Research ArticleT CELLS

T cell receptor–triggered nuclear actin network formation drives CD4+ T cell effector functions

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Science Immunology  04 Jan 2019:
Vol. 4, Issue 31, eaav1987
DOI: 10.1126/sciimmunol.aav1987
  • Fig. 1 TCR signaling induces nuclear actin polymerization.

    (A) Top: Jurkat NCLA cells were placed on TCR-stimulatory coverslips and subjected to live-cell microscopy. Shown are still images at the indicated time after contact with the stimulatory surface (see also movie S1). Bottom: Single-cell analysis of actin dynamics over 10 min after activation. Individual cells were imaged as in (A), and the periods in which polymerized F-actin was detected in the nucleus (green) or at the PM (red) are indicated. p.a., post activation. (B) Primary human CD4+ T cell expressing nuc.lifeact interacting with SEB-pulsed Raji B cells. Top: Representative still images of live-cell movies. Inset: Boxed contact region (cont.) in high contrast (see also movie S2). Bottom: Single-cell analysis of actin dynamics over 50 min after contact with the Raji B cell. (C to E) Nuclear actin polymerization in Jurkat NLA cells in response to surface-bound (C, top, and D) or soluble (C, bottom, and E) stimuli. Shown are representative images (see also movie S3) and relative occurrence of nuclear F-actin filaments (mean and SD from three experiments in which at least 30 cells were analyzed per condition each). Statistical significance relative to the αCD3/28 (D) or PMA/Iono (E) control was assessed by one-sample t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DMSO, dimethyl sulfoxide. Visualization of nuclear F-actin with nuclear lifeact.GFP after (+) or without (−) stimulation with PMA/Iono in primary human CD4+ T cells (F). Visualization of endogenous nuclear actin filaments with Alexa Fluor 488 Phalloidin in A3.01 T cells without stimulation (−) or after stimulation (+) with PMA/Iono (G). Scale bars, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments.

  • Fig. 2 Comparative analysis of the nuclear F-actin networks in A3.01 T cells and NIH3T3 fibroblasts.

    (A) STED microscopy of endogenous nuclear actin filaments with Alexa Fluor 488 and Atto 647N Phalloidin in A3.01 T cells without stimulation (−) or after stimulation (+) with PMA/Iono. (B) Workflow for the quantification of nuclear actin filaments. STED images of endogenous nuclear actin filaments and segmentation based on supervised machine learning for comparative quantification between A3.01 T cells stimulated with PMA/Iono (left) and NIH3T3 (3 T3) cells stimulated by serum (right). (C) Example of NIH3T3 cytoplasmic actin stress fibers. Stimulated A3.01 T cells and NIH3T3 show nuclear filaments that are similar in size (D) and do not differ in the number of junctions per filament (E). ns, not significant. A3.01 show fewer nuclear actin filaments per cell (F) compared with stimulated NIH3T3 cells. The overall size of the nuclear actin filament network (sum of all nuclear actin filaments) is higher in NIH3T3 cells than in A3.01 cells (G), but the number of filaments in the nucleus is comparable (H). When normalized to the nuclear area, the network size between A3.01 and NIH3T3 cells appears to be similar (I). (J and K) Anisotropy of nuclear actin filaments: No orientation is favored by nuclear actin filaments, and all angles in the orientation range are occupied to a similar extent by nuclear actin filaments in stimulated A3.01 (J) and NIH3T3 (K) cells. For comparison, cytoplasmic actin stress fibers (stress) are plotted in (J) and show a characteristic isotropic peak in the range of orientation. Scale bars, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments (B) or stress fibers (C). Statistical significance was determined by unpaired t test (n = 19 A3.01 T and n = 18 NIH3T3 cells quantified from two independent experiments). Only filaments longer than 200 nm are considered in the quantitative analysis. **P ≤ 0.01, ***P ≤ 0.001.

  • Fig. 3 Nuclear calcium signaling drives nuclear actin polymerization in T cells.

    (A) Live-cell imaging of Ca2+ release (top, X-Rhod-1 AM) and nuclear actin polymerization (bottom) in Jurkat NLA cells upon contact with a TCR-stimulatory surface. Shown are still images at the indicated time after exposure to the stimulatory surface (see also movie S4). (B) Quantification of Ca2+ release triggered upon contact with a TCR-stimulatory surface in cells analyzed as in (A). Depicted are means ± SD of total and nuclear Ca2+ levels from eight cells analyzed. FC, fold change. (C) Single-cell analysis over 20 min after activation. Occurrence of actin polymerization at the PM (red) or the nucleus (green) as well as Ca2+ release (yellow) is shown. (D to G) Visualization of Ca2+ release and nuclear actin polymerization upon PMA/Iono stimulation in A3.01 NLA cells. (D and E) Stills of live-cell imaging movies of Ca2+ release of the same cell before (t = 0 s) and after (t = 44 s) stimulation with PMA/Iono. Dashed circles indicate the position of the nucleus. (F and G) Quantification of Ca2+ release (means ± SD from at least six cells each; intensities normalized to the frame before stimulation). Cells were treated with DMSO (D and F) or the Ca2+ chelator BAPTA-AM (E and G). (H) Relative occurrence of nuclear actin filaments in A3.01 NLA cells after treatment with the indicated stimuli and small-molecule inhibitors of Ca2+ signaling (means ± SD from three experiments with at least 30 cells evaluated each per condition). (I and J) Inhibition of nuclear CaM impairs the formation of nuclear F-actin filaments. Representative micrographs (I) and quantification of occurrence of nuclear or PM F-actin (means ± SD of three experiments with 30 cells evaluated each per condition) (J). A3.01 NLA (nuclear F-actin) or Jurkat NLA (F-actin ring) cells transiently expressing mCherry or the nuclear CaM inhibitor CaMBP4.mCherry were analyzed 58 s or 5 min after activation of a TCR-stimulatory surface, respectively. Asterisks indicate mCherry-positive cells. Statistical significance relative to the PMA/Iono (H) or mCherry (J) control was assessed by one-way analysis of variance (ANOVA) or one-sample t test, respectively. Scale bars, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments.

