Anti il 4 antibody

The ELISpot protocol for detecting Th responses after a protein vaccination was developed previously.⁶¹ (link) Briefly, plates were coated overnight with anti-IFNγ, anti-IL-2 or anti-IL-4 antibody (BD Bioscience). 14 days after vaccination with rED44Her2, rED44Her2-FrC or the irrelevant control vaccine (plant expressed 5T33 Ig, not fused to FrC) spleens were processed. Following lymphoprep density centrifugation lymphocytes were plated at 2.5 × 10⁵ per well in RPMI 1640 media supplemented with 10% FCS, 1 mM sodium pyruvate, 2 mM glutamine, 1% non-essential amino acids, 50 U/mL penicillin, 50 µg/mL streptomycin and 50 µM β-mercaptoethanol. The cells were restimulated in vitro with media alone, 10 µg/mL rED44Her2, 10 µg/mL FrC or 10 µg/mL control protein (EndoGrade Ovalbumin, Hyglos GmbH). Following 40 h of restimulation, cytokine secretion was detected using cytokine-specific biotinylated antibodies, and then streptavidin-ALP (MAbtech AB). Spots were revealed using BCIP/NBT substrate (Invitrogen) and enumerated using an ELISpot microplate reader (AID GmbH). The non-specific media alone background was subtracted, and the response considered positive if the number of spots was more than twice that of the control mice or to the control protein, whichever was highest.

Splenic CD4 T cells (1 × 10⁶) isolated from 10-week-old prediabetic female NOD mice were stimulated with plate-bound anti-CD3 antibody (3 μg/ml, clone: 145-2C11, BD Biosciences, USA) and soluble anti-CD28 antibody (3 μg/ml, clone: 37.51, BD Biosciences) in the presence of recombinant mouse IL-12 (10 ng/mL, TONBO Biosciences), recombinant mouse IL-2 (5 ng/mL, Invitrogen, USA), and anti-IL-4 antibody (10 μg/ml, clone: 11B11, BD Biosciences, USA) in 12-well culture plates. After 5 days, cells were harvested and washed, and 1 × 10⁶ differentiated Th1 cells were treated with plate-bound anti-CD3 antibody (1 μg/ml) and soluble anti-CD28 antibody (1 μg/ml) in the presence or absence of recombinant sCD137 (240 μg/mL) for 24 h. In some wells, recombinant mouse IL-2 (25 U/ml) was added. After 24 h, cells were washed and rested in fresh TCM without IL-2 for 48 h; 0.5 × 10⁵ rested living cells were restimulated with plate-bound anti-CD3 antibody (0.5 μg/ml) and soluble anti-CD28 antibody (0.5 μg/ml) for 24 h. Supernatant was collected, and IL-2 concentration was measured by ELISA.

Mannan-induced psoriasis was introduced by a singular intra peritoneal injection of 10 mg mannan from Saccharomyces cerevisiae (Sigma #M7504). The mice were blindly assessed for both arthritic symptoms and psoriatic lesions according to standardized macroscopic scoring system (3 (link), 5 (link), 25 (link)).
As described previously (see text footnote 1), mannan-enhanced collagen antibody-induced arthritis (CAIA) is induced by a cocktail of four monoclonal anti-collagen type II antibodies injected i.v., followed by an i.p. injection of mannan day 5.
Type 2 macrophages (M2) were induced by an injection of 5 μg recombinant mouse interleukin 4 (rmIL4; Peprotech #214-14) and 25 μg anti-IL4 antibody (BD Biosciences #554387) in PBS (Sigma, Helsinki, Finland) on days 0 and 2, mice were then euthanatized on day 4, the protocol was adopted from Jenkins et al. (26 (link)) and Eichin et al. (27 (link)). Peritoneal cells were aseptically collected from naïve or in vivo stimulated mice, washed with sterile PBS, and plated 5 × 10⁵ cells/ml in six-well plates. The cells were incubated with RPMI complemented with 5% FCS, 100 μg/ml streptomycin, and 100 U/ml penicillin for 70 h in +37°C in humidified incubator with 5% CO₂. Cells were scraped, spun down, and washed with PBS.

Naive CD4⁺ T cells from the spleens of 6-8 weeks old male GREAT mice were prepared using magnetic bead cell sorting (Miltenyi Biotec, Bergisch-Gladbach, Germany). The purity of CD4⁺CD44^lowCD62L^high T cell subset was validated by flow cytometry. These naïve CD4⁺ T cells were stimulated with 5 μg/ml pre-coated anti-CD3ϵ antibody (Biolegend) and 1 μg/ml soluble anti-CD28 antibody (Biolegend) in culture medium containing 10 ng/ml IL-12 (Peprotech, Cranbury, NJ, USA), 10 ng/ml IL-2 (Peprotech) and 10 μg/ml anti–IL-4 antibody (BD Biosciences) for 3 days. The culture medium was RPMI 1640 medium (plus 50 μM β-mercaptoethanol) supplemented with 10% FBS, 1% GlutaMax, and 1% Pen/Strep (Gibco, Shanghai, China). 1 × 10⁵ naive CD4⁺ T cells were cultured in 96-well plates with 100 μl culture medium per well.

Human buffy coats were obtained from the Scottish Blood Transfusion Service and peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated from buffy coat samples by density gradient centrifugation according to manufacturer’s protocols (Lymphoprep, STEMCELL Technologies). From each donor, 100 × 10⁶ PBMCs were used for isolation of CD4 +T cells. Cells were decorated with anti-CD4^FITC antibodies (Biolegend, #357406) and isolated by magnetic separation according to manufacturer’s protocols (MACS Miltenyi) to a purity >98% CD4+. Freshly isolated resting CD4⁺ T cells (3 × 10⁷ per donor) were activated under Th-1 polarizing conditions using ImmunoCult Human CD3/CD28 T Cell Activator (StemCell, Cat#10971) following manufacturer instructions for 3 days in RPMI-1640, 10% v/v FBS, 100 U/ml penicillin-streptomycin (Gibco) in the presence of the cytokines IL-2 (Novartis, #709421, 20 ng/ml), anti-IL-4 antibody (10 ng/ml, BD Biosciences, #554481), IL-12 (20 ng/ml, BioLegend, #573002). After 3 days of priming, cells were expanded for another 5 days in the presence of IL-2 (20 ng/ml).