Differential gene expression microarray analysis of Tregs and Teff cells expanded by TCR-dependent Vs independent methods

CD4+ cells from Foxp3.eGFP mice containing Foxp3- Teff and Foxp3+ Treg cells were treated with anti-CD3/CD28 monoclonal antibodies or soluble OX40L and JAG1 for 3 days to induce TCR-dependent vs TCR-independent Treg proliferation. Untreated fresh CD4+ T-cells used as control. Post treatment T-cell proliferation was confirmed by Cell Trace violet dilution and Foxp3+ (Treg) and Foxp3-(Teff) were sorted. Differential gene expression profiling between Tregs and Teff cells among control, anti-CD3/CD28 and OX40L-JAG1 treated cultured was performed using affymetrix mouse gene 2.0 ST micro array. We used microarrays to detail the global programme of gene expression underlying Treg proliferation and identified distinct classes of up-regulated genes during this process under different methods of expansion. Sorted Foxp3- Teff and Foxp3+ Treg cells from fresh (control), anti-CD3/CD28 monoclonal antibodies or soluble OX40L and JAG1 treated cultures were used for RNA extraction followed by cDNA construction. Differential gene expression profiling between Tregs and Teff cells among control, anti-CD3/CD28 and OX40L-JAG1 treated cultured was performed using affymetrix mouse gene 2.0 ST micro array.