Microarray profiling of in vitro expanded FRCs cultured alone or with activated T cells

To investigate whether FRCs express molecules capable of promoting the functions of activated T cells, we expanded FRCs from primary lymph node stromal cell (LNSC) cultures as previously described (Lukacs-Kornek et al., Nature Immunology, 2011), and then cultured them alone or with splenocytes activated with soluble antibody (0.25μg/ml) against CD3 (anti-CD3) and anti-CD28 for 16 hours. FRCs co-cultured with activated T cells upregulated expression of genes encoding molecules known to dampen T cell function such as Arg1, CD274 and Nos2. However, in response to activated T cells, FRCs also upregulated molecules with immunostimulatory capabilities such as Icosl, Cd40 and Il6. In vitro expanded FRCs cultured alone (2 biological replicates) or with splenocytes activated with soluble antibody (0.25μg/ml) against CD3 (anti-CD3) and anti-CD28 (2 biological replicates) for 16 hours. The FRCs were sorted as previously described (Malhotra et al., Nature Immunology, 2012) and processed for microarray analysis.