Journal: Arthritis Research & Therapy

Article Title: Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

doi: 10.1186/ar3824

Figure Lengend Snippet: Cytokine concentrations in ocular fluids from uveitis patients with Behçet's disease during infliximab treatment . The levels of cytokines such as IFN-γ, IL-2, IL-4, IL-10, TNF-α, IL-6, and IL-17 were evaluated by ELISA or CBA. Ocular fluid samples from patients who had Behçet's disease (BD) with active uveitis (n = 6) or inactive uveitis (n = 4; no signs or symptoms of uveitis at the remission stage without treatment) were used for the assay. We also collected ocular fluid samples from BD patients receiving infliximab (IFX, n = 8). The controls consisted of the aqueous humor of patients with age-related cataracts (n = 3) and the vitreous fluid of patients with idiopathic macular holes (n = 3). P value indicates active uveitis group (active UVE) vs. infliximab group (IFX treatment). CBA, cytometric beads array; n.s., not significant.

Article Snippet: For the induction of human Th17 cells, purified CD4 + T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

doi: 10.1186/ar3824

Figure Lengend Snippet: In vitro effects of infliximab on CD4 + T cells from uveitis patients with Behçet's disease . CD4 + T cells from Behçet's disease (BD) patients (black bars) with active uveitis or healthy donors (HD, open bars) were co-cultured with rIL-2 and anti-human CD3/CD28 abs in the presence of infliximab (IFX). As the control abs for infliximab, anti-human TNF-α monoclonal antibody (αTNF-α) or anti-human IL-6 monoclonal antibody (αIL-6) were used. The levels of cytokines such as IFN-γ (A) , IL-4 (B) , IL-10 (C) , TNF-α (D) , IL-6 (E) , and IL-17 (F) in supernatants of T cells were evaluated by ELISA or CBA. Bars, mean ± SEM. Cytokine production by T cells. Asterisks mean values significantly higher than medium only (Abs (-)): * P < 0.05, ** P < 0.005, *** P < 0.0005. Abs, antibodies; CBA, cytometric beads array; n.s., not significant; SEM, standard error of the mean.

Article Snippet: For the induction of human Th17 cells, purified CD4 + T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems).

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Arthritis Research & Therapy

Article Title: Inhibition of Th17 differentiation by anti-TNF-alpha therapy in uveitis patients with Behçet's disease

doi: 10.1186/ar3824

Figure Lengend Snippet: Establishment of IL-17-producing Th17 cells from uveitis patients with Behçet's disease . (A) Purified CD4 + T cells from a Behçet's disease (BD) patient or a sarcoidosis (SAR) patient (black bars) with active uveitis or a healthy donor (HD, open bars) in the presence of anti-CD3/CD28 antibodies, anti-IFN-γ antibody, anti-IL-4 antibody, rIL-1β, rIL-6, rIL-23, and rTNF-α. For ELISA analysis, supernatants of polarized Th17 cell lines or control CD4 + T cell lines in the presence of only anti-CD3/CD28 antibodies were harvested. The graph indicates the amount of IL-17 determined by ELISA (pg/ml). (B) For flow cytometric analysis, harvested Th17 cell lines from BD or HD were stained with anti-IL-17 abs and anti-CD4 Abs after permeabilization. The numbers in the histograms indicate the percentages of cells that were double-positive for IL-17/CD4. Abs, antibodies.

