Journal: Nature

Article Title: An engineered IL-2 partial agonist promotes CD8 + T cell stemness

doi: 10.1038/s41586-021-03861-0

Figure Lengend Snippet: a, Preactivated CD8+ T cells were rested overnight and cultured with PBS, IL-2, H9, or H9T as indicated for 10 min, and western blotted for phospho-STAT5 (pSTAT5) and pERK. Total STAT5 and ERK were included as controls (on the same gel of pSTAT5 and pERK). Data are representative of two independent experiments. Relative densitometry is shown below each panel (normalized to the IL-2 condition). For gel source data, see Supplementary Fig. 1b b, Preactivated CD8+ T cells were rested overnight and stimulated with 10 nM IL-2 or H9T, lysed, and immunoprecipitated with anti-STAT5A or anti-STAT5B antibodies followed with western blotting analysis for pSTAT5 and STAT5A or STAT5B as loading controls (on the same gel). Data are representative of two independent experiments. Relative densitometry is shown below each panel (normalized to the IL-2 condition). For gel source data, see Supplementary Fig. 1c. Data are from two independent repeats. c, Preactivated CD8+ T cells were rested overnight, cultured with PBS, IL-2, H9, or H9T for 6 days, fixed, permeabilized and intracellularly stained for pSTAT5, n= 4 mice. Data are presented as mean values +/− SEM, one-way ANOVA test with Dunnett’s correction. Data are from two independent repeats. d, Pre-activated mouse CD8+ T cells were rested overnight and then cultured in medium containing serial dilutions of IL-2, H9, or H9T for 8 days and analyzed for TIM-3 expression. Data are representative of three independent experiments. e-h, Pre-activated CD8+ T cells were cultured with 10 nM IL-2, H9, or H9T for 8 days, and surface expression of TIM-3 (e), PD-1 (f), LAG-3 (g), and 2B4 (h) was analyzed by flow cytometry. Data are representative of four independent experiments.

Article Snippet: For western blotting analysis, anti-Granzyme B (#674301) was from BioLegend; anti-Perforin (#3693S, 1:1000), anti-H3 (#14269, 1:1000), anti-STAT5-pY694 (#9351, 1:1000), anti-STAT5-pY694 (#9356S, 1:1000), and anti-ERK1/2-pT202-pY204 (D13.14.4E, 1:1000) were from Cell Signaling, anti-STAT5 (#610192) was from BD, and anti-ERK1/2 (MAB15761, 1:1000) was from R & D Systems.

Techniques: Cell Culture, Western Blot, Immunoprecipitation, Staining, Expressing, Flow Cytometry

Journal: Nature

Article Title: An engineered IL-2 partial agonist promotes CD8 + T cell stemness

doi: 10.1038/s41586-021-03861-0

Figure Lengend Snippet: a, b Pre-activated mouse (a) or human (b) CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 6 days, and cell density was counted by beads-based flow cytometry. Data are mean +/− SEM, one-way ANOVA test with Dunnett’s correction. a, n= 6 mice; b, n = 6 donors. Data are representative of two independent experiments. c, Pre-activated mouse CD8+ cells were rested and cultured with 10 nM of the indicated cytokines for 6 days, and TIM-3 expression was examined by flow cytometry. Data are mean +/− SEM, one-way ANOVA test with Dunnett’s correction, n= 4 mice. Data are representative of two independent experiments. d-f, Preactivated mouse CD8+ T cells were rested overnight and cultured with 10 nM of the indicated cytokines for 0, 0.5, 1, 2, or 4 hours. Cells were then fixed, permeabilized, and stained for STAT5-pY694 (d), AKT-pS473 (e) and ERK-pT202/pY204 (f). Data are mean +/− SEM, n= 6 mice, Data are representative of two independent experiments. g-h, Pre-activated mouse CD8+ T cells were rested and cultured with 10 nM of the indicated cytokines for 1 day and collected for RNA-seq. PCA analysis (g) and selected gene expression (h) are shown. Data are combined from two biological repeats.

Article Snippet: For western blotting analysis, anti-Granzyme B (#674301) was from BioLegend; anti-Perforin (#3693S, 1:1000), anti-H3 (#14269, 1:1000), anti-STAT5-pY694 (#9351, 1:1000), anti-STAT5-pY694 (#9356S, 1:1000), and anti-ERK1/2-pT202-pY204 (D13.14.4E, 1:1000) were from Cell Signaling, anti-STAT5 (#610192) was from BD, and anti-ERK1/2 (MAB15761, 1:1000) was from R & D Systems.

Techniques: Comparison, Cell Culture, Flow Cytometry, Expressing, Staining, RNA Sequencing Assay

Journal: Nature

Article Title: An engineered IL-2 partial agonist promotes CD8 + T cell stemness

doi: 10.1038/s41586-021-03861-0

Figure Lengend Snippet: a, ChIP-seq analysis of STAT5A and STAT5B binding sites at the Havcr2 locus from previously published datasets (GSE36890). b-c, TIM-3 expression on CD8+ T cells from Stat5a (b) and Stat5b (c) knock-out mice versus wild-type littermate controls. Cells were expanded for 8 days with 10 nM of IL-2 as described above, and TIM-3 expression analyzed by flow cytometry. Data are mean +/− SEM, n= 4 mice, two-sided t-test. Data are from two independent repeats. d, ChIP-seq analysis of STAT5 binding sites at HAVCR, GZMB, TCF7, SLC2A1, and SLC2A3 loci. Human CD8+ T cells were preactivated with anti-CD3/anti-CD28 beads for 2 days, rested overnight, incubated with 10 nM IL-2 or H9T for 2 hours, and then fixed and lysed for ChIP-Seq. Data are from two independent experiments. e-h, Mouse CD8+ T cells expressing empty vector (EV)-GFP or STAT5A-1*6 vector-GFP were sorted and expanded in H9T-containing medium for an additional four days prior to staining with the indicated antibodies. Data are representative of two independent experiments. i-m, RNA-seq analysis of the effects of STAT5A-1*6 expression. Mouse CD8+ T cells were treated as above, and cells were collected for RNA-seq. PCA analysis (i) and expression of selected genes (j) are shown. GSEA analysis of IL-2-STAT5 signaling (k), PI3K-AKT-MTOR signaling (l) and exhaustion versus memory (m) are also shown, Kolmogorov-Smirnov test. Data are from two biological repeats. n-p, ATAC-seq analysis of the effect of STAT5A-1*6 expression. Mouse CD8+ T cells were treated as described above, and cells were collected for ATAC-seq. Shown are PCA analysis to compare IL-2-, H9-, and H9T-expanded cells (n), and ATAC-seq data at the Havcr2 (o) and Tcf7 (p) loci.

Article Snippet: For western blotting analysis, anti-Granzyme B (#674301) was from BioLegend; anti-Perforin (#3693S, 1:1000), anti-H3 (#14269, 1:1000), anti-STAT5-pY694 (#9351, 1:1000), anti-STAT5-pY694 (#9356S, 1:1000), and anti-ERK1/2-pT202-pY204 (D13.14.4E, 1:1000) were from Cell Signaling, anti-STAT5 (#610192) was from BD, and anti-ERK1/2 (MAB15761, 1:1000) was from R & D Systems.

Techniques: Activation Assay, ChIP-sequencing, Binding Assay, Expressing, Knock-Out, Flow Cytometry, Incubation, Plasmid Preparation, Staining, RNA Sequencing Assay