Gene expression profiles of LAG-3 positive and negative regulatory T cells in human healthy donors

Regulatory T cells (Tregs) are known to exert a critical role in self tolerance. This subset of cells is also implicated in immune evasion mechanisms operated by cancer cells. In humans, it is known that Tregs are a heterogeneous population that includes different subsets of T cells with different phenotype, differentiation state and presumably exerting their suppressor activity with different modalities. We have provided data showing that a subset of human CD4+ Tregs (CD25hiFoxp3+) expressing the Lymphocytes Activation Gene 3 (LAG-3) molecule is amplified in melanoma invaded lymph nodes as compared with their tumor free counterpart, and these cells are endowed with enhanced suppressive functions (Camisaschi et al., J Immunol 2010). To gain insights into the phenotype, functional features and specificity of this population of Tregs expressing LAG-3, we performed microarrays to obtain gene expression profiles of LAG-3+ and LAG-3- Tregs. Tregs were purified from healthy donor PBMCs by immunomagnetic cell separation at a ≥ 96% purity and expanded in vitro according to a previous protocol (Camisaschi et al., J Immunol 2010). After 10 days of in vitro activation, cells were stained with anti-LAG-3 mAb (Clone 17b4; Apothech) and sorted using a FACSAria (BD Biosciences). From each healthy donor PBMCs we obtained 2 independent preparations of Tregs, with a total of 11 couples of LAG-3+ and LAG-3- Tregs analyzable. Total RNA was extracted with Agencourt RNA advance cell V2 system (Beckman Coulter), which allows the high recovery of pure RNA from few cells. Isolated RNA was quantified with Nanodrop (ThermoScientific) and its quality analyzed using the Agilent 2100 bioanalyzer for the RNA integrity Number (RIN) calculation. Gene expression profiles were then generated using Illumina HumanHT12_v4 WG-DASL (Illumina).