Anti il 4 antibody

Purified CD4⁺ T cells were obtained from the spleens of naïve DBA/1 mice and stimulated with 1 μg/ml plate-coated anti-CD3 (clone 2C11) and 1 μg/ml soluble-form anti-CD28 (clone PV1.17) under Th1-polarizing conditions [5 ng/ml IL-2, 10 ng/ml IL-12 and 2 μg/ml anti-IL-4 antibody (Biolegend, San Diego, CA, United States)] or Th17-polarizing conditions (20 ng/ml IL-6, 2.5 ng/ml TGF-β, 10 μg/ml anti-IFN-γ and 10 μg/ml anti-IL-4), and the cells treated with or without KPs (4.4 mg/ml) starting at the beginning of the induction. The cells were collected on day 4 of KPs treatment, and intracellular cytokine analysis was performed.

Naïve CD4⁺ T cells were cultured for 7 days with plate‐bound anti‐CD3ε and anti‐CD28 antibodies (both from TONBO Bioscience) in the presence of polarizing cytokines as follows: 10 ng·mL⁻¹ IL‐12 (PeproTech) and 10 µg·mL⁻¹ anti‐IL‐4 antibody (BioLegend) for Th1 cells and 20 ng·mL⁻¹ IL‐4 (PeproTech) and 10 µg·mL⁻¹ anti‐IL‐12 antibody (BioLegend) for Th2 cells.

Naïve CD4⁺ T cells from lymph nodes or spleen were purified by CD4⁺CD62L⁺ T cell isolation MACS kit (Miltenyi Biotech) over 95% purity. Naïve T cells were activated for 72 h with plate-bound 1 μg ml⁻¹ of anti-CD3e and -CD28 antibody (BD Biosciences) in a 96-well flat-bottom plate. For the differentiation to each subset of T helper (Th) cells, the following condition was applied for each Th subset: Th1 cells for IL-12 (10 ng ml⁻¹; Peprotech), anti-IL-4 antibody (1 μg ml⁻¹; BioLegend), Th2 cells for IL-4 (20 ng ml⁻¹; Peprotech), anti-IFNγ antibody (1 μg ml⁻¹; BioLegend), Th17 cells for TGF-β1 (1 ng ml⁻¹; Peprotech), IL-6 (30 ng ml⁻¹; Peprotech), anti-IFNγ and IL-4 antibody (1 μg ml⁻¹) and iT_reg cells for TGF-β1 (0.5, 1 or 5 ng ml⁻¹), and IL-2 (20 ng ml⁻¹; Peprotech).

Male C57BL/6N mice (6–8 weeks) were purchased from Janvier (Le Genest, France) and treated according to the International Association for the Study of Pain guidelines. For all experiments the ethics guidelines for investigations in conscious animals were obeyed and the procedures were approved by the local ethics committee (Regierungspräsidium Darmstadt). The animals had free access to food and water and were maintained in climate‐ (23 ± 0.5°C) and light‐controlled rooms (light from 6.00 a.m. to 6.00 p.m.).
Inflammation was induced by injection of 10 μl zymosan (3 mg/ml in PBS; Merck, Darmstadt, Germany) subcutaneously into the plantar side of one hind paw. Eosinophil depletion was achieved by intraperitoneal (i.p.) injection of anti‐Siglec F antibody (0.883 mg/kg; clone 238,047; R&D Systems, Minneapolis, MN) 24 h prior zymosan injection. As control purified rat IgG2a (Biolegend, San Diego, USA) was used. IL‐4c was prepared using IL‐4 (Peprotech, Hamburg, Germany) and anti‐IL4 antibody (Biolegend, San Diego, USA) (Finkelman et al, 1993 (link); Milner et al, 2010 (link); Jenkins et al, 2011 (link)) and administered i.p. (0.166 mg/kg IL‐4 and 0.883 mg/kg anti‐IL4 antibody) 24 h prior zymosan injection. (Finkelman et al, 1993 (link); Milner et al, 2010 (link); Jenkins et al, 2011 (link)).