  • Fig. 4 Nuclear Arp3 mediates formation of TCR-induced nuclear actin filaments.

    (A and B) Silencing of Arp3 expression impairs formation of TCR-induced nuclear actin filaments. (A) Representative images of Jurkat NLA cells expressing the indicated shRNAs after PMA/Iono stimulation. (B) Relative occurrence of cells with nuclear F-actin (top) and Western blot expression analysis (bottom). GAPDH, glyceraldehyde phosphate dehydrogenase. (C and D) Pharmacological inhibition of Arp2/3 complex impairs formation of TCR-induced nuclear actin filaments. Jurkat NLA cells were treated with DMSO or increasing amounts of the Arp2/3 inhibitor CK-869, stimulated with PMA/Iono, and analyzed for formation of nuclear actin filaments. (C) Representative images. (D) Relative occurrence of cells with nuclear F-actin. (E to G) Inhibition of nuclear Arp2/3 impairs formation of TCR-induced nuclear actin filaments at the IS. Primary human CD4+ T cells expressing mCherry or nucleus-targeted dn Arp2.mCherry (nuc.dnArp2.mCherry) together with nuc.lifeact.GFP were incubated with SEB-loaded Raji B cells (red) analyzed for the formation of F-actin in the nucleus and at the cell-cell contact. (E) Representative micrographs. Scale bar, 5 μm. Yellow arrowheads indicate F-actin at the IS, and white arrowheads indicate F-actin in the nucleus (see also movie S9). (F) Quantification of cells with nuclear F-actin. (G) Quantification of cells with F-actin at the IS. D1, donor 1. Silencing of NIK (H and I) or NWASP (J and K) expression impairs formation of TCR-induced nuclear actin filaments. (H and J) Representative images of Jurkat NLA cells expressing the indicated shRNAs after PMA/Iono stimulation. Quantifications (B, D, I, and K) depict means ± SD from three experiments with at least 30 cells evaluated each per condition. Statistical significance relative to shC (B, I, and K) and DMSO-treated (D) controls was assessed by one-sample t test.

  • Fig. 5 Arp2/3-mediated formation of nuclear actin networks is essential for CD4+ T cell effector functions.

    (A and B) Presence of the Arp2/3 inhibitor CK-869 during 10 min of PMA/Iono stimulation inhibits nuclear actin network formation in primary human CD4+ T cells. (A) Representative images of primary human CD4+ T cells expressing nuc.lifeact.GFP. (B) Occurrence of nuclear F-actin relative to the PMA/Iono control (mean ± SD of cells from four different donors with 30 cells evaluated each per condition). (C) Primary human CD4+ T cells isolated from three healthy donors as depicted in fig. S7A were either left unstimulated or stimulated by PMA/Iono in the presence or absence of CK-869 for 24 hours. Shown are cytokine concentrations detected in the supernatants of the respective treatments that were potently blocked by the presence of CK-869 during T cell stimulation. n.d., not detected. (D) Schematic overview and experimental workflow of the in vivo analysis of T cell help. Transferred cells expressed transgenic BCR and TCR recognizing HEL and OVA antigens, respectively. (E) Cytokine production of murine CD4+ T cells expressing nuc.dnArp2.mCherry relative to mCherry control shown as log2 fold changes for cells from three animals. (F and G) Expression of nuc.dnArp2.mCherry in CD4+ T lymphocytes impairs production of antigen-specific antibodies. CD4+ T cells were transduced to express mCherry or nuc.dnArp2.mCherry, and mCherry-positive cells were adoptively transferred together with B cells into recipient mice, which were later immunized with HEL-OVA antigen (see legend to fig. S10 for details). At the indicated time points after HEL-OVA immunization, IgG and IgM antibody production was quantified (F and G). At day 12 or 14 after immunization, cells were isolated from the lymph node of recipient animals to determine the frequency of mCherry-positive cells among the lymphocyte population (H). Ex vivo proliferation assay of murine OT-II cells expressing mCherry or nuc.dnArp2.mCherry activated by coculture with SW-HEL B cells with different concentrations of HEL-OVA (I). Statistical significance relative to DMSO-treated (B) or mCherry (F) controls was assessed by one-sample t test (B) or Mann-Whitney test (F; mean ± SD from six mice per group and two independent experiments). Scale bar, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/31/eaav1987/DC1

    Materials and Methods

    Fig. S1. Visualization of nuclear and cytoplasmic actin dynamics upon TCR stimulation.

