disease status: renal cell carcinoma (RCC) gender: male age: 62 years cell type: CD8+ T lymphocytes time point: day 35 of culture
Treatment protocol
For MLTC experiments, PBMCs from RCC patients or HLA-matched healthy donors were co-cultured in 24-well plates (Costar, Corning, USA) at 10^6 cells/well with irradiated RCC stimulator cells (10^5 cells/well) in AIM-V medium (Life Technologies, Invitrogen, Italy) supplemented with 10% heat-inactivated pooled human serum (Sigma (medium Mb)). Recombinant human IL-2 was added on day 3 (250 IU/mL; Proleukin, Chiron, and Emeryville, CA). Responder lymphocytes were restimulated weekly with 10^5 irradiated tumor cells in IL-2-containing medium for 2 weeks prior to responder CD8+ T cells isolation. Isolated CD8+ T cells were cultured for an additional 2 weeks. On day 0 and day 35 of culture, CD8+ T cells were analyzed for gene expression profiles.
Growth protocol
CD8+ T cells were isolated from the PBMCs of autologous and allogeneic normal, healthy donors by negative selection using specific MACS magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) on MiniMACS columns, following the manufacturer's protocol and subsequently assessed for their functional and phenotypic profiles.
The One-Cycle cDNA Synthesis Kit (Affymetrix) was used. Starting from 1.5 micrograms of total RNA, we performed the in vitro transcription (IVT) reaction for complementary RNA (cRNA) amplification and biotin labeling, according to the manufacturer’s instructions. The biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip brand expression arrays.
Hybridization protocol
Total RNA was hybridized on to the GeneChip Human Genome U133A Array (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions.
Scan protocol
We used the default settings of GCOS software to scan the chips.
Description
CD8+_TC_T35_1 Male patient, 62 years old, affected by renal cell carcinoma, G1, T3aN0M0.
Data processing
We used the default settings of Affymetrix Microarray Suite software version 5 (MAS 5.0; Affymetrix) to calculate scaled gene expression values. R 2.0.1 statistical software was used to perform the normalization of the data according to R. A. Irizarry et al., Biostatistics, 2003. Differentially expressed genes were identified using GeneSpring GX software (Agilent, Palo Alto, CA), by applying a moderated t-test with Storey with bootstrapping multiple testing correction. We selected the differentially expressed genes using a q-value cut-off of 0.05.
