurkat T cell migration.
    • Movie S2 (.avi format). P2X4 and P2Y11 receptors regulate T cell migration.
    • Movie S3 (.avi format). P2Y11 receptors direct cell protrusions during T cell migration.
    • Movie S4 (.avi format). P2Y11 receptors restrict P2X4 receptor signaling and mitochondrial activation to the front of migrating T cells.
    • Movie S5 (.avi format). Proposed model of how P2X4 and P2Y11 receptors synergize to regulate T cell migration.
    • Movie S6 (.avi format). Extracellular ATP impairs T cell migration.

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