Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) Schematic rendition of OMCPmutIL-2 binding to cell surface and variability in common γ chain cytokine surface receptor capture, relying on high-affinity α chain in cis for IL-2 and in trans for IL-15. ( B ) Experimental design and expansion of peripheral blood lymphocyte–derived (PBL-derived) naive CD8 + CD44 lo 62L hi murine T cells as measured by fold expansion and viability. Apoptotic cells defined as Annexin viability dye – and nonviable cells defined as Annexin viability dye + . ( C ) Expression of exhaustion markers on cells expanded in respective cytokines. * P < 0.05; ** P < 0.01; *** P < 0.001; t test. CTLA-4, cytotoxic T lymphocyte–associated protein 4; Lag-3, lymphocyte activating 3; PD-1, programmed cell death 1.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Binding Assay, Derivative Assay, Expressing

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) Heatmap showing relative gene expression of memory-driving transcription factors. ( B ) Relative proportion and number of CD44 lo 62L hi naive, CD44 hi 62L hi central memory (Tcm), or CD44 hi 62L lo effector cells (Teff) after 2 weeks of expansion in IL-2 (blue), IL-15 (green), or OMCPmutIL-2 (red). Representative FACS plots (top), quantitative percentage (bottom left), and total cell count (bottom right) from a starting population of 1 × 10 6 flow cytometrically sorted naive cells. ( C ) Analysis of spleen 50 days after adoptive transfer of CD45.2 congenic CD8 + T cells expanded in wild-type IL-2 (blue), IL-15 (green), or OMCPmutIL-2 (red) prior to transfer. ( D ) In vitro cytotoxicity by murine pmel anti-GP100 TCR-transgenic CD8 + T cells as determined by the ability to lyse B16 melanoma (nonviable) and T cell degranulation measured by surface CD107a expression with 1:1 B16/pmel CD8 + T cell ratio. ( E ) Effector cytokine (IFN-γ and TNF-α) production by murine pmel anti-GP100 TCR-transgenic CD8 + T cells expanded in respective cytokines, upon maximal stimulation. ** P < 0.01; *** P < 0.001; t test.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Expressing, Cell Counting, Adoptive Transfer Assay, In Vitro, Transgenic Assay

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) Canonical signaling pathways described for common γ chain cytokines. ( B ) JAK1 and JAK3 total and phosphorylated levels by Western blot analysis after in vitro activation with anti-CD3/28 stimulation and in the presence of IL-2, IL-15, OMCPmutIL-2, or control (saline). ( C ) Phospho-STAT5 levels in CD8 + T cells activated with IL-2, IL-15, OMCPmutIL-2, or saline control in the presence of anti-CD3/28. * P < 0.05; ** P < 0.01; *** P < 0.001; t test.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Western Blot, In Vitro, Activation Assay

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) STAT5 localization by confocal microscopy after in vitro activation with anti-CD3/28 stimulation in the presence of IL-2, IL-15, OMCPmutIL-2, or saline control. Original magnification, 20× (top) and 63× (bottom). ( B ) Gene enrichment analysis for STAT5-dependent genes in memory T cells generated from naive cells in vitro in the presence of IL-2 or IL-15 versus OMCPmutIL-2. ( C ) Principal component analysis between IL-2 (blue), IL-15 (green), and OMCPmutIL-2 (red) and differential gene expression in murine splenic CD8 + T cells expanded in IL-2, IL-15, or OMCPmutIL-2 as expressed via Venn diagram and Kyoto Encyclopedia of Genes and Genomes pathway analysis comparing top differential pathways. OMCPmutIL-2 versus IL-2 and OMCPmutIL-2 versus IL-15 are presented in separate graphs (purple for OMCPmutIL-2 vs. IL-2 and orange for OMCPmutIL-2 vs. IL-15). ( D ) Phosphorylated (inactive) NFAT quantification in CD8 + T cells after expansion in various cytokines. ( E ) Change in CD8 + T cell expansion and cytotoxicity in the presence or absence of NFAT inhibitor FK506. ( F ) Change in CD8 + T cell proliferation and cytokine production in the presence or absence of NFAT inhibitor FK506. * P < 0.05; ** P < 0.01; *** P < 0.001; t test.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Confocal Microscopy, In Vitro, Activation Assay, Generated, Expressing

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) Canonical pathway of NFAT activation through TCR signal transduction. ( B ) TCR signaling, as measured by Nur77 upregulation (representative FACS plot, left) in the presence of cytokines and variable concentrations of TCR agonistic antibody (anti-CD3) as well as 2 μg/mL of anti-CD28 (MFI, quantification from 5 individual Nur77GFP mice, right). ( C ) Relative gene expression of the TCR signaling pathway as defined by heatmap analysis in splenic murine CD8 + T cells cultured in the presence of anti-CD3/28 and various cytokines. ( D ) Phosphorylation of TCR signaling components of murine CD8 + T cells in the presence of wild-type IL-2 (blue), IL-15 (green), or OMCPmutIL-2 (red) or no cytokine (black) with anti-CD3/CD28 cross-linking antibodies. ( E ) Phosphorylation levels of CD3ζ and STAT-5 levels as measured by MFI in murine CD8 + T cells in the presence or absence of JAK1/3 inhibitor as compared with CD8 + T cells derived from Jak3 –/– mice. ( F ) Graphical representation of OMCPmutIL-2–mediated activation of NFAT activation at the expense of the canonical STAT5/AKT signaling pathways. ( G ) TCR signal transduction, as measured by Nur77 expression, in OT-1 clonotypic CD8 + T cells incubated with dendritic cells loaded with various SIINFEKL mutants with variable TCR binding avidity. * P < 0.05; ** P < 0.01; *** P < 0.001; t test.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Activation Assay, Transduction, Expressing, Cell Culture, Derivative Assay, Incubation, Binding Assay

