Journal: Nature immunology

Article Title: Sestrins induce natural killer function in senescent-like CD8 + T cells

doi: 10.1038/s41590-020-0643-3

Figure Lengend Snippet: a) Representative flow cytometry plots showing T cell gating and NKR expression on peripheral blood lymphocytes, specifically focusing on CD8+ T cell subsets stratified by the expression of CD27/CD45RA in healthy donors. Defined subsets are CD27+CD45RA+ TN, CD27+CD45RA− TCM, CD27−CD45RA− TEM, and CD27−CD45RA+ TEMRA cells. b) Confirmation of expression of CD57, KLRG1, CD244, NKG2D, NKG2C, and KIR2DL on TN (naïve), TCM, TEM, and TEMRA CD8+ T cell subsets. Numbers in quadrants represent percentages of cells in each subset. Numbers above the histograms indicate the MFI. c) Flow cytometry gating of CD8+ T cells to confirm CD27 and CD28 expression in subpopulations based on CD27/CD45RA gating.

Article Snippet: Where indicated, freshly purified human CD8 + T cells were transfected with small interfering RNA (siRNA) for NKG2D (Santa Cruz Biotechnology, sc-42948) or DAP12 (sc-35172) by electroporation using the Amaxa Human NK Cell Nucleofector Kit and Nucleofector technology (Lonza), according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry

Journal: Nature immunology

Article Title: Sestrins induce natural killer function in senescent-like CD8 + T cells

doi: 10.1038/s41590-020-0643-3

Figure Lengend Snippet: a) Spleen weight following mBSA-driven DTH response in young wild-type (Y WT, n = 4 mice), old wild-type (O WT, n = 8 mice), old Sesn1−/− (O Sesn1−/−, n = 5 mice), and old Sesn2−/− (O Sesn2−/−, n = 4 mice). Bars represent means and s.d.. b) Representative gating strategy to identify NK1.1+ NK cells (violet), TCRβ+CD1d tetramer reactive iNKT cells (purple), TCRβ+CD3+ CD4+ (blue) and CD8+ (red) T cells in mice. Similar results were obtained in all mice (n = 3 per group). c) Quantification of these cell types in the spleen (means and s.e.m., n = 3 mice per group). d) Dot plots showing relative frequencies of CD44−CD62L+ naïve (grey), CD44+CD62L+ central (blue), and CD44+CD62L− effector (red) CD8+ T cells. e) Quantification of these cell types as a proportion of total splenic CD8+ T cells (means and s.e.m., n = 3 per group). f) Enumeration of NKG2D, NKG2A/C/E, KLRG1, Ly49, and NKp46 expression on CD8+ T cells from Y WT, O WT, O Sesn1−/−, and O Sesn2−/− mice (means and s.e.m., n = 3 per group). One-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Where indicated, freshly purified human CD8 + T cells were transfected with small interfering RNA (siRNA) for NKG2D (Santa Cruz Biotechnology, sc-42948) or DAP12 (sc-35172) by electroporation using the Amaxa Human NK Cell Nucleofector Kit and Nucleofector technology (Lonza), according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Nature immunology

Article Title: Sestrins induce natural killer function in senescent-like CD8 + T cells