The naive CD4⁺ T cells were cultured in 96-well plates with 1 × 10⁵ cells, in 24-well plates with 5 × 10⁵ cells, or in 6-well plates with 5 × 10⁶ cells (precoated with anti-CD3, 1 μg/ml, Thermo Fisher Scientific Co., Ltd, Xian, China) and were then transfected with miR-126a-5p mimics (60 nM, QIAGEN, Germany) and inhibitor (150 nM, QIAGEN, Germany) using HiPerFect transfection reagent (QIAGEN, Germany) or DLK1-siRNA (56 nM, Shanghai GenePharma Co., Ltd, China) using Lip3000 (Shanghai GenePharma Co., Ltd, China) or OE-DLK1 using polybrene (Solarbio Science & Technology Co., Ltd, Beijing, China) or DAPT (γ-secretase inhibitor, 20 μM, Sigma-Aldrich LLC). After 4 h, soluble anti-CD28 (0.2 μg/ml, Thermo Fisher Scientific Co., Ltd, Xian, China) was added along with cytokines that differentiate naive CD4⁺ T cells into different T-cell subtypes. For Th1 cells, IL-2 (20 ng/ml, BioLegend, Inc, California, USA.), IL-12 (50 ng/ml, BioLegend, Inc, California, USA.), and anti-IL-4 antibody (10 ng/ml, BioLegend, Inc.) were added to the well plates for 72 h. To differentiate the cells into Th2 cells, IL-2 (20 ng/ml, BioLegend, Inc, California, USA.), IL-4 (10 ng/ml, BioLegend, Inc, California, USA.), and anti-IL-IFN-γ antibody (10 ng/ml, BioLegend, Inc, California, USA.) were added to the well plates for 72 h.

Total CD4⁺ T cells from spleens of MRL/lpr mice were purified with negative selection using a Mouse T Cell Isolation Kit (Stem Cell Technologies Inc) according to the manufacturer’s instructions. The purity was determined by FACS analysis (>98% CD4⁺). For T cell differentiation, the above purified naïve CD4⁺ T cell were cultured in RPMI-1640 with 10% heat-inactivated FBS and 2 mM L-glutamine in the presence of 1 μg/mL anti-mouse CD3 (Biolegend) and 0.5 μg/mL anti-mouse CD28 (Biolegend), followed by treatment with or without NCTD. Then, 2 ng/mL transforming growth factor-1 (TGF-β1) (Peprotech), 20 ng/mL IL-6 (Peprotech), 10 μg/mL anti-IL-4 antibody (Biolegend) and 10 μg/mL anti-IFN-γ antibody (Biolegend) were added to drive Th17 cell polarization. After 4 days, cells were collected for FACS analysis and cell culture supernatants were also collected to detect the IL-17 production as described above.

Spleen and lymph nodes from C57BL/6 mice were surgically removed and single cells were obtained mechanically using a 70μm cell strainer (BD Falcon). Red blood cells were lysed by ACK lysis buffer (Lonza) before CD4⁺ isolation, using the CD4⁺ T cell isolation Kit (Miltenyi). 0.5 ×10⁶ CD4⁺ cells/mL were suspended in 10% FBS RPMI-1640 (ThermoFisher) supplemented with 5 ng/mL IL-2 (PeproTech) and 0.5 μg/mL anti-CD28 antibody (clone:37.51, BioLegend) and seeded into 2 μg/mL anti-CD3 antibody (clone: 145–2C11, BioLegend) pre-coated 24-well plates. Th1 differentiation was achieved using 1 μg/mL anti-IL-4 antibody (clone: 11B11, Biolegend), 5 ng/mL IL-2 (PeproTech), and 10 ng/mL IL-12 (PeproTech) in the culture media. Th2 differentiation was achieved using 1 μg/mL anti-IFN-γ antibodies (clone: XMG1.2, Biolegend), 5 ng/mL IL-2(PeproTech), and 10 ng/mL IL-4 (PeproTech) in the culture media. Cells were incubated for 96 hours in 5% CO2 at 37°C. Cell Activation Cocktail with Brefeldin A (Biolegend) was added to the culture media 5 hours prior to harvesting and staining the cells. For PLX5622 treatment, different concentrations of PLX5622 were added to the culture media during the stage of differentiation to Th1/Th2.