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) Time course analysis (0–60 minutes) of phosphorylation of CD3ζ and STAT5 in murine CD8 + T cells stimulated with OMCPmutIL-2 (red), wild-type IL-2 (blue), and IL-15 (green). ( B ) (Left) High-resolution confocal microscopy of human CD8 + T cell surface receptors IL-2Rβ (CD122) (green), CD3ζ (red), and NKG2D (pink) after 1 hour in the presence of plate-bound anti-CD3/28 with different cytokines. Visible colocalization of receptors is shown by arrows (yellow). Original magnification, ×63. (Right) Percentage of T cells demonstrating visible clustering of CD3ζ and IL-2Rβ per high-power field. n = 13. * P < 0.05; ** P < 0.01; *** P < 0.001; t test.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Confocal Microscopy

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) High-resolution confocal microscopy of human CD8 + T cells evaluating receptor localization/clustering of CD3ζ (red) and IL-2Rβ/CD122 (pink) with lipid rafts (stained by Cholera Toxin B, CTB, in green). Clustering of receptors is shown by yellow arrow. ( B ) High-resolution confocal microscopy of human CD8 + T cell surface receptors (left panel) IL-2Rβ (CD122) (green), CD3ζ (red), and lipid rafts (stained by CTB in green) after lipid raft disruption by Methyl-β-cyclodextrin (MBCD) demonstrates no visible colocalization of receptors. Percentage clustering (right panel) shows little clustering and no difference between groups after MBCD treatment. Original magnification, ×63. ( C ) Flow cytometric analysis of CD3ζ and STAT5 phosphorylation after 1 hour of stimulation in various cytokines in the presence or absence of MBCD. ( D ) Phospho-CD3ζ and phospho-STAT5 expression in human PBL-derived CD8 + T cells after culture with OMCPmutIL-2 versus IL-2. ( E ) Phospho-CD3ζ and phospho-STAT5 expression in human PBL-derived CD8 + T cells after culture with mutIL-2–redirected constructs with different NKG2D ligands having different binding affinity. ** P < 0.01; t test ( B – D ) or ANOVA ( E ).

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Confocal Microscopy, Staining, Expressing, Derivative Assay, Construct, Binding Assay

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) Quantification of mitochondria using high-resolution confocal microscopy using MitoTracker Deep Red dye (upper 2 panels) and electron microscopy (lower panel) depicting increased number of fused mitochondria in OMCPmutIL-2–treated CD8 + T cells. Original magnification, ×63 (middle panel) and ×1000 (lower panel). ( B ) Gene expression analysis showing increased NRF1 transcription factors in OMCPmutIL-2–expanded CD8 + T cells (upper panel). Flow cytometric analysis of mitochondrial membrane potential (middle) and oxygen consumption rate by Seahorse assay (lower) in murine CD8 + T cells expanded in IL-2 (blue), IL-15 (green), or OMCPmutIL-2 (red). ( C ) Analysis of differential expression of genes involved in oxidative phosphorylation as expressed by enrichment scores and heatmaps. ( D ) Flow cytometric quantification of PGC-1α and mitochondrial mass and Tcm after inhibition of PGC-1α. ( E ) PGC-1α levels, mitochondrial mass, and relative proportion of Tcm in the presence of constitutively active AKT (myrAKT mice) or pharmacologic AKT inhibition. ( F ) PD-1 expression as measured by MFI of murine CD8 + T cells in the presence of constitutively active AKT (myrAKT) or AKT inhibition. * P < 0.05; ** P < 0.01; *** P < 0.001; t test.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: Confocal Microscopy, Electron Microscopy, Expressing, Inhibition

Journal: JCI Insight

Article Title: A reengineered common chain cytokine augments CD8 + T cell–dependent immunotherapy

doi: 10.1172/jci.insight.158889

Figure Lengend Snippet: ( A ) In vitro cytotoxicity of anti-CD19 CAR T cells, expanded in various cytokines, against NALM6 CD19-expressing tumors. ( B ) Experimental design, tumor growth, and survival of B16 melanoma–bearing mice treated with adoptive transfer of pmel anti-GP100 TCR-transgenic CD8 + T cells expanded in the presence of IL-2 (blue), IL-15 (green), or OMCPmutIL-2 (red). ( C ) Experimental design, tumor growth, and survival of mice bearing CT26 colon cancer tumors. ( D ) Tumor growth and survival percentage of mice bearing tumor antigen ovalbumin expressing Lewis lung carcinoma (LLC ova ) in combinatorial immunotherapy with cytokines and checkpoint inhibitors CTLA-4 and PD-1. ( E ) Tumor growth in surviving mice after rechallenge with LLC ova . * P < 0.05; ** P < 0.01; *** P < 0.001; t test. s.q., subcutaneous.

Article Snippet: Both wild-type human IL-2 and OMCPmutIL-2 were produced through transient transfection in CHO cell line (Celltheon) based on previously described methods ( ).

Techniques: In Vitro, Expressing, Adoptive Transfer Assay, Transgenic Assay