Research ArticleImmunodeficiency

Inhibition of diacylglycerol kinase α restores restimulation-induced cell death and reduces immunopathology in XLP-1

See allHide authors and affiliations

Science Translational Medicine  13 Jan 2016:
Vol. 8, Issue 321, pp. 321ra7
DOI: 10.1126/scitranslmed.aad1565
  • Fig. 1. DGKα silencing or inhibition restores RICD in XLP-1 patient T cells.

    (A and B) Activated T cells from normal donors (Ctrl) or indicated XLP-1 patients were transfected with control (Cntrl) or DGKα-specific siRNA, and then restimulated 4 days later with OKT3 Ab. After 24 hours, % cell loss was evaluated by propidium iodide (PI) staining. Data are means ± SD of two experiments (A) or one experiment (B) performed in triplicate, representative of two independent experiments using different control donors. (C) DGKα relative expression (rel exp) in siRNA-transfected cells from (A) measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (upper panel, mean ± SEM, n = 4) or by Western blotting, with tubulin as loading control (lower panel). (D to F) Ctrl or XLP patient T cells were restimulated with OKT3 Ab after pretreatment with DGK inhibitor R59949 or R59022 (5 to 10 μM) or dimethyl sulfoxide (DMSO). After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SD of three experiments (E) or one experiment (D and F) performed in triplicate representative of two independent experiments using different control donors. (G) Cells used in (D) were pretreated with R59949 (10 μM) or DMSO and restimulated with OKT3 (100 ng/ml) for 0, 6, and 12 hours. The % of apoptotic cells was measured by AnnexinV staining. Representative histograms are shown; marker numbers denote % AnnexinV+ cells. The net increase in AnnexinV+ cells at 12 hours is shown at the right. Data are means ± SD of six independent experiments using four separate controls and two XLP patients. Asterisks denote statistical significance by two-way analysis of variance (ANOVA) with Sidak correction (A, B, D, and F) or paired t test (C and G).

  • Fig. 2. DGKα silencing or inhibition restores RICD in SAP-silenced T cells.

    (A) Activated normal donor T cells were transfected with control or SAP siRNA and restimulated 4 days later with OKT3 Ab. After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SEM of three experiments performed in triplicate. (B) SAP expression in siRNA-transfected T cells from (A) was measured by qRT-PCR (upper panel, mean ± SEM of four experiments) or by Western blotting, with actin as a loading control (lower panel). (C and D) siRNA-transfected cells (A) were restimulated with OKT3 Ab after pretreatment with DMSO and DGK inhibitor R59949 or R59022 (5 to 10 μM). After 24 hours, the % cell loss was evaluated by PI staining. Data are means ± SEM of five experiments (C) or five (control) and eight (SAP siRNA) independent experiments (D) performed in triplicate. (E) siRNA-transfected cells as in (A) were pretreated with DMSO or R59022 (10 μM) and restimulated with OKT3 (10 ng/ml). After 12 hours, the % apoptotic cells was evaluated by AnnexinV staining. Representative histograms are shown; marker numbers denote % AnnexinV+ cells. The net increase in AnnexinV+ cells at 12 hours is shown at the right. Data are means ± SD of four experiments. (F) siRNA-transfected cells (A) were treated with C8-DAG (50 μM) and restimulated with OKT3 Ab. After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SEM of five experiments performed in triplicate. Asterisks denote statistical significance by two-way ANOVA with Sidak correction (A and C to F) or paired t test (B and E). (G) Schematic cartoon: Proapoptotic TCR signaling is governed by DGKα inhibition in activated T cells.

  • Fig. 3. DGKα silencing or inhibition restores synapse formation in SAP-deficient T cells.

