Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: In general, non-receptor interacting loops were deleted from the WT-IL7 sequence and loops connecting the adjacent helices were modeled using Rosetta Loop Remodeler and Rosetta fix backbone design function. The sequence of the designed model was extracted and submitted to AlphaFold (monomer and multimer mode for structure and protein-receptor binding prediction respectively) as a preliminary validation of the Rosetta-remodeled protein. Iterations of the bad models (models that do not fold into the expected structure or models that did not predict to bind to the receptors) back to the design stage were performed. Models that passed the AlphaFold validation proceeded to subsequent in vitro assay using yeast display system and flow cytometry to determine their relative binding affinity to IL-7 receptors in comparison to WT-IL7.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Sequencing, Binding Assay, Biomarker Discovery, In Vitro, Flow Cytometry, Comparison

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: Blueprint of the WT-IL7 was shown on the left of the figure. The connectivity of the functioning helixes was connected in a manner that requires extremely long protein loops by design (i.e. helices were not connected to the closest adjacent helixes but to the opposite helix). Loops that were not interacted with the IL-7 receptors were deleted and the helixes were reconnected in a clockwise manner via new protein linkers connecting to the adjacent helixes. The blueprint of the redesigned protein was shown at the right side of the figure. Protein structures are colored as rainbow (from N-to-C terminus with the order of Blue-Green-Yellow-Red).

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques:

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) AlphaFold validation of the first loop design version of Neo-7 (Neo-7 LDv1) using the default (left) and single sequence mode (right). ( B ) AlphaFold validation of the second loop design version of Neo-7 (left; Neo-7 LDv2) and Neo-7 LDv2 with mutations (right) favored by Rosetta fix backbone design. ( C ) Crystal structure of human IL-7 in complexation to human IL-7 receptor alpha (PDB ID = 3DI2). ( D ) Superimposition of Neo-7 structures (with or without additional disulfide bridge) predicted by AlphaFold. ( E ) Yeast display and flow cytometry validation of IL-7/Neo-7 bindings towards the IL-7 receptors. The yeast-displayed protein (different redesigned IL-7s) carries a HA-tag while the recombinant IL-7 receptors carry either a HIS tag (IL-7 receptor alpha) or a FC-tag (common-IL-2 family receptor gamma; for detection of IL-2Rγ binding, yeast cells were first incubated with recombinant IL-7Rα, washed, and subsequently incubated with IL-2Rγ.) The signal intensity of the X-axis (conferred by the binding of anti-HA mab) correlates with the expression level of the displayed protein while the signal intensity of the Y-axis (conferred by the binding of the anti-HIS/anti-FC mAb to the recombinant receptors bound to the displayed proteins) correlates with the binding affinity of the displayed proteins towards the IL-7 receptors.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Biomarker Discovery, Sequencing, Flow Cytometry, Recombinant, Binding Assay, Incubation, Expressing

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: ( A ) Inspection of structural and binding interactions of residue mutations Q6P and T45I on Neo-7 towards the murine IL-7R alpha. ( B ) Yeast display and flow cytometry validation of the binding ability of IL-7/Neo-7 variants toward the IL-7 receptors.

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Binding Assay, Residue, Flow Cytometry, Biomarker Discovery

Journal: eLife

Article Title: Targeted computational design of an interleukin-7 superkine with enhanced folding efficiency and immunotherapeutic efficacy

doi: 10.7554/eLife.107671

Figure Lengend Snippet: FPLC profile of E. coli expressed ( A ) WT-IL7 ( B ) refolded WT-IL7 ( C ) Neo-7-Q6P and ( D ) Neo-7-Q6P-T45I. Percentage of purity is calculated from the SEC-FPLC peak profile via Cytiva Unicorn 7 software after affinity chromatography purification. SPR (Biacore) characterization of the binding kinetics of ( E ) Neo-7-Q6P ( F ) Neo-7-Q6P-T45I and ( G ) WT-IL7 towards murine IL-7R alpha. ( H ) 2E8 proliferation assay to investigate the biological activity of the IL-7/Neo-7s expressed by E. coli . Error bars represent standard deviation (n=3).

Article Snippet: Receptor binding was done by incubation of yeast with a final concentration of 50 nM IL-7 receptor-α (IL7Rα; Sino Biological Cat: 50090-M08H) consisting of a C-terminal polyhistidine tag for 30 min at 4°C on a mini shaker agitated at 600 rpm.

Techniques: Software, Affinity Chromatography, Purification, Binding Assay, Proliferation Assay, Activity Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: IL-33 upregulates IL-2 receptor alpha chain (CD25) expression in IL-3 primed human basophils. Basophils isolated from PBMCs of healthy donors were cultured alone or in the presence of various stimuli with or without IL-3 (10 ng/ml) priming. (A) Each of the indicated stimulatory agents was added either alone or after 1 hr of IL-3 priming, and CD25 expression was evaluated by FACS after 24 hrs. Summarized data (% positive cells and MFI) for n = 6 independent donors. (B) Dose response of IL-33 cytokine on the expression of CD25 (MFI, n = 3). (C) Time kinetics of IL-3 and IL-33 on the expression of CD25 up to 96 hrs compared to time 0. IL-33 was added at 5 ng/ml after 1 hr of priming with IL-3 (10 ng/ml). The culture was supplemented every 24 hrs with additional IL-33 (5 ng/ml) (MFI, n = 3). (D) Representative FACS histogram for CD25 expression on basophils compared to isotype control. (E) Levels of soluble CD25 in the culture supernatants of basophils either cultured alone or stimulated with IL-3 and IL-33 at different time points (n = 6). Data are represented as mean ± SEM. * P < 0.05 , ** P < 0.01 , *** P < 0.001 , **** P < 0.0001 ; ns, not significant by one-way ANOVA (A, B) or 2-way ANOVA (C, E) followed by Tukey’s multiple comparison test. Abbreviation: MFI, median fluorescence intensity.

