Anti cd3 and anti cd28 antibody

Briefly, to isolate MDSCs from SA penile tumors, tumors were harvest and digested by Mouse Tumor Dissociation kit (Miltenyi Biotec) to obtain the single cells. Total MDSCs were isolated using Mouse Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec). Total spleen T cells were isolated from spleen of 2-month-old male C57BL/6 mice (Jackson Laboratory) using the mouse Pan T Cell Isolation Kit II (Miltenyi Biotec). To assess the suppressive activities of MDSCs on T cell proliferation, we first labeled the T cells with 5 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen). The labeled T cells were stimulated in an antigen-nonspecific manner with anti-CD3 and anti-CD28 antibodies following eBioscience’s protocol. MDSCs were added at different ratios to T cells. CFSE intensity was quantified 72 h later with BD LSRFortessa Cell Analyzer. Viable CD3⁺ T cells with CFSE peaks located left of the highest peak (i.e. no proliferation, thus no decline of CFSE intensity) were counted as CFSE^low (i.e. proliferative) cells.

Cell migration assay was performed using a 96-well plate based chemotaxis system (Neuroprobe, Gaithersburg, MD), as described previously [27 (link)], in three independent experiments. CD4⁺ cells were isolated from mouse spleen cells using magnetic bead negative selection (Stemcell® Mouse CD4⁺ T Cell Enrichment Kit), were pre-activated with anti-CD3 and anti-CD28 antibodies (eBioscience) for 3 h in RPMI medium containing 10 % fetal calf serum and resuspended at 1 × 10⁷ cells/ml. Lower chambers were loaded with 29 μl of the chemokine CXCL12 in the indicated concentration (ranging from 1 μg/ml to 0.01 ng/ml; ImmunoTools, Friesoythe, Germany) and overlaid with a 5-μm pore-size filter membrane. The cells (20 μl, i.e. 2 × 10⁵ cells) were loaded on the hydrophobic surface surrounding each well on the membrane (upper chamber). CGS21680 was added in different concentrations in order to assess its effect on migration (upper and lower chambers always containing the same CGS21680 concentration). After 3 h of incubation, cells in the lower chamber were transferred to a 96-well plate and counted automatically using a Becton Dickinson FACSCanto II with a 96-well high throughput sampler (HTS) device.

A peroxidase-labeled mouse anti-human IgE Fc antibody and peroxidase-labeled goat anti-mouse IgE, IgG1 and IgG2a Fc antibodies were obtained from SouthernBiotech, USA (9160-05, 1110-05, 1070-05 and 1155-05); tetramethylbenzidine (TMB) was purchased from Solarbio, China (R1200); aluminum hydroxide was obtained from Thermo Fisher, USA (77161); and LPS was purchased from Sigma, USA (L3012). ELISA kits for IL-4, IFN-γ and TNF-α were obtained from Ebioscience, USA (88-7044, 88–7314 and 88–7324); ELISA kits for IL-5 and IL-13 were purchased from 4 A Biotech, China (CME0003, CME0009); an IRF4 antibody was obtained from Cell Signaling, USA (4964); an antibody against GAPDH was procured from Proteintech, China (10494-1-AP); a TLR4 signaling inhibitor was purchased from Invivogen, USA (CLI-095); an anti-mouse TLR2 Ab was obtained from Biolegend, USA (121802); the PE-CD80, PE-CD83, FITC-MHCII and FITC-CD40 antibodies were obtained from Ebioscience, USA (12–0801, 12–0831, 11–5321 and 11–0402); and mouse GM-CSF and IL-4 were purchased from Sino Biological, China (51048-M07H, 51084-M08B). Anti-CD3 and anti-CD28 antibodies were obtained from Ebioscience, USA (16-0031-82, 16-0281-82).