Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: Computationally designed IL-21 mimic recapitulates receptor interactions with superior stability and human-mouse cross-reactivity. (A) The optimized IL-21 mimic, 21h10, is designed by recapitulating the helical bundle structure of the native hIL-21. Unstructured regions in the hIL-21 are removed, and short helices are extended to accommodate improved intramolecular packing. Some of the interface residues are conserved from the native hIL-21 for the design of initial hits. Other residues from the de novo scaffolds are further optimized, as shown. (B) Association and dissociation of 21h10 to human and murine IL-21R and 𝛾 c show concentration-dependent binding curves of 21h10. 21h10’s K D against hIL-21R is 482 pM, hIL-21R/h𝛾 c is 86.7 nM, mIL-21R is 7.87 nM, and mIL-21R/m𝛾 c is 72.8 nM. For measuring K D against hIL-21R and mIL-21R, IL-21R was immobilized on the biolayer interferometry (BLI) tip. For measuring K D against hIL-21R/h𝛾 c and mIL-21R/m𝛾 c , 𝛾 c was immobilized on the BLI tip and IL-21R was provided in solution with 1.5-fold molar excess to 21h10. Dotted lines show curve fits of raw data using a 1:1 Langmuir binding model. (C) The optimized IL-21 mimic, 21h10, exhibits monodispersed distribution when sized through size-exclusion chromatography using Superdex 75 10/300 GL column. (D) Wavelength scan from 200 nm to 250 nm confirmed 𝛼-helical secondary structure present in 21h10. Thermal melt from 25°C to 95°C with 222 nm scan revealed outstanding thermal stability of 21h10, with a T m around 75°C. (E) Crystal structure of 21h10 in complex with hIL-21R and h𝛾 c (PDB: XXXX). (F) 21h10 conserves key molecular interactions in the site I interface (left) and site IIa interface (right) observed in the structure of the human IL-21 receptor complex. (Human IL-21 receptor complex, PDB ID: 8ENT).

Article Snippet: To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 2-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Concentration Assay, Binding Assay, Size-exclusion Chromatography

Journal: bioRxiv

Article Title: Potent antitumor activity of a designed interleukin-21 mimic

doi: 10.1101/2024.12.06.626481

Figure Lengend Snippet: ( A ) Pre-activated human and murine CD8 + T cells were treated with increasing doses of native IL-21 and 21h10 for 20 minutes and stained with AF488-phospho-STAT1 and AF647-phospho-STAT3 for flow cytometry analysis. ( B ) Genes related to cellular signaling and phenotype are compared for expression levels between murine CD8 + T cells treated with PBS, mIL-21, or 21h10 at 100 pM or 1 nM. * indicates memory-related genes, and ** indicates effector-related genes. Murine CD8 + T cells were pre-activated with TCR signals and treated with the cytokines at different concentrations for 24 hours, and cells were harvested for RNA-sequencing. ( C ) The percentage of IFN-𝛾-secreting cells within isolated CD8 + T cells upon treating PBS, mIL-21, or 21h10 at 1 nM, was quantified using flow cytometry after 5 hours of stimulation with PMA/Ionomycin in the presence of a protein transport inhibitor. ( D ) LCMV was inoculated into healthy mice along with daily injections of PBS, mIL-21 (2.5 µg per mouse), or 21h10 (2.5 µg per mouse). LCMV-specific CD8 + T cells from PBMC and spleen were analyzed for granzyme B expression. ( E ) Computational model of 21AT36, which is redesigned from 21h10 using ProteinMPNN, to bind IL-21R but not bind 𝛾 c . The mutated residues are highlighted in red, which abolish interactions with 𝛾 c . The 𝛾 c is included in the figure to show where the interface is positioned, not to imply that 21AT36 binds to 𝛾 c . ( F ) Western blot for STAT and pSTAT with vehicle (PBS), mIL-21, 21h10, and 21AT36 in TRP1 high CD8 + T cells. ( G ) MC38-bearing mice were treated with PBS, 21AT36, 21h10, or anti-PD-1 for 14 days. ( H ) Mice were inoculated with MC38 and treated with PBS, mIL-21, 10-fold dose mIL-21, and 21h10. ( I ) Western blot for STAT and pSTAT in the spleens of mice treated with vehicle (PBS), mIL-21, and 21h10 ( J ) Normalized pSTAT3/STAT3 in spleens of treated mice over 24 hours from ( I ). ( K ) MC38 rechallenges with MC38-survivor mice from previous 21h10 or anti-PD-1 treatment.

