Mice. Male C3H (H-2k, I-Ak) and C57BL/10J (B10; H-2b, I-Ab) mice, 8 to 10-week-old were purchased from the Animal Laboratory of National Institute Taipei, Taiwan, and maintained in the pathogen-free facility of Change-Gung Memorial Hospital. Some B10 mice were maintained to 12 months old to be employed as aged mice. Experimental use of these mice was approved by Animal Care Committee of Chang-Gung Memorial Hospital (No. 105-0293C).
Bone marrow-derived dendritic cells propagation. To propagate bone marrow-derived dendritic cells (DC), bone marrow cells were harvested from femurs and tibias of C3H or B10 mice, and cultured in 24-well plates (2 × 106 cells/well) in 1 ml of RPMI-1640 medium (Life Technologies, Graitherburg, MD), supplemented with 4 ng/ml recombinated mouse GM-CSF (R&D Systems Inc. Minneapolis, MN) and 1000u/ml mouse IL-4 (R&D Systems Inc. Minneapolis, MN). The propagation of large numbers of DC from mouse bone marrow with minor modification herein was similar to that described initially by Inaba et al.(26) The DC progenitor clusters were selected after 2 days of culture by gently swirling the plates and depleting the non-adherent granulocytes. Half of the cultural medium was refreshed. The DC was harvested after 5 days of culture.
Enriched T-cells. Enriched T-cells were obtained from splenocytes passing through nylon wool column. Nylon wool column was prepared by packing 0.5 gram nylon wool in a 10-c.c. syringe. The column was equilibrated by running cultural medium and incubated at 37oC for an hour before splenocytes were loaded into nylon wool column. Splenocyte-loaded nylon wool column was incubated at 37oC for one hour. Then, the non-adherent cells, enriched T-cells, were collected.
Cell lines. Murine cancer cell lines were employed in in vitro studies. R1.1, P815, Yac-1 cell lines were obtained from the Cell Collection and Research Center (CCRC, Hsin-Chu, Taiwan), maintained in antibiotic-free Dulbecco‘s minimal essential Medium (DMEM, Life Technologies, Gaithersburg, MD) and supplemented with 10% v/v fetal bovine serum.
Phenotypes of immune cells. The phenotypes of the cells were classified by the expression of surface molecules. After the surface molecules were stained directly by a panel of fluorescenced monoclonal antibodies, the cell surface molecular expressions of DC, myeloid-derived suppressor cells (MDSC) and regulatory T-cells (Treg) were analyzed by cytofluorography employing a Beckman Coulter NAVIOS flow cytometer (Beckman Coulter Co., Indianapolis, IN).
Quantitative T-cell proliferation. The allogeneic stimulatory capacities of DC were determined via an one-way mixed lymphocyte reaction (MLR) employing colorimetric tetrazolium (MTT) assay.(27, 28) Enriched T-cells were stimulated by irradiated DC triplicatedly in 96-well plates for 3 days. In the last 4 hours of the procedure, sterilized MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/ml, Sigma, St Louis, MO) was added to each well (10 ul/well) to produce blue formazan. Upon termination of MLR, acid-isopropanol (0.04N HCL-isopropanol, 100 ul/well) was added to each well and mixed thoroughly to dissolve the blue crystals. After 10 minutes at room temperature to ensure that all crystals were dissolved, the plates were read on a Dynatech MR580, microelisa reader, employing a test wavelength of 570 nm to measure the optical densities (OD) of formazan formation.
Cell-mediated cytotoxicity. T-cells activated by DC for three days were applied as effector cells. P815 (H-2d), R1.1 (H-2k), and YAC-1 (natural killer sensitivity) cells were applied as targets. Cell-mediated cytotoxicity was performed at various effector-to-target ratio triplicatedly in 96-well plates, and assessed by flow cytometry. To assess the cell-mediated cytotoxicity by flow cytometry, the targets were labeled with PKH-26 and 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) according to the manufacturer’s instruction (Sigma. St. Louis, MO). Briefly, target cells, 1 × 106/ml, were stained with PKH-26 (final concentration of 2.5 × 10− 6 M) at room temperature for 5 minutes and washed by PBS once. Then, these PKH-26 labeled target cells were stained with CFSE (final concentration of 5 × 10− 6 M). After 4 hours of cytotoxic reaction, all cells were collected and analyzed by flow cytometry. The target cells were identified by PKH-26 positive gate. The cells contained within the PKH-26 and CFSEhigh double positive gate represent viable target cells. Therefore, percent survival of target cells = (CFSEhigh percent of test well / CFSEhigh percent of spontaneous release) x 100%. Percent specific cytotoxicity= (1-% survival) x100%.
Cytokine measurement. The serum of young and old mice was collected. The product of cytokines was measured by enzyme-linked immunoabsorbent assay. The procedure was conducted as the instructions of producers. (PharMingen, San Diego, CA).
Skin transplantation. Under adequate anesthesia, an incision was made on the flank of B10 mice. A 1 × 1 cm skin flap taken from C3H mice was attached to the flank of B10 mice and fixed to adjacent skin with 3 − 0 Dexon sutures. The skin flap was protected by circulated gauge for 3 days. The skin flap were observed very 2–3 days. Rejection was diagnosed when the skin grafts were fully detached.
Statistical analysis. Unpaired Student’s t-test was used to analyze continuous variables. Categorical variables were analyzed by either Chi-square test or Fisher’s exact test. All pairwise multiple comparisons were done by Holm-Sidak method. The survival rates were calculated using the Kaplan-Meier method. The statistical analyses were all performed with SigmaPlot 12.3 for Window software (Systat Software, Inc., San Jose, CA, USA). P < 0.05 was considered statistically significant.