    ShCNTRL or shSAP Jurkat T cells were transiently transfected as indicated. (A to J) After 48 hours (C, D, and G to J) or 96 hours (A, B, E, and F), T cells were challenged with SEE-loaded Raji B cells, and confocal live-cell images were captured during T cell–antigen presenting cell (APC) conjugation. In (C), (D), (G), (H), (I), and (J), T cells were pretreated for 30 min with 10 μM R59949 or DMSO. (A and C) Top row: Enhanced green fluorescent protein (EGFP)–tagged PKCθ-CRD (pseudocolor) together with the perimeter of the APC (dotted line). Bottom row: Phase-contrast images with APC denoted by *. Scale bar, 10 μm. (B and D) Quantification of EGFP-PKCθ-CRD accumulation at the IS. Mean ± SEM of >20 conjugates per condition from three experiments. (E and G) Top row: LifeAct–GFP (green fluorescent protein) (green). Bottom row also shows Raji B cells stained with CellTracker Red CMTPX (red). Scale bar, 10 μm. (F and H) Quantification of LifeAct-GFP accumulation at the IS. Mean ± SEM of >20 conjugates per condition from three experiments. (I) Top row: GFP-tubulin (green). Bottom row also shows Raji B cells stained with CellTracker Red CMTPX (red). Scale bar, 10 μm. (J) Quantification of MTOC polarization index. Mean ± SEM of >35 conjugates per condition from two experiments. Asterisks in all panels denote statistical significance by one-way ANOVA with Sidak correction.

  • Fig. 4. DGKα silencing or inhibition restores PKCθ and RasGRP1 recruitment to the IS in SAP-deficient cells.

    (A, C, E, and G) Activated T cells were transfected with the indicated siRNA and after 72 hours were incubated with SEE-loaded Raji B cells (denoted with *) for 15 min and fixed and stained for PKCθ (A and C) or RasGRP1 (E and G). Top rows: Target protein (green); bottom rows also show phase contrast. Scale bar, 5 μm. (B) Percentage of cells displaying PKCθ at the IS. Data are means ± SEM of six replicates from two independent experiments. (D) Percentage of cells displaying PKCθ at the IS. Data are means ± SEM of three experiments. (F) Percentage of cells with RasGRP1 at the IS. Data are means ± SD of one representative experiment performed in quadruplicate. (H) Percentage of cells displaying RasGRP1 at the IS. Data are means ± SEM of three experiments. Asterisks in all panels denote statistical significance by two-way ANOVA + Sidak correction.

  • Fig. 5. DGKα silencing restores TCR-induced PKCθ and Ras-mediated signaling pathways to drive RICD in SAP-deficient cells.

    (A and B) Activated normal donor T cells were transfected with the indicated siRNA and restimulated 4 days later with OKT3 Ab (10 ng/ml). After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SEM of seven (A) or six (B) experiments performed in triplicate. Right panels: Expression of PKCθ (A) or RasGRP1 (B) was measured by Western blotting, with actin as a loading control. (C and D) qRT-PCR for IL2 mRNA in T cells pretreated with R59949 (10 μM) (C) or transfected with DGKα siRNA (D) after restimulation with OKT3 (10 μg/ml, 4 hours). GUSB served as the reference gene. Graphs represent mean ± SEM of six (C) or seven (D) experiments. (E) Left: Representative flow cytometric histograms showing CD25 surface expression on siRNA-transfected T cells from (A) ±OKT3 restimulation (24 hours). Right: Graph depicts mean fluorescence intensity (MFI) of CD25 expression. Data are means ± SEM of four experiments. Asterisks in all panels denote statistical significance by two-way ANOVA with Sidak correction. IgG, immunoglobulin G. (F) Schematic cartoon: SAP-mediated inhibition of DGKα activity ensures a sufficient pool of DAG required for proper IS organization and recruitment of PKCθ and RasGRP, which mediates downstream signaling for RICD.

  • Fig. 6. Silencing or inhibition of DGKα restores RICD sensitivity in SAP-deficient T cells via induction of proapoptotic molecules NUR77 and NOR1.

