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, IL-2/IL-2R diagram showing the mutations to generate H9T. b, Binding of H9-IL-2Rβ and H9T-IL-2Rβ complexes to IL-2Rγ, analyzed by surface plasmon resonance (see Methods). c, d, Proliferation of preactivated CD8+ T cells expanded with 10 nM IL-2, H9, or H9T, n= 8 mice (c) or with a range of concentrations of these cytokines for 8 days, n= 3 mice (d). e-f, CD8+ T cells were treated as in panel c to assess TIM-3 expression (e, n= 4 mice), or treated as in panel d (f, n= 3 mice). g-j, Expression of TIM-3 (g), PD-1 (h), LAG-3 (i), and 2B4 (j) on CD8+ T cells treated with 10 nM of each cytokine for 8 days. One-way ANOVA test with Dunnett’s correction. g, h, j, n= 3 mice; i, n= 6 mice. k-n, pmel-1 CD8+ T cells were expanded as in panel g and stimulated with 100 nM gp100 or control peptide for 1 hour followed with for 5 μg/ml Brefeldin A for 6 h intracellular staining of IFN-γ (k), TNF-α (l), IL-2 (m), and IL-10 (n). o, Cells were treated as in panel m but stained for TIM-3 and IL-2. p, Pre-activated CD8+ T cells were expanded as in panel g, and analyzed by western blotting (on the same gel). Uncropped gel in Supplementary Fig. 1a. Data are mean +/− SEM (c-j). Data are from two (c, d, k-n, p), three (e-j), or four (o) independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Binding Assay, SPR Assay, Expressing, Staining, Western Blot

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

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Cell Culture, Western Blot

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-d, RNA-seq analysis of IL-2-, H9-, or H9T-expanded CD8+ T cells. PCA analysis (a), differentially expressed genes (b), heat map of selected genes (c), and GSEA (d) with Kolmogorov-Smirnov test are shown. e-g, Level of TCF-1, CXCR3, and BLIMP1 protein on IL-2-, H9-, or H9T-expanded CD8+ T cells, n= 3 mice. Data are mean +/− SEM, one-way ANOVA test with Dunnett’s correction. h-j, PCA of ATAC-seq (h); ATAC-seq at the Tcf7 (i) or Havcr2 (j) loci are shown. Data are from two (a-d, h-j) or three (e-g) independent repeats.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: 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, Volcano plots of RNA-Seq data from preactivated CD8+ T cells that were expanded for 6 days with IL-2, H9, or H9T. Shown are gene expression differences between cells expanded with IL-2 versus H9T (a) or H9 versus H9T (b). Data are representative of two independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: RNA Sequencing Assay, Expressing

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, Heat map (a) and PCA (b) analysis of metabolomic data prepared with 8 day expanded CD8+ T cells (see Methods). c, Seahorse-based ECAR analysis, n= 7 mice, one-way ANOVA test with Dunnett’s correction. Shown is mean +/− SEM. d, 2-NBDG uptake assay prepared with 8 day expanded CD8+ T cells. e-f, qPCR analysis of Slc2a1 (e) and Slc2a3 (f) expression. g, TMRM assay showing mitochondrial membrane potential. h, Seahorse-based mitochondrial stress test , n= 3 mice, data are mean +/− SEM. i, Spare respiratory capacity, n= 7 mice. One-way ANOVA test with Dunnett’s correction, data are mean +/− SEM. j-l, Level of TCF-1, CD62L and pSTAT5 after 2 day 2-DG treatment. m-n, RNA-seq analysis (3 biological replicates) of 2-DG treated CD8+ T cells (m) and GSEA of PBS versus 2-DG treated cells (n), Kolmogorov-Smirnov test. Data are from two (a-g, j-l) or four (h-i) independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Expressing, 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, 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.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Cell Culture, 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, Pre-activated human CD8+ cells were rested and cultured with 10 nM indicated cytokines for 6 days, and TIM-3 expression was examined. Data are mean +/− SEM, one-way ANOVA test with Dunnett’s correction, n= 6 donors. Data are from two independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Cell Culture, Expressing

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, Expression of CD62L and the percentage of memory-like cells (CD62L+CD44+). Pre-activated CD8+ T cells were cultured with 10 nM IL-2, H9, or H9T for 8 days, and stained for CD44 and CD62L. Data are mean values +/− SEM, n= 6 mice, one-way ANOVA test with Dunnett’s correction. Data are representative of three independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Functional Assay, Expressing, Cell Culture, Staining

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, Photograph showing media color of CD8+ T cells expanded for 8 days with IL-2, H9, or H9T. Data are representative of two independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques:

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-c, Dose response of IL-2, IL-15, and H9T in human CD8+ T cells. Pre-activated human CD8+ cells were rested and cultured with 0-100 nM of IL-2, IL-15, or H9T for 6 days and stained for surface expression of TIM-3 (a) or permeabilized and stained for intracellular granzyme B (b). Cell expansion rate (c) was assessed using flow cytometry based counting beads. Data are mean +/− SEM, n= 2 donors. Data are representative of two independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Cell Culture, 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-c, Splenocytes from mice receiving cytokine-expanded CD8+ T cells were analyzed for persistence of donor cells after transfer. n= 5 mice, Shown is mean +/− SEM, one-way ANOVA test with Dunnett’s correction. d-f, CD45.1+ or CD90.1+ pmel-1 CD8+ T cells were expanded with IL-2 or H9T separately for 8 days, mixed at a 1:1 ratio, transferred into B16KVP tumor bearing mice, and infiltration efficacy assessed. n = 4 mice, shown is mean +/− SEM with two-sided t-test. g-j, Expanded pmel-1 CD8+ T cells were transferred to treat B16KVP tumor-bearing mice (g). Representative measured tumor area (h) is shown, n= 5 mice with two-sided t-test (day 27). Mouse survival analyzed with Log-rank test (i) and percentage of cured mice from 3 independent repeats were assessed (j); shown are mean +/− SEM with two-sided t-test. k, Wild-type mice (1st challenge, n= 10 mice) or cured mice (as in j) (2nd challenge, n= 7 mice) were inoculated with B16KVP melanoma tumor. Tumor size was measured one week later, shown are mean +/− SEM with two-sided t-test. l, Phenotype of pmel-1 cells 7 days after transfer. m-n, CD8+ T cells with or without chimeric anti-CD19 receptor transduction were expanded with IL-2, H9 or H9T for 8 days and transferred into E2a-PBX leukemia-bearing mice for survival analysis. n= 5 mice with Log-rank test. Data are from two (a-f, k, m-n), three (g-j, l) independent experiments.

Article Snippet: Primary cell isolation, activation, culture, and retroviral transduction Mouse CD8 + T lymphocytes were isolated using the EasySep Mouse CD8 + T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 μM β-mercaptoethanol.

Techniques: Transduction