Article Snippet: For the induction of human Th17 cells, purified CD4 + T cells from BD patients or healthy donors were co-cultured with anti-human CD3 antibody (2 μg/ml, BD PharMingen, San Diego, CA, USA), anti-human CD28 antibody (2 μg/ml, BD PharMingen), anti-human IFN-γ antibody (5 μg/ml, R&D Systems, Minneapolis, MN, USA), anti-human IL-4 antibody (5 μg/ml, R&D Systems), and recombinant human proteins such as IL-1β (20 ng/ml, Peprotech, Rocky Hill, NJ, USA), IL-6 (20 ng/ml, R&D Systems), IL-23 (20 ng/ml, R&D Systems), and TNF-α (20 ng/ml, R&D Systems).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS Pathogens

Article Title: Coincident airway exposure to low-potency allergen and cytomegalovirus sensitizes for allergic airway disease by viral activation of migratory dendritic cells

doi: 10.1371/journal.ppat.1007595

Figure Lengend Snippet: For experimental design and codes of experimental groups, see and . Frequencies of epitope-specific cells among immunomagnetically-purified CD4 + and CD8 + T cells, retrieved from lung tissue at 48 hrs after the last challenge exposure to OVA aerosol, were determined by IFN-γ-based ELISPOT assay after sensitization with synthetic antigenic peptides. (A) Frequencies of CD4 + and CD8 + T cells recognizing the indicated viral epitopes. (B) Frequencies of CD4 + and CD8 + T cells recognizing the OVA epitopes OVA 323-339 (ISQAVHAAHAEINEAGR) and OVA 257-264 (SIINFEKL), respectively. Bars represent most probable numbers calculated by intercept-free linear regression analysis of data from graded numbers of effector cells each tested in triplicate cultures. Error bars indicate the 95% confidence intervals.

Article Snippet: Frequencies of IL-4 producing CD4 + Th2 cells were determined by using the IFN-γ/IL-4 double-color ELISPOT assay, according to the manufacturer’s protocol (CTL, Shaker Hights, OH).

Techniques: Purification, Aerosol, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Isolation and characterization of human Abs directed against IL-4Rα. ( a ) Binding activity of the isolated anti-IL-4Rα Abs to plate-coated human IL-4Rα or GST, as determined by ELISA. Data represented as mean ± SD (n = 3). ( b ) Schematic diagram of the reporter HEK-Blue TM IL-4/IL-13 cell line to monitor the biological activity of anti-IL-4Rα Abs. The details are described in the text. ( c ) IL-4Rα-blocking activity of the indicated Abs, as determined by SEAP secretion levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) in the presence of the Abs (40 and 200 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to PBS-treated samples. ( d ) Binding isotherms of the immobilized anti-IL-4Rα Abs to soluble antigen IL-4Rα, measured by bio‐layer interferometry on OctetRED96 (Fortebio). The concentrations of IL-4Rα analysed are indicated (colored).

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Isolation, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Affinity maturation of 4 R34 and characterization of the isolated clones. ( a ) Scheme of library construction and screening of 4R34 in the format of scFab using yeast surface display technology. The indicated residues in VL-CDR3, VH-CDR2, and VH-CDR3, highlighted by “X,” were randomly mutated, while maintaining the original amino acids at each residue of 4R34 at a frequency of approximately 50%, using designed spiked oligonucleotides. Numbering is according to the Kabat definition. ( b ) Flow cytometric analysis of antigen binding and expression levels of 4R34-based scFab yeast library in each round screening by FACS. The screening conditions of antigen and sorting gate used in each round are indicated. ( c ) Comparison of association and dissociation of soluble IL-4Rα antigen at 10 nM to immobilized anti-IL-4Rα antibodies (Abs), as measured by bio‐layer interferometry. ( d ) IL-4Rα-blocking activity of the indicated Abs, as determined by SEAP secretion levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) in the presence of the Abs (20 and 100 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to PBS-treated samples.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Isolation, Clone Assay, Binding Assay, Expressing, Blocking Assay, Activity Assay

