normalization data transformation protocol
Gene expression profiling data was extracted from the Affymetrix Microarray Suite 5.0 (MAS 5.0) software and R/Bioconductor package was used for QC of microarray data. The data was processed using MAS5 (Affymetrix) with a trimmed mean value scaled to 100. Fresh samples and expanded samples were then further normalized separately by lowess method, using each group median as the respective baseline reference and a fraction parameter of 10%. A filter was applied to remove the poorly detected probe sets. To be retained for further analysis, a probe set had to be expressed at an average value of 20 or more and have a detection p-value less than 0.065 in 80% of the samples of any one group. This filter resulted in 23,805 retained probe sets. Lowess normalized expression signal is provided in the sample data table for each sample replicate with MAS5 detection p-value. ID_REF = VALUE = Lowess normalized; MAS5 detection p-value ABS_CALL = DETECTION P-VALUE =
array scanning protocol
Scanning protocol was performed according to the protocol described in the GeneChip Expression analysis manual (Affymetrix).
GeneChip® Human Genome U133 Plus 2.0 arrays (Affymetrix) were hybridized by Pfizer to 5 μg labeled, amplified cDNA, washed, stained, and scanned according to the protocol described in the GeneChip Expression analysis manual (Affymetrix).
For each sample, 25 ng total RNA were amplified using the Ovation® Pico WTA System (NuGen) and labeled with Encore Biotin Module V2 (NuGen).
Human Treg and Tconv were FACS isolated from blood samples of five healthy subjects and divided in two groups: first set was treated immediately, second set was subjected to a 14 day expansion protocol before treatment.
nucleic acid extraction protocol
Total RNA was extracted by Pfizer using the RNeasy Micro Kit (Qiagen) including on-column DNase (Qiagen) digestion as described by the manufacturer's protocol. RNA quantification was done using a Nanodrop Spectrophotometer 2000 (Thermo Fisher Scientific) and the quality was confirmed on an Agilent 2100 bioanalyzer (Agilent Technologies).
sample treatment protocol
Both fresh and expanded cells were stimulated for 4 hours with PMA/ionomycin and labeled with the IFNg cytokine capture kit (Miltenyi Biotech) followed by FACS isolation of IFNg- and IFNg+ populations, which were pelleted and flash frozen before RNA isolation and processing.