doi: 10.1038/s41590-020-0643-3

Figure Lengend Snippet: a) Box-and-whisker plot of cell-surface CD107a expression of CD27+CD28+CD8+, CD27+CD28−CD8+, CD27−CD28−CD8+, and CD3−CD56+ NK cells isolated from healthy donors co-cultured with K562 cells at E:T 2:1 (median and range, n = 5). b) CD107a expression on CD28−CD8+ T cells transfected with NKG2D siRNA (siNKG2D) or control siRNA (siCtrl) and cultured with C1R-MICA or C1R (E:T ratio 2:1) for 6 hours (mean and s.e.m., n = 4). c) Immunoblots (representative of five independent experiments with similar results) and d) flow cytometry (means and s.e.m., n = 12) showing DAP12 expression on CD27+CD28+CD8+, CD27+CD28−CD8+, CD27−CD28−CD8+, and CD3−CD56+ NK cells. g) Expression of DAP12 in lysed CD28+CD8+ T cells and CD28−CD8+ T cells co-immunoprecipitated with antibody against NKG2D. Loading control, light-chain IgG (IgGL). Whole-cell lysate immunoblot shown as a control. Representative of two independent experiments. f) Phosphorylation of Zap70(Tyr319) and Syk(Tyr352) in freshly isolated CD27−CD28−CD8+ T cells after treated with CD3 mAb (OKT3, 10 μg/mL, 15 minutes) and/or NKG2D Ab (1D11, 5 μg/mL, 15 minutes). Numbers indicate relative expression normalized to total Zap70. Representative of 2 experiments. g) Granzyme B expression (left) and IFN-γ secretion (right) in CD27+CD28+CD8+, CD27+CD28−CD8+, and CD27−CD28−CD8+ T cells after stimulation with NKG2D Ab (5 μg/mL, means and s.e.m., n = 15 donors). h) CD107a expression in human CD28−CD8+ T cells transfected with DAP12 siRNA (siDAP12) or control siRNA (siCtrl) cultured with C1R-MICA*008 or C1R cells for 6 hours (E:T ratio 2:1; means and s.e.m., n = 4). Statistical significance determined with Kruskal-Wallis test in a) Friedman test with Dunńs correction in d), two-way ANOVA with Bonferroni correction in b, j) and one-way ANOVA with Tukey’s in g) (*p <0.05, **p <0.01, ***p <0.001).

Article Snippet: Where indicated, freshly purified human CD8 + T cells were transfected with small interfering RNA (siRNA) for NKG2D (Santa Cruz Biotechnology, sc-42948) or DAP12 (sc-35172) by electroporation using the Amaxa Human NK Cell Nucleofector Kit and Nucleofector technology (Lonza), according to the manufacturer’s instructions.

Techniques: Whisker Assay, Expressing, Isolation, Cell Culture, Transfection, Western Blot, Flow Cytometry, Immunoprecipitation

Journal: Nature immunology

Article Title: Sestrins induce natural killer function in senescent-like CD8 + T cells

doi: 10.1038/s41590-020-0643-3

Figure Lengend Snippet: a) Titration curve of varying effector to target (E:T) ratios on cytotoxicity measured as specific lysis of K562 cells using a calcein-release of CD27−CD28−CD8+ (DN) T cells and NK cells isolated by FACS. Non-linear regression (5-parameter asymmetric) was performed (means and s.d., n = 3 donors). b) Calcein-release cytotoxicity assay of K562 cells by CD27+CD28+ (DP), CD27+CD28− (SP), DN CD8+ T cells, and NK cells at E:T 20:1. Cytotoxicity was assessed over a period of six hours (means and s.d., n = 3 donors). c) Representative dot plot of MICA/B expression in C1R and C1R-MICA*008 cells. d) Representative histogram of NKG2D expression on CD28−CD8+ T cells after transfection with NKG2D siRNA (siNKG2D, black) or scrambled siRNA (siCtrl, grey), determined 36 hours after transfection. Numbers indicate MFI. e) Expression of DAP10 on human NK cells, and DP, SP, and DN CD8+ T cell subsets. Mean fluorescence intensity is shown (means and s.d., n = 4 for T cell, n = 3 NK cells). f) Sestrin 2 on CD8+ T cells from young (<35 years, n = 5) and old (>65 years, n = 4) donors. MFIs are shown (geometric means and geometric s.d. factor). Two-tailed, unpaired Welch’s t test, ** p < 0.01.

Article Snippet: Where indicated, freshly purified human CD8 + T cells were transfected with small interfering RNA (siRNA) for NKG2D (Santa Cruz Biotechnology, sc-42948) or DAP12 (sc-35172) by electroporation using the Amaxa Human NK Cell Nucleofector Kit and Nucleofector technology (Lonza), according to the manufacturer’s instructions.

Techniques: Expressing, Titration, Lysis, Isolation, Cytotoxicity Assay, Transfection, Fluorescence, Two Tailed Test