    Fig. S2. Calcium and actin dynamics during T cell stimulation.

    Fig. S3. Screen for actin nucleators involved in nuclear actin polymerization upon T cell stimulation.

    Fig. S4. Effect of CK-869 on Ca2+ and actin dynamics.

    Fig. S5. Effect of T cell activation on subcellular distribution of Arp3.

    Fig. S6. Influence of nuc.dnArp2.mCherry on actin and Ca2+ dynamics upon T cell stimulation.

    Fig. S7. Cytokine release upon T cell stimulation in the presence of CK-869.

    Fig. S8. Differentially expressed genes upon T cell stimulation and inhibition of Arp2/3 or nuclear calcium.

    Fig. S9. Gene expression of cytokines is affected early after activation in the presence of CK-869.

    Fig. S10. In vivo model system used for analysis of T cell help.

    Table S1. Time-dependent differential gene expression of primary human CD4+ T cells upon PMA/Iono activation and CK-869 treatment.

    Table S2. Raw data of all figure graphs.

    Movie S1. Nuclear actin polymerization precedes formation of circumferential F-actin rings at the PM triggered by TCR engagement.

    Movie S2. Nuclear actin polymerization upon immune synapse formation.

    Movie S3. TCR signaling induces nuclear actin polymerization.

    Movie S4. Calcium release and nuclear actin polymerization coincide.

    Movie S5. Inhibition of nuclear calcium signaling inhibits nuclear actin polymerization.

    Movie S6. Three different shRNAs targeting Arp3 expressed in Jurkat NLA impair formation of PMA/Iono-induced nuclear actin filaments.

    Movie S7. Pharmacological inhibition of Arp2/3 complex impairs formation of TCR-induced nuclear actin filaments.

    Movie S8. Inhibition of nuclear Arp2/3 impairs formation of TCR-induced nuclear actin filaments.

    Movie S9. Specific inhibition of nuclear Arp2/3 and the effect on nuclear actin polymerization on immune synapse formation in T cells.

    Movie S10. Pharmacological inhibition of Arp2/3 inhibits nuclear actin polymerization in primary T cells.

    References (4352)

  • Supplementary Materials

    The PDF file includes:

    • Materials and Methods
    • Fig. S1. Visualization of nuclear and cytoplasmic actin dynamics upon TCR stimulation.
    • Fig. S2. Calcium and actin dynamics during T cell stimulation.
    • Fig. S3. Screen for actin nucleators involved in nuclear actin polymerization upon T cell stimulation.
    • Fig. S4. Effect of CK-869 on Ca2+ and actin dynamics.
    • Fig. S5. Effect of T cell activation on subcellular distribution of Arp3.
    • Fig. S6. Influence of nuc.dnArp2.mCherry on actin and Ca2+ dynamics upon T cell stimulation.
    • Fig. S7. Cytokine release upon T cell stimulation in the presence of CK-869.
    • Fig. S8. Differentially expressed genes upon T cell stimulation and inhibition of Arp2/3 or nuclear calcium.
    • Fig. S9. Gene expression of cytokines is affected early after activation in the presence of CK-869.
    • Fig. S10. In vivo model system used for analysis of T cell help.
    • Legends for tables S1 and S2
    • Legends for movies S1 to S10
    • References (4352)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Time-dependent differential gene expression of primary human CD4+ T cells upon PMA/Iono activation and CK-869 treatment.
    • Table S2 (Microsoft Excel format). Raw data of all figure graphs.
    • Movie S1 (.mov format). Nuclear actin polymerization precedes formation of circumferential F-actin rings at the PM triggered by TCR engagement.
    • Movie S2 (.mov format). Nuclear actin polymerization upon immune synapse formation.
    • Movie S3 (.mov format). TCR signaling induces nuclear actin polymerization.
    • Movie S4 (.mov format). Calcium release and nuclear actin polymerization coincide.
    • Movie S5 (.mov format). Inhibition of nuclear calcium signaling inhibits nuclear actin polymerization.
    • Movie S6 (.mov format). Three different shRNAs targeting Arp3 expressed in Jurkat NLA impair formation of PMA/Iono-induced nuclear actin filaments.
    • Movie S7 (.mov format). Pharmacological inhibition of Arp2/3 complex impairs formation of TCR-induced nuclear actin filaments.
    • Movie S8 (.mov format). Inhibition of nuclear Arp2/3 impairs formation of TCR-induced nuclear actin filaments.
    • Movie S9 (.mov format). Specific inhibition of nuclear Arp2/3 and the effect on nuclear actin polymerization on immune synapse formation in T cells.
    • Movie S10 (.mov format). Pharmacological inhibition of Arp2/3 inhibits nuclear actin polymerization in primary T cells.

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