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Expressing, Isolation, Cell Culture, Control, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: Expression of IL-2 receptor alpha (CD25), beta (CD122) and gamma (CD132) chains on IL-3 + IL-33 stimulated basophils. CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ) expression was analyzed on basophils after 24 hrs of stimulation with or without IL-3 and IL-33 cytokines as indicated. (A-C) Representative FACS histogram and summarized data for the expression level of CD25, CD122, and CD132 (% positive cells and MFI). (D) Percentage of cells expressing the trimeric IL-2 receptor was identified by successive gating on each of the receptor chains. Data are represented as mean ± SEM (n = 8). * P < 0.05 , ** P < 0.01, ***P < 0.001, and ****P < 0.0001 ; ns, not significant by one-way ANOVA followed by Tukey’s multiple comparison test. Abbreviation: CA, cells alone; MFI, median fluorescence intensity.

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Expressing, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: Binding of IL-2 to CD25-expressing basophils neither induces downstream signaling nor limits IL-2 availability to Treg cells. Isolated basophils were cultured alone or in the presence of IL-3 and IL-33 for 24 hrs to induce the expression of CD25. (A) Cultured basophils were treated with 5 μg/mL biotinylated-IL-2 for 30 min and stained with PE-streptavidin to detect extracellular IL-2 binding to CD25. Representative FACS histograms and summarized data for binding of IL-2 to basophils (% positive cells and MFI, n = 4). (B) Cultured basophils were treated with IL-2 (3000 U/ml) for 30 min, followed by intracellular staining for phosphorylated STAT5 (pSTAT5). IL-3 (10 ng/ml) was added for 30 min on untreated basophils as a positive control for STAT5 activation on basophils. Representative FACS histograms and summarized data showing pSTAT5 expression (% positive cells, n = 3). (C) Representative histogram showing the p-STAT5 expression in isolated regulatory T cells following stimulation with IL-2 (3000 U/ml). (D) Basophils were treated with IL-3 and IL-33 for 72 hrs, and IL-2 (20 U/ml) was added during last 24 hrs. Subsequently, Treg cells were cocultured with these basophils at a 1:1 ratio, and the viability of Treg cells was analyzed by Annexin-V and PI staining after 48 hrs. Representative dot plots and summarized data from different donors are presented (n = 6). Data are represented as mean ± SEM. * P < 0.05 , ** P < 0.01 , **** P < 0.0001 ; ns, not significant by One-way ANOVA followed by Tukey’s multiple comparison test. Abbreviation: CA, cells alone; MFI, median fluorescence intensity; PI, propidium iodide.

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Binding Assay, Expressing, Isolation, Cell Culture, Staining, Positive Control, Activation Assay, Comparison, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-33 and IL-3 synergistically induce CD25 expression on human basophils without functional IL-2 signaling: a potential marker of severe COVID-19

doi: 10.3389/fimmu.2025.1718240

Figure Lengend Snippet: Basophils from severe COVID-19 patients display enhanced expression of IL2RA and IL2RG . (A) Expression of IL2 receptor subunit transcripts in BALF basophils of healthy controls, mild, and severe COVID-19 patients. Violin plots showing the normalized expression levels of IL2RA , IL2RB , and IL2RG in BALF basophils from healthy controls (Cnt, blue), mild (mild, green), and severe (Sev, red) COVID-19 patients. Individual dots indicate single-cell expression values (B) Global Expression of IL3 and IL33 in cells from BALF of healthy controls, mild, and severe COVID-19 patients. Violin plot showing the normalized expression of IL3 and IL33 in BALF cells from healthy controls (Cnt, blue), mild (mild, green), and severe (Sev, red) COVID-19 patients. Individual dots indicate single-cell expression values. Statistical significance was assessed using Wilcoxon rank-sum test; **** P < 0.0001 , * P < 0.05 . (C) Graphical abstract. IL-3 and IL-33 cytokines synergistically induce CD25 expression in basophils. Transient IL-2 sequestration by CD25 + basophils or basophil-derived soluble CD25 could increase the bioavailability of low-dose IL-2 for Treg cells and thereby have a positive impact on their survival. On the other hand, the expression of CD25 and CD132 on basophils might serve as potential biomarkers of severe inflammation like COVID-19. Figure created at BioRender.com .

Article Snippet: CD4 + CD25 + CD127 dim/– regulatory T Cell Isolation Kit II (Miltenyi Biotec) was used for the isolation of CD4 + CD25 + CD127 dim/– Treg cells from human PBMCs.

Techniques: Expressing, Derivative Assay