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Engineering and characterization of 4 R34.1.19. ( a ) Library construction scheme of 4 R34.1, where the indicated residues in the VL-CDR1 and VH-CDR1 were randomized with NNK degenerate codon that encodes all 20 amino acids. ( b ) Flow cytometric analysis of antigen binding and expression levels of 4 R34.1-based scFab yeast library in each round screening by FACS. The screening conditions of antigen and sorting gate used in each round are indicated. ( c ) Binding isotherms of the immobilized anti-IL-4Rα Ab 4R34.1.19 to soluble antigen IL-4Rα, measured by bio‐layer interferometry. The concentrations of IL-4Rα analysed are indicated (colored). ( d , e ) IL-4Rα blocking activity of the indicated Abs, as determined by SEAP levels from HEK-Blue TM IL-4/IL-13 cells after stimulation with rhIL-4 (100 pM) ( d ) or rhIL-13 (1 nM) ( e ) in the presence of the Abs (20 and 100 nM) for 24 h. Data are presented as percentage (mean ± SD (n = 3)) in SEAP levels relative to phosphate buffer saline (PBS)-treated samples. Statistical analysis was performed using a two-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus dupilumab analogue. ( f ) Binding specificity of the indicated Abs (20 and 100 nM) for cell surface expressed IL-4Rα, as analysed in IL-4Rα-expressing THP-1 cells and IL-4Rα-deficient Molt-4 cells by flow cytometry. Representative histograms from three independent experiments are shown.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Binding Assay, Expressing, Blocking Assay, Activity Assay, Flow Cytometry

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Inhibitory effects of anti-IL-4Rα Abs on IL-4-stimulated T cell proliferation and T H 2 differentiation. ( a ) Dose-dependent blocking effects of anti-IL-4Rα Abs on the proliferation of T cells among PHA-activated PBMCs in response to rhIL-4 (500 pM), determined by CTG assay after 72 h culture. Data are represented as mean ± SD (n = 3). ( b,c ) Inhibitory effects of anti-IL-4Rα Abs (100 nM) on the T H 2 differentiation of naïve CD4 + CD45RO − T cells from healthy donors or asthmatic patients after 7 days culture in T H 2-skewing conditions in the presence of rhIL-4 (500 pM) and anti-IL-4Rα Abs (100 nM). The number of IL-4-producing T H 2 cells were determined by ELISPOT. Quantification of spot forming T cells ( b ) and representative image both healthy donor and asthmatic patient ( c ) are shown. In ( b ), error bars, ± SD (n = 3). Statistical analyses were performed using a one-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus PBS-treated group.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Blocking Assay, CTG Assay, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Engineering of anti-human interleukin-4 receptor alpha antibodies with potent antagonistic activity

doi: 10.1038/s41598-019-44253-9

Figure Lengend Snippet: Epitope mapping of anti-IL-4Rα Abs by alanine scanning mutagenesis. ( a ) Competitive ELISA showing the percentage of bound IL-4Rα (10 and 50 nM) to plate-coated hIL-4-mFc in the presence of the indicated Abs (20, 100, and 500 nM) compared to that without the Ab competitor. Data are represented as mean ± SD (n = 3). Statistical analyses were performed using a two-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant versus dupilumab analogue. ( b ) Overall structure of the human IL-4Rα:IL-4 complex (PDB: 1IAR). Magnified section shows the residues of IL-4Rα putatively involved in IL-4 binding. ( c ) The percent relative binding of the indicated hIL-4-mFc (5 nM) and anti-IL-4Rα Abs (2.5 nM of 4 R34 and 100 pM of 4 R34.1, 4 R34.1.19 and dupilumab analogue) to IL-4Rα alanine mutants compared to that of wild-type IL-4Rα. Data are represented as mean ± SD (n = 3). Statistical analyses were performed using a one-way ANOVA followed by the Newman-Keuls post-test. * P < 0.05, ** P < 0.01, *** P < 0.001 versus binding to wild-type IL-4Rα.

Article Snippet: For hIL-4-mFc expression, the cDNA plasmids carrying human IL-4 (residues 1–153) genes (Sino biological Inc.,HG-11846-CM) were subcloned in frame into pcDNA3.4 vector to be expressed in the C-terminal mouse immunoglobulin Fc (hinge-CH2-CH3) fused form .

Techniques: Mutagenesis, Competitive ELISA, Binding Assay