Human peripheral blood samples from self-reported healthy donors under 40 years of age were acquired by venipuncture after informed consent, and in accordance with the UCLA IRB. After centrifugation, the layer of peripheral blood mononuclear cells (PBMC) was carefully removed and washed twice in “complete RPMI” (10% fetal bovine serum, 10 mM Hepes, 2 mM glutamine, 50 IU/mL penicillin/streptomycin). The EasySep CD8 enrichment kit (Stemcell) was used to isolate CD8+ T cells by negative selection, and purity of the cells was verified by flow cytometry. Cell cultures were established as described previously [18
]. Briefly, CD8 T cells were exposed to T cell activation microbeads (anti-CD2/3/28, Miltenyi) with 10 μl microbead cocktail added for every 1
cells, and stimulation repeated every 14 days. Cultures were supplemented with recombinant IL-2 (20 U/mL). Every 3–4 days, viable cell concentration was determined by trypan blue exclusion, and when the concentration reached ≥8
/ml, cells were subcultivated to a density of 5
cells/ml. Population doublings (PD) were determined according to the formula:
CD28 Gene Transduction
The pBABE retroviral supernatants containing either CD28-puromycin, or empty vector-puromycin were provided by the UCLA Vectorcore (Los Angeles, CA). On day 4 following the first stimulation, 1
CD8 T cells were isolated and the media was removed by centrifugation. Cells were resuspended in either CD28 or empty vector viral supernatants, supplemented with 8 μg/ml polybrene, and centrifuged at 1,800×g
for 3 h. Cells were incubated in the viral supernatant overnight, at which point they were washed with complete RPMI 1640. A second round of transduction was performed after 24 h to ensure optimal gene transfer. Transduced cells were selected with pre-titrated concentration of puromycin (Sigma-Aldrich, St. Louis, MO) for 3 days. Transduction efficiency ranged from 5% to 15% and gene expression remained stable for the life of the culture.
Flow Cytometry Surface expression of CD4, CD28, CD8, and CD3 was examined by immunostaining and flow cytometry. Cells were incubated with FITC-conjugated anti-CD4, PE-conjugated anti-CD28, allophycocyanin (APC)-conjugated anti-CD3, and PerCp-conjugated anti-CD8 (BD Biosciences, San Jose, CA) at 4°C for 20 min, washed, and fixed in PBS containing 1% paraformaldehyde. Parallel samples were incubated with Ig isotype control antibody or secondary Abs (BD Biosciences). All samples were analyzed on a FACS Calibur flow cytometer (Beckton Dickson). Fluorescence data from at least 25,000 cells were acquired. Analysis of data was performed using Cell Quest Pro (BD Biosciences).
Real-Time Quantitative Polymerase Chain Reaction RNA was isolated using the Qiagen RNAeasy kit (Qiagen, Valencia, CA), and quantified using the Quant-iT Ribogreen RNA Assay Kit (Molecular Probes). cDNAs were synthesized with the iScript cDNA synthesis kit (Bio-Rad) using 500 ng RNA. Real-time quantitative polymerase chain reaction assays were performed using the iQ SYBER Green SuperMix and IQCycler (Bio-Rad). GAPDH was used as an internal control. The sequences were designed with the aid of Beacon Designer software and synthesized at Integrated DNA Technologies, Inc.
CTLA-4 sense—TGAGTTGACCTTCCTAGATGATTCC, anti-sense—CTGGGTTCCGTTGCCTATGC; IL-2 sense—TCACCAGGATGCTCACATTTAAGTTTTAC, anti-sense—TTCCTCCAGAGGTTTGAGTTCTTCTTC; interferon gamma (IFN-γ)—GAGTGTGGAGACCATCAAGGAAGAC, anti-sense—GCGTTGGACATTCAAGTCAGTTACC; adenosine deaminase (ADA) sense—CAGGCTAACTACTCGCTCAACACAG, anti-sense—TGTCCCGTTTGGTCATCTGGTAATC; GAPDH sense—GGTCATGAGTCCTTCCACGATACCA, anti-sense—CCTCAAGATCATCAGCAATGCCTCCT.
Samples were run in triplicate in a 96-well plate at the following settings: 95º C for 15 s, 61°C for 30 s, 72°C for 30 s, using single fluorescence measurement.
Cytokine Measurements Culture supernatants were harvested 96 h post-stimulation and analyzed for IL-6 and TNF-α in an ELISA Ready-Set-Go (eBioscience, San Diego, CA). All measurements were performed in triplicate wells and in accordance to manufacturer's recommendations.
Telomerase Activity Measurements
Telomerase activity was determined using the telomere repeat amplification protocol (TRAP), as previously described in Saldanha et al. [19
], with minor modifications.
p21 Quantification p21 concentration was determined through the use of a p21 quantification assay (Invitrogen, San Diego, CA). All measurements were performed in triplicate wells and in accordance to manufacturer's recommendations.
Statistical Analysis Mean values and standard deviation were calculated for each time-point. Significance was established by using a two-tail Student's t test, and a p value of <0.05 is considered significant.