Sugar-carrying Polystyrenes Facilitate Harvesting of APCs from MLRs: Possible Application of Sugar-carrying Polystyrenes to Immunotherapy

Materials and Methods

Mice. Male C57BL/6 (H-2^b) and BALB/c (H-2^d) mice between 6 and 7 weeks of age were obtained from Oriental Yeast Co., ltd. (Tokyo, Japan). Animals were specific pathogen-free and had free access to food and water in the animal care facility under the Institutional guidelines for the usage of experimental animals.

Mononuclear leukocytes (MNLs). C57BL/6 (H-2^b) and BALB/c (H-2^d) mice were sacrificed and their spleens and lymph nodes were removed aseptically. MNLs were obtained by mincing lymphoid organs, followed by passing through a cotton wool column and washing with RPMI-1640 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (JRH BioSciences, Tokyo, Japan), 5×10⁻⁵ M 2-mercaptoethanol (Sigma–Aldrich, St. Louis, MO, USA,), 10 mM HEPES (Sigma Chemical Co. St. Louis, MO, USA), 100 μg/ml penicillin (Meijiseika, Tokyo, Japan), and 100 μg/ml streptomycin (Meijiseika) (referred to as MLR medium).

Target MNLs as antigens. MNLs from C57BL/6 (H-2^b) or BALB/c (H-2^d) mice were treated with concanavalin A (20 μg/ml; Sigma Chemical Co.) for 48 h at 37°C and then mitomycin C (MMC, 50 μg/ml; Sigma Chemical Co.) for 1 h at 37°C to stop proliferation. The cells were then used as target cells. Target MNLs from C57BL/6 (H-2b) mice were used as allogeneic antigens and those from BALB/c (H-2d) mice were used as syngeneic antigens.

Induction of APCs. Naive MNLs from BALB/c (H-2^d) mice (6×10⁷ cells/ml) and C57BL/6 (H-2^b) mice (6×10⁷ cells) were co-cultured in T-75 flasks (Becton Dickinson, Franklin Lakes, NJ, USA) containing 24 ml MLR medium at 37°C for 12 days. Other flasks (Becton Dickinson) were coated with PVGlcNAc or a PVLA derivative co-polymer consisting of sulfonylurea linked to PVLA [P(VLA-co-SU)]. APCs attached to conventional flasks were harvested by three methods: i) mechanical shaking on ice (4°C), ii) enzymatic digestion, and iii) pipetting. Cells attached to PVGlcNAc- and P(VLA-co-SU)-coated flasks were harvested by mechanical shaking at 4°C.

Mixed leukocyte reaction (MLR). To obtain T-helper (Th) cells, we performed a primary MLR. The techniques of primary and secondary MLRs have been described previously (6). Briefly, the MLR was performed for 10-14 days using naive MNLs from BALB/c mice (6×10⁷ cells/ml) and target MNLs (6×10⁷ cells/ml) with APCs in T-75 flasks containing MLR medium supplemented with interleukin-2 (20 ng/ml; Sigma Chemical Co.). Then non-adherent primed cells were harvested. These primed cells were re-stimulated at approximately 5×10⁴/ml in the presence of irradiated stimulator spleen cells of the appropriate haplotype at 4×10⁶/ml in 15 ml MLR medium supplemented with 20 ng/ml interleukin-2 and used as primed Th cells.

Secondary MLR was performed to obtain cytotoxic T-lymphocytes (CTLs). Primed Th1 cells obtained by primary MLR and naive MNLs from BALB/c mice were co-cultured for 10-14 days in T-75 flasks containing MLR medium supplemented with 20 ng/ml interleukin-2. Then non-adherent cells were harvested as CTLs.

7-Aminoactinomycin D (7-AAD) assay. CTLs from BALB/c (H-2^d) mice and target MNL from C57BL/6 (H-2^b) mice were co-cultured in MLR medium for 2 days. Then, samples (1×10⁶ cells) were stained with fluorescein isothiocyanate (FITC)-anti mouse H-2D^b antibody that specifically bound with target MNL from C57BL/6 mice for 30 min at 4°C. After washing once, 5 μg/ml of 7-AAD (Sigma Chemical Co.) were added. The percentage specific cytotoxicity was calculated as following; % specific cytotoxicity =100 × [(H-2^b/7-AAD double positive cell number)^{test cell} − (H-2^b/7-AAD double positive cell number)^{negative control}]/total cell number.

Fluorescence-activated cell sorting (FACS). To analyze the expression of CD80, CD86, CD11c and I-A^d on APCs, the cells were incubated for 1 h with FITC-conjugated antibody to CD11c, biotin-conjugated antibody against CD80, biotin-conjugated antibody against CD86 and phycoerythrin-conjugated-I-A^d antibody (BD Pharmingen, San Diego, CA, USA). Mouse IgG was used as an isotype control (BD Pharmingen). For staining, cells were washed twice with phosphate-buffered saline (PBS) and were incubated in PBS containing 3% bovine serum albumin (Sigma, St. Louis, MO, USA) and 0.1% NaN₃ (FACS buffer; Sigma) and the appropriate concentration of labeled for 1 h at 4°C. After washing with FACS buffer, the expression-positive cells were measured using a FACSCalibur flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA) by 3-color analysis and analyzed with CELLQuest software (BD Bioscience).