Journal: bioRxiv

Article Title: A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

doi: 10.64898/2026.09.18.752703

Figure Lengend Snippet: a, Structure of WT IL-2 model, based on Protein Data Bank (PDB) ID 1M47 (cartoon representation), bound to IL-2Rαβγ from PDB ID 2B5I; IL-2Rα (CD25) shown in gray (cartoon representation), IL-2Rβ (CD122) and IL-2Rγ (CD132) shown in light gray (surface representation). Rosetta Design was used to replace the regions in red with short, structured loops (light blue) that reconnect α-helices H1-H4, generating relooped designs R1.1-38. b, IL-2Rβγ signaling activity (HEK-Blue reporter assay) and expression level from Expi293 supernatants diluted 1:400; designs R1.1-38 normalized to WT IL-2 levels. c, Optimization of design R1.1 by extending IL-2Rγ-interacting helices and redesigning the H2-H3 loop (“Round 2 optimization”) to produce design R2.1, which was further optimized by mutating two IL-2Rβ-interacting residues, R88N and I99L, to produce design R2.2. d, Binding affinity to IL-2Rβγ-Fc and IL-2Rα as measured by biolayer interferometry (BLI): design R2.2 binds IL-2Rβγ with a K D close to that of WT IL-2 (0.80 nM and 0.44 nM, respectively), consistent with previously reported measurements for WT IL-2 , . The purified proteins measured here contain STII tags; NB = no binding. e, Circular dichroism (CD) wavelength scan at 25℃ ( left ) and thermal melt monitoring helical signal at 208 nm ( right ). f, STAT5 phosphorylation (pSTAT5) in resting T cell subtypes after treatment with the indicated designed cytokines or WT IL-2. NC = not calculated.

Article Snippet: To measure interactions between designed cytokines and IL-2Rβγ, Fc-tagged IL-2Rβγ (Acro Biosystems) or biotinylated Fc-tagged IL-2Rβγ (Acro Biosystems) were captured on ProA or SA biosensors to ∼3 nm (SA biosensors were used for all VHH fusion proteins due to VHH binding to ProA); binding was measured with analyte serially diluted from 100 to 0.4 nM, using 120 s association and 480 s dissociation.

Techniques: Activity Assay, Reporter Assay, Expressing, Binding Assay, Purification, Circular Dichroism, Phospho-proteomics

Journal: bioRxiv

Article Title: A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

doi: 10.64898/2026.09.18.752703

Figure Lengend Snippet: Designs were expressed in Expi293 cell and supernatants collected and evaluated for binding to IL-2Rα by ELISA ( top , normalized to WT IL-2) and IL-2Rβγ signaling activity by HEK-Blue assay ( bottom , levels normalized to that of WT IL-2). NTC is a non-transfected control.

Article Snippet: To measure interactions between designed cytokines and IL-2Rβγ, Fc-tagged IL-2Rβγ (Acro Biosystems) or biotinylated Fc-tagged IL-2Rβγ (Acro Biosystems) were captured on ProA or SA biosensors to ∼3 nm (SA biosensors were used for all VHH fusion proteins due to VHH binding to ProA); binding was measured with analyte serially diluted from 100 to 0.4 nM, using 120 s association and 480 s dissociation.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Transfection, Control

Journal: bioRxiv

Article Title: A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

doi: 10.64898/2026.09.18.752703

Figure Lengend Snippet: a, Schematic of OUTSMART dIL-2 system. In response to antigen-stimulated T cell activation, engineered T cells secrete the dIL-2 designed cytokine, which is targeted to CD8α to favor the engagement of IL-2Rβγ on endogenous NK and T cells in addition to the engineered T cells. b, dIL-2 is encoded in a single lentiviral vector that is broadly compatible with engineered CAR-T and TCR-T therapies. The dIL-2 protein is expressed under the control of a regulated promoter, and consists of c, Designed cytokine R2.2 (teal) fused to an anti-CD8α VHH (blue) via a 3xG4S linker. The VHH shown is a model for illustrative purposes (there is no crystal structure of the VHH used in this work). CD8α (PDB ID 8EW6) and IL-2Rβγ receptors (PDB ID 2B5I) are shown in surface representation. d, pSTAT5 activity in resting and activated T cell subtypes after treatment with recombinant protein of the indicated cytokines. e, Schematic of regulated promoter screen. Concatenated enhancer motifs were engineered into CAR lentiviral backbones utilizing either a dual-promoter (constitutive MND CAR with inducible cytokine) or single-promoter (CAR and cytokine expressed from the same inducible promoter) architecture. CAR-T cells were evaluated for inducible cytokine production in the presence or absence of ROR1 antigen. f, Regulated promoter-driven cytokine production at rest (unstimulated) versus after antigen stimulation. Illustrations in a were created using BioRender ( https://biorender.com , Fontana, T. 2024).