    (A to D) qRT-PCR for NR4A1 (A and B) or NR4A3 (C and D) from activated normal donor T cells transfected with control or SAP-specific siRNA ± DGKα-specific siRNA (A and C) or 5 μM R59949 (B and D) and restimulated with OKT3 (10 μg/ml) for 4 hours. GUSB served as the reference gene. Data are means ± SEM of eight (A), five (B), seven (C), or six (D) experiments. (E) Activated T cells from normal donor (Ctrl) or XLP-1 patient 7 were pretreated for 30 min with R59022 or R59949 (10 μM), then restimulated with OKT3 (100 ng/ml). Cell lysates were analyzed by Western blotting for NUR77, NOR1, and β-actin content. Data are representative of two independent experiments using different donors. (F and G) Activated T cells from normal donors (Ctrl) or XLP-1 patients were pretreated for 30 min with DMSO, SL0101 (90 μM), R59949 (10 μM), or both, followed by restimulation with OKT3 (100 ng/ml). After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SD of one experiment each performed in triplicate using different donors. (H) Activated donor T cells were transfected with the indicated siRNA and treated 4 days later with SL0101 (50 μM) for 30 min, followed by OKT3 (10 ng/ml). After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SEM of five experiments performed in triplicate. (I and J) Activated T cells from normal donors (Ctrl) or XLP-1 patients were transfected control or NUR77 + NOR1 siRNA and treated 4 days later with DMSO or R59022 (10 μM) for 30 min, followed by OKT3 (100 ng/ml). After 24 hours, % cell loss was evaluated by PI staining. Data are means ± SD of one experiment each, performed in triplicate using different donors. Asterisks in all panels denote statistical significance by two-way ANOVA with Sidak correction. (K) Western blot for NUR77 and NOR1 expression in OKT3-restimulated, siRNA-transfected T cells from (I). Actin served as a loading control. (L) Schematic cartoon: Mechanism of proapoptotic TCR signaling governed by SAP-dependent DGKα inhibition in activated T cells. n.s., not significant.

  • Fig. 7. In vivo DGKα inhibition reduces the number of activated virus-specific CD8+ T cells in LCMV-infected Sh2d1a−⁄− mice.

    (A) Images of spleens from uninfected [phosphate-buffered saline (PBS), “P”] and LCMV-infected mice without (LCMV, “L”) or with R59022 treatment (LCMV + R59022, “L + R”). Representative spleens from each cohort from B6 wild-type (WT) (top panel) and Sh2d1a−⁄− mice (lower panel) are shown. (B and C) Ratio of spleen over body weight (B) and total splenocyte count (C) for animals in each group are presented. B6 WT mice, red symbols; Sh2d1a−⁄− mice,  blue symbols. (D to I) Representative flow cytometric (density) plots showing the percentages of CD8+ CD44+ (top) and LCMV-specific CD8+ gp33+ (bottom) in the spleens (D) and livers (G) of B6 WT and Sh2d1a−⁄− mice. Percentages (E and H) and absolute numbers (F and I) of CD8+ CD44+ and CD8+ CD44+ gp33+ cells in the spleens (E and F) and livers (H and I) of B6 WT (red symbols) and Sh2d1a−⁄− (blue symbols) mice. Data are from one of two experiments in which a total of 6 to 10 mice in each cohort were examined. Error bars represent SD. Asterisks denote statistical significance that was determined by two-way ANOVA with Sidak correction.

  • Fig. 8. In vivo DGKα inhibition reduces the number but not incidence of virus-specific CD8+ cytokine-producing cells of LCMV infected Sh2d1a−⁄− mice.