Journal: Nature immunology

Article Title: Sestrins induce natural killer function in senescent-like CD8 + T cells

doi: 10.1038/s41590-020-0643-3

Figure Lengend Snippet: a) Measurement of paw size (normalized to the contralateral, PBS control paw) over time (0h, 4h, 24h, 36h, 2, 3, 4, 5, 6, 7 days) in young wild-type (young WT; n = 4), old WT (n = 10), old Sesn1−/− (n = 5) and old Sesn2−/− (n = 4) mice following intra-plantar treatment with mBSA. b) Area under the curve integration of the time course data shown in a) (means and s.d.) c) Representative pseudo-colour density plots showing CD44 vs NKG2D expression on TCRβ+CD3+CD8+ T cells isolated from spleens of young WT, old WT, old Sesn1−/−, and old Sesn2−/− mice. Frequencies of parent gates are shown in the top right-hand corner. d) Cumulative data of NKG2D expression in splenic TCRβ+CD3+CD8+ T cells from young WT, old WT, old Sesn1−/−, and old Sesn2−/− mice (n = 3 mice per group). e) Representative histogram of DAP12 expression from splenic TCRβ+CD3+CD8+ T cells from young WT, old WT, old Sesn1−/−, and old Sesn2−/− mice. FMO control is shown. Cumulative data shown (n = 3 per group, n = 1 young WT). f) NKG2D expression in splenic NK- and iNKT cells from the same mice as in c-d) (means and s.d., n = 3 mice per group). g) Retrieval of Rae-1+ 5TGM1 cells as a fraction of injected cells (left panel) and killing and specific lysis of injected Rae-1+ 5TGM1 cells (right panel) from the spleens of NK-depleted (24h, anti-NK1.1, i.p.) old WT and old Sesn1−/−Sesn2−/−Sesn3+/− mice 6 hours after i.v. challenge (means and s.d., n = 3 mice per group). Statistical significance determined with one-way ANOVA with Tukey’s multiple comparisons test in b,d-g); two-tailed unpaired Student’s t tests in h-i). (*p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).

Article Snippet: Where indicated, freshly purified human CD8 + T cells were transfected with small interfering RNA (siRNA) for NKG2D (Santa Cruz Biotechnology, sc-42948) or DAP12 (sc-35172) by electroporation using the Amaxa Human NK Cell Nucleofector Kit and Nucleofector technology (Lonza), according to the manufacturer’s instructions.

Techniques: Expressing, Isolation, Injection, Lysis, Two Tailed Test

Journal: Nature immunology

Article Title: Sestrins induce natural killer function in senescent-like CD8 + T cells

doi: 10.1038/s41590-020-0643-3

Figure Lengend Snippet: a) Expression of DAP12, sestrin 2 and p-Jnk (T183/Y185) in lysed CD28+CD8+ T cells and CD28−CD8+ T cells immunoprecipitated with NKG2D. Loading control: IgG light chain (IgGL). Results are representative of two independent experiments. b) Cellular localization of Sesn2 (AF488, green), DAP12 (PE, red) and P-Jnk (T183/Y185, AF647, yellow) in CD27+CD28+CD8+ and CD27−CD28−CD8+ T cells. “Sesn2”, “DAP12”, and “p-Jnk” denote single stain controls in CD27−CD28−CD8+. Nuclei are stained with DAPI (blue). Scale bars – 7 μm. c) Overlap of Sesn2 and DAP12 or P-Jnk in CD27+CD28+CD8+ and CD27−CD28−CD8+ T cells. Bright detail similarity scores exceeding 2 were considered to be overlapping. Data are normalized to the CD27+CD28+CD8+ T cell subset for each donor (means and s.d., n = 6). d-e) Isolated human CD8+CD28− T cells were transduced with control (shCtrl) or anti-sestrin (shSesn) vectors. d) Representative immunoblot for Sesn2 and DAP12 (representative of two experiments). e) Representative contour plots and summary data of NKG2D expression. Results are presented relative to cells transduced with shCtrl for each donor, set as 1 (means and s.d., n = 3 donors). f) Frequency of NKG2D and CD28 in isolated CD28−CD8+ T treated with siJnk (means and s.d., n = 6 donors). g) Lck phosphorylation in CD28−CD8+ T cells pre-treated with Jnk inhibitor (SP-600125, 10 μM) prior to anti-CD3 stimulation (OKT3, 10 μg/ml, 15 minutes; means and s.d., n = 8 donors). Two-tailed paired Student’s t tests in c,f-g) (*p <0.05, **p <0.01, ***p <0.001).

Article Snippet: Where indicated, freshly purified human CD8 + T cells were transfected with small interfering RNA (siRNA) for NKG2D (Santa Cruz Biotechnology, sc-42948) or DAP12 (sc-35172) by electroporation using the Amaxa Human NK Cell Nucleofector Kit and Nucleofector technology (Lonza), according to the manufacturer’s instructions.

Techniques: Expressing, Immunoprecipitation, Staining, Isolation, Transduction, Western Blot, Two Tailed Test