Cell Isolation and Culture
UCB and PB were obtained from healthy donors. Mononuclear cells (MNC) were prepared by density gradient centrifugation using lymphocyte separation medium (Mediatech, Inc). T cells were isolated using CD3 microbeads (Miltenyi Biotec, Auburn, CA). Purity was assessed by flow cytometry and was >97% (not shown). Isolated T cells were cultured at 1 × 106 cells/ml in Ham's F12 + DMEM (1:2 ratio) with 10% male AB-human serum (Seracare Life Sciences, Oceanside, CA), ethanolamine (50 μmol/L), ascorbic acid (20 mg/L), 5μg/L sodium selenite (Na2SeO3), β-mercaptoethanol (24μmol/L), and penicillin (100U/ml)-streptomycin (100U/ml). Recombinant cytokines IL-2 (Chiron, Emeryville, CA), IL-15 (PeproTech Inc., Rocky Hill, NJ), IL-4 (R&D systems, Minneapolis, MN) and IL-7 (gift from National Institute of Health) were added as indicated.
Flow cytometry analysis
Prior to staining, cells were washed in PBS with 2% fetal bovine serum and 0.2% NaN3 and then stained with monoclonal antibodies (mAb) for 30 minutes at 4°C. Samples were analyzed on a FACScalibur using CellQuest software. Flowjo 5.7.0 software was used for data analysis. For intracellular staining, the cells were fixed and permeabilized with BD Cytofix/Cytoperm™ solution and washed with BD Perm/Wash™ buffer and stained according to manufactures specifications. The following mouse anti-human antibodies were used: CD3-FITC, CD4-FITC, CD8-FITC, CD62L-PE, CD4-APC, CD8-APC, CD56-APC (BD Biosciences, San Jose CA), NKp30-PE, NKp44-PE, NKp46-PE and TCRζ-PE (Beckman Coulter, Inc., Miami, FL), CCR7-APC (R&D systems, Minneapolis, MN), CD45RA-FITC (Ebioscience, San Diego, CA).
Cell proliferation assay
Freshly isolated UCB T cells were washed and resuspended in prewarmed PBS at 5×106/ml containing 5μM carboxyfluorescein diacetate, succinimidyl ester (CFSE) (Molecular Probes Inc., Eugene, OR) and incubated for 15 minutes at 37°C. Following this, cells were washed again with prewarmed PBS, then resuspended in prewarmed medium and incubated for another 30 minutes and again washed prior to cell culture.
24-well plates were coated with mAbs against human NKp30 (clone 210847), NKp44 (clone 253415), NKp46 (clone 195314) (R&D systems, Minneapolis, MN), CD3 (OKT3) or isotype control (Sigma, St Louis, MO) all at 5μg/ml in PBS for 4 hours at 37°C. Plates were washed and UCB or PB T cells at day 14 of culture were added at 0.3×106 in 300 μl medium. Anti-CD107a-FITC (BD Biosciences, San Jose, CA) was added to the culture prior to incubation. After 8 hours of incubation at 37°C 5% CO2, cells were harvested, and analyzed for CD107a expression.
96-well plates were coated with antibodies against human NKp30, NKp44, NKp46, CD3 (OKT3) mAbs or isotype control antibody at 5μg/ml in PBS for 4 hours at 37 °C. Plates were washed and cells were added (0.2×106 cells in 200 μl medium). After 16 hours of incubation at 37°C 5% CO2, cells were harvested and washed with cold PBS, resuspended in 1X Binding Buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and stained with Annexin V-FITC (BD Biosciences, San Jose CA) for 15 minutes at room temperature and and PI (Sigma, St Louis, MO) was added. Cells were analyzed by flow cytometry.
0.2 ×106 cells in 200μl medium were stimulated with plate-bound Ab coated 96-well plates for 24 hours. Supernatants were harvested and assayed for IFN-γ production by ELISA (R&D systems, Minneapolis, MN) according to the protocol by the manufacturer.
Redirected killing assay
The FcγR-positive cell line, P815 (murine mastocytoma) was used as a target for redirected killing assays. P815 cells were labeled with 51Cr (DuPont-NEN, Boston, MA) by incubating 1 × 106 cells in 300 μCi (11.1 MBq) 51Cr for 1 hours at 37°C 5% CO2. Cells were then washed 3 times with PBS, resuspended in culture medium, and added in triplicate to 96-well plates at 104 cells/well. Effector cells were added at the specified ratios and incubated at 37°C 5% CO2. Effector cells were preincubated in PBS in the presence of soluble antibodies (5 μg/1×106 cells in 100μL PBS) for 30 minutes and washed once with PBS prior to coincubation with tumor cell targets. After 4 hours, 100μl of supernatant was counted using a γ counter. The percentage of specific lysis was calculated using the following equation: % specific 51Cr release = 100 × [(test release) - (spontaneous release)]/[(maximal release) - (spontaneous release)].
Polymerase Chain Reaction
Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Santa Clarita, CA) and reverse transcribed to cDNA (iScript, Bio-Rad, Hercules, CA). 2ng of cDNA was amplified using recombinant Taq Polymerase (Gibco BRL) for 30 cycles. The following primers were used: DAP12 forward CCGCA AAGAC CTGTA CGCCA, reverse TGGAC TTGGG AGAGG ACTGG; FcεRIγ. forward ATGAT TCCAG CAGTG GTCTT G, reverse GTGCT CAGGC CCGTG TAAA; CD3ζ forward GCACAG TTGCC GATTA CAGA, reverse GGTTC TTCCT TCTCG GCTTT. PCR products of DAP12 (650bp), CD3ζ (273bp) and FcεRIγ (209bp) were resolved on 2% agarose gel and bands were visualized with ethidium bromide.
UCB T cells at day 14 of culture were lysed using freshly prepared lysis buffer (10mMTris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% deoxycholate, 0.1% SDS [sodium dodecyl sulfate], protease inhibitor cocktail (complete protease inhibitor; Roche, Indianapolis, IN), PMSF, and 1mM Na3VO4. Samples were separated on a 15% polyacrylamide gel and transferred to a PVDF membrane. Membranes were blocked with 5% nonfat dry milk and probed with anti-actin (SantaCruz Biotechnology, Santa Cruz, CA), anti-FcεRIγ (Upstate USA Inc., Charlottesville, VA) or anti-DAP12 (generous gift from Dr. Paul Leibson, Rochester, MN) followed by a species-specific secondary horseradish peroxidase (HRP)-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Blots were developed using chemiluminescence (Pierce, Rockland, IL).
Statistical analyses were performed with Statistical Analysis System statistical software version 9.1 (SAS Institute, Inc., Cary, NC). For non-normally distributed data the Mann-Whitney rank sum test was used in the evaluation of the statistical differences between UCB and PB T cells, CD4+ vs. CD8+ and CD56- vs. CD56+ subpopulations. One-way ANOVA was used when the data was approximately normally distributed with the general linear models procedure (PROC GLM). Adjustments for multiple comparisons were done with the Tukey's method. Groups with values of p ≤ 0.05 were considered to be statistically different.