    B6 WT (red symbols) and Sh2d1a−⁄− mice (blue symbols) were either uninfected (PBS, “P”) or infected with LCMV without (LCMV, “L”) or with R59022 treatment (LCMV + R59022, “L + R”). (A) Serum IFNγ levels were assayed on day 8 after infection by enzyme-linked immunosorbent assay (ELISA). Data are compiled from two experiments in which a total of 6 to 10 mice in each cohort were examined. Error bars represent SEM. (B to D) Hematoxylin and eosin–stained liver sections from mice in each group were analyzed for the number of inflammatory foci (B) and area of the inflammatory infiltrate (C). For each sample, five random fields were captured at ×20 magnification and scored. Histology of the livers from representative mice in each group under ×20 magnification (top row) is shown (D). Arrows point to the inflammatory foci. Micrographs in the bottom row are the respective computer analyzed images shown in the top row. (E to G) Splenocytes (2 × 106) from PBS (P), LCMV-infected (L), and LCMV-infected mice with R59022 treatment (L + R) groups were left unstimulated or stimulated with gp33 peptide (0.4 ng/ml) in the presence of monensin (1000 μg/ml) for 5 hours. Cells were then analyzed for intracellular cytokine production and degranulation. Representative flow cytometric (density) plots gated on CD8+ CD44+ splenocytes showing the percentages of CD8+ IFNγ+ (top), IFNγ+ TNFα+ (middle), and IFNγ+ CD107a+ (bottom) cells from B6 WT and Sh2d1a−⁄− mice (E). Percentages (F) and absolute numbers (G) of CD8+ IFNγ+, IFNγ+ TNFα+, and IFNγ+ CD107a+ cells. Absolute numbers were calculated by multiplying the percentages with the respective absolute numbers of CD8+ gp33+ cells. Error bars in (B), (C), (F), and (G) represent SD. Asterisks denote statistical significance that was determined by two-way ANOVA with Sidak correction.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/321/321ra7/DC1

    Methods

    Fig. S1. Activity of DGKα and DGKζ contributes to RICD resistance in T cells.

    Fig. S2. DGKα silencing restores RICD in SAP-deficient cells through PKCθ- and Ras-mediated signaling pathways.

    Fig. S3. DGKα blockade fails to rescue TCR-induced up-regulation of proapoptotic mediators FASL and BIM in SAP-deficient cells.

    Fig. S4. Major steps of the automated segmentation and fluorescence quantification algorithm.

    Fig. S5. DGKα inhibition enhances SAP-deficient CD8+ T cell cytotoxicity against autologous B cell targets.

    Fig. S6. R59022 DGKα inhibitor does not impair viral clearance in the livers and spleens of LCMV infected Sh2d1a−⁄− mice.

    Fig. S7. Gating strategies used in Figs. 7 and 8.

    Table S1. Reagents.

    Table S2. siRNA sequences.

    Table S3. TaqMan gene expression arrays.

    Table S4. Spot densitometry analysis for Western blotting.

    Table S5. Statistical analyses.

    Unmodified Western blot images.

    Source data (excel).

  • Supplementary Material for:

    Inhibition of diacylglycerol kinase α restores restimulation-induced cell death and reduces immunopathology in XLP-1

    Elisa Ruffo, Valeria Malacarne, Sasha E. Larsen, Rupali Das, Laura Patrussi, Christoph Wülfing, Christoph Biskup, Senta M. Kapnick, Katherine Verbist, Paige Tedrick, Pamela L. Schwartzberg, Cosima T. Baldari, Ignacio Rubio, Kim E. Nichols, Andrew L. Snow, Gianluca Baldanzi, Andrea Graziani*

    *Corresponding author. E-mail: graziani.andrea{at}hsr.it

    Published 13 January 2016, Sci. Transl. Med. 8, 321ra7 (2016)
    DOI: 10.1126/scitranslmed.aad1565

    This PDF file includes:

    • Methods
    • Fig. S1. Activity of DGKα and DGKζ contributes to RICD resistance in T cells.
    • Fig. S2. DGKα silencing restores RICD in SAP-deficient cells through PKCθ- and Ras-mediated signaling pathways.
    • Fig. S3. DGKα blockade fails to rescue TCR-induced up-regulation of proapoptotic mediators FASL and BIM in SAP-deficient cells.
    • Fig. S4. Major steps of the automated segmentation and fluorescence quantification algorithm.
    • Fig. S5. DGKα inhibition enhances SAP-deficient CD8+ T cell cytotoxicity against autologous B cell targets.
    • Fig. S6. R59022 DGKα inhibitor does not impair viral clearance in the livers and spleens of LCMV infected Sh2d1a−⁄− mice.
    • Fig. S7. Gating strategies used in Figs. 7 and 8.
    • Table S1. Reagents.
    • Table S2. siRNA sequences.
    • Table S3. TaqMan gene expression arrays.
    • Table S4. Spot densitometry analysis for Western blotting.
    • Table S5. Statistical analyses.
    • Unmodified Western blot images.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

Navigate This Article