Article Snippet: To measure interactions between designed cytokines and IL-2Rβγ, Fc-tagged IL-2Rβγ (Acro Biosystems) or biotinylated Fc-tagged IL-2Rβγ (Acro Biosystems) were captured on ProA or SA biosensors to ∼3 nm (SA biosensors were used for all VHH fusion proteins due to VHH binding to ProA); binding was measured with analyte serially diluted from 100 to 0.4 nM, using 120 s association and 480 s dissociation.

Techniques: Activation Assay, Plasmid Preparation, Control, Activity Assay, Recombinant

Journal: bioRxiv

Article Title: A Dynamically Regulated Designed IL-2 for Tumor-localized Signaling

doi: 10.64898/2026.09.18.752703

Figure Lengend Snippet: a, Binding affinity to IL-2Rβγ-Fc and CD8α measured by biolayer interferometry (BLI) for design R2.2 (untargeted), dIL-2 protein (R2.2 fused to anti-CD8α VHH), and anti-CD8α VHH alone. b, Representative flow cytometry plots from resting ( left ) and activated ( right ) CD8 T cells (corresponds to ) measuring pSTAT5 (Alexa Fluor 647) signal after treatment with the indicated cytokine at a final concentration of 0.065 nM. dIL-2 exhibits superior pSTAT5 of resting T cells and similar pSTAT5 of activated T cells compared to WT IL-2. CD8 T cells are defined by gating on CD3+CD4-signal. c, Compared to WT IL-2, dIL-2 exhibits less pSTAT5 in resting CD4+Foxp3-cells after treatment with the indicated cytokine. d , Fold proliferation of T cells that were rested or stimulated for 24h with anti-CD3/anti-CD28 (TransAct) before incubation with indicated recombinant cytokines at 0.065 nM; cells were counted and split into fresh cytokine media every 3-4 days. e , Frequency of CD8+ cells (defined as CD3+CD4-) in cells stimulated for 24h and grown with the indicated recombinant cytokines at 0.065 nM. By day 14, 81.3% of cells were CD8+ after addition of dIL-2 protein. f , GS linker does not impact dIL-2 protein function: pSTAT5 elicited by dIL-2 protein with or without 3XG4S linker between the CD8ɑ binder and cytokine elements; representative pSTAT5 staining in CD8+ T cells treated with 0.065 nM cytokine ( left ), and pSTAT5 in resting T cell subtypes after treatment with the indicated designed cytokines ( right ).

Article Snippet: To measure interactions between designed cytokines and IL-2Rβγ, Fc-tagged IL-2Rβγ (Acro Biosystems) or biotinylated Fc-tagged IL-2Rβγ (Acro Biosystems) were captured on ProA or SA biosensors to ∼3 nm (SA biosensors were used for all VHH fusion proteins due to VHH binding to ProA); binding was measured with analyte serially diluted from 100 to 0.4 nM, using 120 s association and 480 s dissociation.

Techniques: Binding Assay, Flow Cytometry, Concentration Assay, Incubation, Recombinant, Staining

Journal: Frontiers in Physiology

Article Title: Interleukin-13 R130Q aggravates airway smooth-muscle phenotypic switching and promotes airway remodeling in severe asthma

doi: 10.3389/fphys.2026.1906184

Figure Lengend Snippet: Effects of IL-13 130R and IL-13 130Q on pulmonary inflammatory responses. (A) Total cell counts in bronchoalveolar lavage fluid (BALF; n = 8 per group); differential leukocyte counting was not performed. (B–D) Relative mRNA expression levels of IL-4 (B) , IL-5 (C) , and IL-13 (D) in lung tissue (n = 8 per group). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01. IL, interleukin; OVA, ovalbumin; SEM, standard error of the mean.

