Anti cd3 fitc clone 17a2

The following murine antibodies (Bio-Legend) specific to the surface markers were used: anti-CD3-FITC (clone 17A2) and anti CD90.2-AlexaFluor 700(clone 30-H12) for T cells, anti-CD4-Pacific Blue and APC (clone GK1.5) for CD4+ T cells, anti-F4/80-PE/Cy7 (clone BN8) and anti-CD11b-APC (clone M1/70) for macrophages, anti-Ly-6G/Ly-6C (Gr-1)-PE (RB6-8C5) for neutrophils, cytotoxic T-lymphocyte antigen 4 (CTLA-4)-APC (clone UC10-4B9) and programmed death 1(PD-1)-APC (clone 29 F.1A12) as negative co-stimulatory molecules, anti-CD44-APC/Cy7 (clone IM7) for memory T cells, anti-CD62L-PE (clone MEL-14) for naive T cells, anti-IL-2-PE (clone JES6-5H4) for IL-2, and CD25-FITC (clone BC69) as an activation marker for T cells. Anti-APC Microbeads were used for CD4+ cell purification (Miltenyi Biotec, Auburn, CA, USA).

For Th1/Th2 cytokine analysis, bilateral groin lymph nodes were harvested at 3 days after sensitization by DNFB. Cells were then activated using 25 ng/ml of Phorbol 12-Myristate 13 Acetate and 1 μg/ml of ionomycin with GolgiStop (BD Biosciences, San Diego, CA) in culture medium for 4 h. After activation, cells were stained with anti-CD4 allophycocyanin (APC) (clone GK1.5, BioLegend), anti-CD3 APC cyanin 7 (Cy7) (clone 17A2, BioLegend). Intracellular staining was performed using fixation and permeabilization kit (BD Bioscience) with anti-IFN-γ fluorescein isothiocyanate (FITC) (clone XMG1.2, BioLegend) and anti-IL-4 peridinin chlorophyll protein (PerCP) (clone 11B11, BioLegend). For determination of CD4⁺CD25⁺ T cells, mice were fed with 100 ppm of nickel water freely for two weeks, and then, splenic single-cell suspensions were prepared using BD lysing buffer (BD Bioscience). Cells were incubated with anti-CD16/CD32 mAb for Fc blocking (clone 2.4G2, BD Bioscience), anti-CD3 FITC (clone 17A2, BioLegend), anti-CD25 phycoerythrin (PE) (clone 3C7, BD Bioscience), and anti-CD4 APC (GK1.5, BioLegend). Cells were acquired on a FACS CantoII (BD Biosciences). Data were analyzed with FlowJo v10 software (FlowJo LLC, Ashland, OR).

Tumor tissues were fixed in 4% paraformaldehyde for 4 h at 4°C and then processed through a graded series of sucrose in PBS (10% for 1 h, 20% for 1 h and 30% overnight) at 4°C. After being embedded in optimum temperature cutting compound, samples were frozen on dry ice and stored at −80°C. Thick frozen sections (10 μm) were cut sagittally and placed onto slides. Sections were incubated in PBS containing 5% normal goat serum, 1% bovine serum albumin (BSA) and 0.5% Triton X-100 for 1 h, followed by incubation with primary antibodies (anti-CD3 FITC, Clone 17A2 and anti-CD8 PE, Clone 53–6.7, BioLegend, San Diego, CA, USA) overnight at 4°C. After washing with PBS, sections were mounted with VECTASHIELD mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) and visualized under a fluorescence microscope (Olympus, Shinjuku, Japan).