Isolation and Activation of Mouse T Cells

Primary T cells were isolated from spleen and lymph node cell suspensions as previously described (29 (link)) using a CD4 isolation kit (Stemcell technologies). For T cell activation studies, 1 × 10⁵ cells were cultured and activated as previously described (30 (link)) using plate bound anti-CD3 (145-2C11, Biolegend) and soluble anti-CD28 (37.51, Biolegend) at concentrations of 2 and 1 μg/ml, respectively. Bone marrow derived dendritic cells were differentiated as previously described (28 (link)) Briefly, 10 ×10⁶ bone marrow cells were cultured in complete T cell medium (RMPI 1640, 10% FBS, 100 U/ml Streptomycin, 50 μM βme, 2 mM glutamine, and 1 mM sodium pyruvate) for 7 days in the presence of 10 ng/ml mouse GM-CSF and 2 ng/ml IL-4 (Peprotech). On day 3 and 5 fresh medium with cytokines was added. For co-culture experiments 2 ×10⁴ BMDCs were incubated with 1 μg/ml anti-CD3 antibody (145-2C11, Biolegend) and 1 ×10⁵ naïve CD4 T cells.