Article Snippet: According to the instructions of the manufacturer for the IL-4 and IL-5 ELISA kits (RayBiotech, Peachtree Corners, GA, USA), 100 μL of each sample was added to pre-coated wells and incubated at room temperature for 2.5 h. The wells were then washed three times for 5 min each, and blank wells served as negative controls.

Techniques: Expressing

Journal: Frontiers in Physiology

Article Title: Interleukin-13 R130Q aggravates airway smooth-muscle phenotypic switching and promotes airway remodeling in severe asthma

doi: 10.3389/fphys.2026.1906184

Figure Lengend Snippet: Effects of IL-13 130R and IL-13 130Q on human bronchial smooth muscle cell (hBSMC) phenotypes (n = 4 per group). (A) CCK-8-derived proliferative/metabolic activity. (B) Representative Fluo-3/AM flow-cytometry histograms and median fluorescence intensity quantification of the acetylcholine-induced intracellular calcium response. (C, D) IL-5 (C) and IL-4 (D) concentrations in culture supernatants. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01. IL, interleukin; SEM, standard error of the mean.

Article Snippet: According to the instructions of the manufacturer for the IL-4 and IL-5 ELISA kits (RayBiotech, Peachtree Corners, GA, USA), 100 μL of each sample was added to pre-coated wells and incubated at room temperature for 2.5 h. The wells were then washed three times for 5 min each, and blank wells served as negative controls.

Techniques: CCK-8 Assay, Derivative Assay, Activity Assay, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Complex Modulation of IL-6 Signaling by Apelin and Elabela in HTR-8/SVneo Cells Under Cobalt Chloride-Induced Chemical Hypoxia

doi: 10.64898/2026.08.20.746041

Figure Lengend Snippet: The effect of CoCl 2 on the levels of elabela (ELA), apelin, and apelin receptor APJ. The data were obtained using ELA or apelin ELISA assays in HTR-8/SVneo cell homogenates (Panels A and C) or conditioned media (Panels B and D), western blotting (Panel E), or real-time RT-PCR (Panel F) for APJ. Data are mean ± SEM, n=5-6.

Article Snippet: Individual ELISA kits were used for IL-6 (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-IL6), sIL-6R (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-IL6sR), and sgp130 (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-sgp130) protein quantification in the conditioned media 24 hours after treatment.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Complex Modulation of IL-6 Signaling by Apelin and Elabela in HTR-8/SVneo Cells Under Cobalt Chloride-Induced Chemical Hypoxia

doi: 10.64898/2026.08.20.746041

Figure Lengend Snippet: Elabela (ELA) or apelin treatment decreased IL-6 mRNA in CoCl 2 -treated HTR-8/SVneo cells. Data are mean ± SEM, n=3-6.

Article Snippet: Individual ELISA kits were used for IL-6 (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-IL6), sIL-6R (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-IL6sR), and sgp130 (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-sgp130) protein quantification in the conditioned media 24 hours after treatment.

Techniques:

Journal: bioRxiv

Article Title: Complex Modulation of IL-6 Signaling by Apelin and Elabela in HTR-8/SVneo Cells Under Cobalt Chloride-Induced Chemical Hypoxia

doi: 10.64898/2026.08.20.746041

Figure Lengend Snippet: Schematic of proposed interactions between IL-6 signaling and apelin and elabela in HTR-8/SVneo trophoblastic cells under CoCl₂-mimicked hypoxia. We hypothesize that apelin/elabela/APJ axis regulates IL-6 signaling via its actions on various components of the IL-6 classical and trans-signaling pathway.

Article Snippet: Individual ELISA kits were used for IL-6 (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-IL6), sIL-6R (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-IL6sR), and sgp130 (RayBiotech Inc., Norcross, GA, USA; cat. No. ELH-sgp130) protein quantification in the conditioned media 24 hours after treatment.

Techniques: