Effects of Stimulating Interleukin-2/Anti-Interleukin-2 Antibody Complexes on Renal Cell Carcinoma

Kyu-Hyun Han; Ki Won Kim; Ji-Jing Yan; Jae-Ghi Lee; Eun Mi Lee; Miyeon Han; Eun Jin Cho; Seong Sik Kang; Hye Jin Lim; Tai Yeon Koo; Curie Ahn; Jaeseok Yang

Disclosures

BMC Urol. 2016;16(2) 

In This Article

Methods

Cells and Mice

The RENCA, a murine RCC cell line from a BALB/c mouse background was purchased from Korean Cell line Bank (Seoul, Korea), and cultured in Eagle's Minimum Essential Medium (Gibco/Invitrogen, Grand Island, NY, USA) containing 10 % fetal bovine serum (Gibco/Invitrogen) at 37 °C and 5 % CO2. BALB/c mice were purchased from Orient Bio Inc. (Seongnam, Korea) and maintained at the Biomedical Research Institute of Seoul National University Hospital. Mouse experimental protocols were approved by the Animal Ethics Committee of Seoul National University College of Medicine.

Preparation of IL-2/Anti-IL-2 Antibody Complex

Recombinant murine IL-2 was purchased from eBioscience (San Diego, CA, USA) and the S4B6 anti-mouse IL-2 monoclonal antibodies was provided by Dr. Charles D. Surh (La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA). S4B6 (7.5 μg) was mixed with IL-2 (1.5 μg, equivalent to 8555 IU) and incubated at 37 °C for 30 min before use. To evaluate the immune-enhancing effects of IL-2 under normal conditions, IL-2C or phosphate-buffered saline (PBS) was administered daily to mice by intraperitoneal injection for 5 days, before the spleen was harvested for immune cell analysis.

In Vivo Tumor Model

Eight-week old BALB/c mice were subcutaneously injected with RENCA cells (1 × 105) in 0.1 ml of 1× PBS to induce syngeneic RCC formation. IL-2C (treatment group) or PBS (control group) was intraperitoneally administered to mice every other day from day 0 to 28. Tumor size (length × width) was measured every other day using calipers. IL-2C with S4B6 (7.5 μg) and IL-2 (1.5 μg) or phosphate-buffered saline (PBS) was administered every 2 days to mice by intraperitoneal injection until 28 days. In high-dose IL-2 group, higher dose of IL-2 (35 μg, 200,000 IU) was administered to mice with the same schedule. Spleen, lung and tumor tissues were harvested 28 days after injection of RENCA cells. Tumor weight was measured after harvest. Pulmonary edema was assessed by lung weight, which was calculated by subtracting the dry weight from the wet weight.

Flow Cytometry

Splenocytes were labeled with the following antibodies: anti-CD4-allophycocyanin (APC), anti-CD8-fluorescein isothiocyanate (FITC), anti-CD44-APC, anti-CD45-FITC, anti-CD49-phycoerythrin (PE), and anti-F4/80-PE and the vital dye 7-aminoactinomycin D (7-AAD) (BD Biosciences, San Jose, CA, USA). Forkhead homeobox protein 3 (Foxp3) was labeled using the anti-mouse Foxp3-FITC staining kit (eBioscience) according to the manufacturer's instructions. For analysis of tumor-infiltrating cells, tumors were dissociated with 200U/ml collagenase IV at 37 °C for 30 min. Flow cytometric analysis was carried out on a Canto II Instrument (BD Biosciences).

Enzyme-linked ImmunoSPOT (ELISPOT) Assay

Interferon (IFN)-γ- or IL-10-producing T cells were detected with the ELISPOT assay. Spleens were harvested 28 days after mice were injected with RENCA cells. A 96-well plate was coated with anti-IFN-γ or -IL-10 capture antibodies using ELISPOT mouse IFN-γ or mouse IL-10 kits (BD biosciences). For IFN-γ ELISPOT, splenocytes (1 × 105/well) were incubated with 5 ng/ml phorbol 12-myristate 13-acetate (Sigma, St. Louis, MO, USA) and 500 ng/ml of inomycin (Sigma) at 37 °C for 8 h. For IL-10 ELISPOT, splenocytes (5 x 105/well) were incubated with 1 μg/ml of lipopolysaccharide (Sigma) for 24 h. Detection antibodies were then added, along with horseradish peroxidase (HRP)-streptavidin (BD Biosciences). After adding 3'-amino-9-ethylcarbazole substrate (BD Biosciences) for development, colored spots were measured with an ELSPOT reader (Cellular- Technology, Cleveland, OH, USA).

Immunohistochemistry

Tumor tissue with overlying skin was harvested on day 28. Anti-CD4, anti-CD8, anti-CD49b and anti-F4/80 antibodies (eBioscience) were incubated with tissue sections at 4 °C overnight. Sections were then treated sequentially with secondary antibody (ZytoChem Plus HRP One-Step Polymer anti-mouse; Zytomed, Berlin, Germany) and substrate solution (ImmPACT NovaRED Peroxidase Substrate Kit; Vector, Burlingame, CA, USA). Pulmonary edema was assessed by hematoxylin and eosin staining.

Statistical Analysis

Continuous variables were compared between the IL-2C and the PBS groups using the Student's t-test. RCC growth over 4 weeks was compared between the two groups with the linear mixed model. A P value < 0.050 was considered statistically significant. Analyses were carried out using SPSS v.22.0 software (SPSS Inc., Chicago, IL, USA).

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