Article Snippet: To measure the binding affinity to hIL-21R or mIL-21R, biotinylated hIL-21R (R&D Systems Cat# AVI9249) or biotinylated mIL-21R (Acro Biosystems Cat# ILR-M82E3), respectively, were immobilized on streptavidin-coated biosensors (SAForteBio) at 5 μg/mL in the octet binding buffer, with 2-fold serial dilutions of the ligand starting at 100 nM, with 300 seconds for association and another 300 seconds for dissociation.

Techniques: Staining, Flow Cytometry, Expressing, RNA Sequencing Assay, Isolation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Elevated IL8 transcript levels in the course of NAFLD progression. ( A ) mRNA expression levels of the IL8 gene in the livers of patients with fatty liver ( n = 47) and NASH ( n = 51) from a publicly available RNA sequencing database (GSE167523). Statistical significance was assessed using Student’s t -tests (** p < 0.01). ( B ) GSEA plots showing the enrichment of genes associated with neutrophil chemotaxis and neutrophil activation in gene rank based on differential expression between NASH vs. NAFL livers. ( C ) mRNA expression levels of the IL8 gene in the liver among different stages of NAFLD obtained from a publicly available RNA sequencing database (GSE135251). F0–F1, F2, F3, and F4 represent different NASH groups classified by the severity of fibrosis. TPM refers to transcripts per million base pairs. For comparisons involving more than three groups, a one-way analysis of variance (ANOVA) was conducted, followed by post-hoc Tukey’s tests to determine specific group differences. (* p < 0.05, ** p < 0.01). IL8, interleukin 8; GSEA, gene set enrichment analysis; NAS, NAFLD activity score; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Expressing, RNA Sequencing, Chemotaxis Assay, Activation Assay, Quantitative Proteomics, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: IL8 overexpression induces increased hepatic neutrophil infiltration in the livers of HFD-fed mice. C57BL/6 mice fed an HFD for 3 months were infected with an adenovirus overexpressing the human IL8 gene (Ad- IL8 ) or an adenovirus expressing green fluorescence protein (Ad- GFP ) via the tail vein and sacrificed for analysis 2 weeks post-infection. ( A ) Schematic illustration of the experimental design. ( B ) Serum IL8 levels measured using ELISA analysis. ( C ) Hepatic IL8 transcript levels were calculated using the ΔCt method (Ct IL8 – Ct Gapdh ). ( D ) Paraffin-embedded liver sections were subjected to immunohistochemical analysis of MPO and LY6G ( n = 5/group). Red arrows indicate MPO- or LY6G-positive cells. Scale bars indicate 200 μm. The number of MPO- or LY6G-positive cells per 100× field was counted and illustrated in the graph on the right-hand side. Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (** p < 0.01). ELISA, enzyme-linked immunosorbent assay; HFD, high-fat diet; IL8, interleukin 8; MPO, myeloperoxidase.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, Infection, Expressing, Fluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: IL8 overexpression enhances liver injury and oxidative stress in the livers of HFD-fed mice. ( A ) Serum ALT and AST levels ( n = 5/group). ( B ) Body weight and liver weight ( n = 5/group). ( C , D ) Paraffin-embedded liver sections were subjected to the TUNEL assay (panel C ) and immunohistochemical analyses of MDA and 4-HNE (panel D ) ( n = 5/group). Representative images of each staining are presented ( left ). Scale bars indicate 200 μm. Quantification of the area positive for each staining is illustrated in graphs ( right ). ( E ) Mouse liver homogenates were subjected to RT-qPCR analysis of the genes involved in NOX2 complex formation and neutrophil oxidative burst. ( n = 5/group). ( F ) Immunoblot analysis of mouse liver homogenates. Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (** p < 0.01). 4-HNE, 4-hydroxynonenal; ALT, alanine transaminase; AST, aspartate transaminase; HFD, high-fat diet; IL8, interleukin 8; MDA, malondialdehyde; NOX2, NADPH oxidase 2; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, TUNEL Assay, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: IL8 overexpression enhances hepatic inflammation in the livers of HFD-fed mice. ( A ) Paraffin-embedded liver sections were subjected to immunohistochemical analysis of F4/80 ( n = 5/group). Representative images of F4/80 staining are presented ( left ). Scale bars indicate 200 μm. Quantification of the area positive for F4/80 staining is illustrated in graphs ( right ). ( B , C ) Mouse liver homogenates were subjected to RT-qPCR analysis of proinflammatory genes (panel B ) and chemokine genes (panel C ) ( n = 5/group). Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (* p < 0.05, ** p < 0.01). HFD, high-fat diet; IL8, interleukin 8; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, Immunohistochemical staining, Staining, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Fibrogenic effects of IL8 overexpression in the livers of HFD-fed mice. ( A ) Paraffin-embedded liver sections were subjected to Sirius Red staining and immunohistochemical analysis of α-SMA ( n = 5/group). Representative images of each staining are presented ( left ). Scale bars indicate 200 μm. Quantification of the area positive for each staining is illustrated in graphs ( right ). ( B ) Mouse liver homogenates were subjected to RT-qPCR analysis of fibrogenic genes ( n = 5/group). Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using Student’s t -tests (* p < 0.05, ** p < 0.01). α-SMA, alpha-smooth muscle actin; HFD, high-fat diet; IL8, interleukin 8; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Over Expression, Staining, Immunohistochemical staining, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Effects of recombinant human IL8 protein on stress kinase activation and fibrogenic gene induction in vitro. ( A ) Schematic illustration of neutrophil chemotaxis assay. Mouse bone marrow (BM)-derived neutrophils were placed on the Transwell insert (pore size: 3 µm), and mouse Cxcl1 or human IL8 recombinant protein (100 ng/mL) was added in the bottom chamber. The number of neutrophils that migrated to the lower chamber over 6 h was counted and illustrated graphically (right). ( B ) AML12 mouse hepatocytes were treated with human IL8 recombinant protein (100 ng/mL) for 24 h and subjected to cell viability measurements using the CCK-8 reagent. ( C ) AML12 mouse hepatocytes were treated with human IL8 recombinant protein (100 ng/mL) for 15 min or 30 min and lysed in RIPA buffer for immunoblot analyses of stress kinases. ( D ) LX-2 human hepatic stellate cells were treated with human IL8 recombinant protein (100 ng/mL) for 24 h, and the total RNA was isolated for RT-qPCR analysis of fibrosis-related genes. Values represent the mean ± standard error of the mean (SEM). Statistical evaluation was performed using a one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons or Student’s t -tests (** p < 0.01). HFD, high-fat diet; IL8, interleukin 8; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques: Recombinant, Activation Assay, In Vitro, Chemotaxis Assay, Derivative Assay, Pore Size, CCK-8 Assay, Western Blot, Isolation, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice

doi: 10.3390/ijms242015489

Figure Lengend Snippet: Primer sequences for quantitative polymerase chain reactions.

Article Snippet: Upon reaching 80% confluency, the cells were treated with human IL8 recombinant protein (R&D Systems, Minneapolis, MN, USA) for the indicated time to perform cell viability measurements, immunoblot analysis, and RT-qPCR.

Techniques:

Journal: eLife

Article Title: Kinetics of cytokine receptor trafficking determine signaling and functional selectivity

doi: 10.7554/eLife.49314

Figure Lengend Snippet: ( a ) Human CD4 T cells were isolated from whole PBMCs and treated with Treg polarizing conditions in the presence of saturating concentrations of the different IL-6 variants for five days. Percentage of Treg cells were calculated by counting number of events in the CD4+CD25+FoxP3+ population obtained by flow cytometry. The control condition was defined as 100% response and the other conditions normalized accordingly. Data are mean + /- SEM from five independent replicates. ( b ) Human CD4 T cells were isolated from whole PBMCs and treated with Th17 polarizing conditions in the presence of saturating concentrations of the different IL-6 variants for fourteen days. Percentage of Th17 cells were calculated by counting number of events in the CD4+IL-17A+ population obtained by flow cytometry. The control condition was defined as 100% response and the other conditions normalized accordingly. Data are mean + /- SEM from five independent replicates. ( c ) Human CD4 T cells were isolated from whole PBMCs and treated with Th1 polarizing conditions in the presence of saturating concentrations of the different IL-6 variants for five days. Percentage of Th1 cells were calculated by counting number of events in the CD4+IFNγ+ population obtained by flow cytometry. The control condition was defined as 100% response and the other conditions normalized accordingly. Data are mean + /- SEM from four independent replicates. ( d ) Triangular representation of data from ( a–c ). As the affinity for gp130 decreases (C7 and A1 variants) the different IL-6 activities are differentially affected with Th17 differentiation being the most robust activity to changes in affinity and Treg inhibition being the most sensitive activity.

Article Snippet: Briefly, resting human CD4 + T cells freshly isolated were activated using ImmunoCult Human CD3/CD28 T Cell Activator (StemCell, Cat#10971) following manufacturer instructions for 3 days in the presence of the cytokines required for the different CD4 + T cells populations: Th1 (IL-2 (20 ng/ml), anti-IL-4 (10 ng/ml), IL-12 (20 ng/ml)), Th17 (IL-1β (10 ng/ml, R and D Systems, Cat#201-LB/CF), IL-23 (10 ng/ml, R and D Systems, Cat#1290-IL), anti-IL-4 (10 ng/ml), anti-IFNγ (10 ng/ml, BD Biosciences, Cat#554698)) or Tregs (IL2 (20 ng/ml), TGF-β (5 ng/ml, Peprotech, Cat#100–21), anti-IL-4 (10 ng/ml), anti-IFNγ (10 ng/ml)) in the presence or absence of saturating concentrations of the different variants of IL6 described in this manuscript.

Techniques: Isolation, Flow Cytometry, Control, Activity Assay, Inhibition

Journal: eLife

Article Title: Kinetics of cytokine receptor trafficking determine signaling and functional selectivity

doi: 10.7554/eLife.49314

Figure Lengend Snippet: ( a–b ) Representative FACS plots and population strategy to define Treg cell numbers in response to IL-6 variants. ( c–d ) Representative FACS plots and population strategy to define Th17 cell numbers in response to IL-6 variants. ( e–f ) Representative FACS plots and population strategy to define Th1 cell numbers in response to IL-6 variants.

Article Snippet: Briefly, resting human CD4 + T cells freshly isolated were activated using ImmunoCult Human CD3/CD28 T Cell Activator (StemCell, Cat#10971) following manufacturer instructions for 3 days in the presence of the cytokines required for the different CD4 + T cells populations: Th1 (IL-2 (20 ng/ml), anti-IL-4 (10 ng/ml), IL-12 (20 ng/ml)), Th17 (IL-1β (10 ng/ml, R and D Systems, Cat#201-LB/CF), IL-23 (10 ng/ml, R and D Systems, Cat#1290-IL), anti-IL-4 (10 ng/ml), anti-IFNγ (10 ng/ml, BD Biosciences, Cat#554698)) or Tregs (IL2 (20 ng/ml), TGF-β (5 ng/ml, Peprotech, Cat#100–21), anti-IL-4 (10 ng/ml), anti-IFNγ (10 ng/ml)) in the presence or absence of saturating concentrations of the different variants of IL6 described in this manuscript.

Techniques: