Positive bead selection

Primary CD4⁺ T cells utilized in these studies were freshly isolated from healthy donor PBMCs and separated by Lymphocyte separation medium (MP Biomedicals, Santa Clara, CA). Purified CD4⁺ T cells, were obtained by negative selection (Stem Cell Technologies, Vancouver, Canada) to > 95% purity, as determined by flow cytometry. CD4⁺ T cells were cultured in complete RPMI 1640 medium with 2% L-glutamine-penicillin-streptomycin (Gibco Laboratories, Gaithersburg, MD) and 10% FBS (Gibco Laboratories, Gaithersburg, MD). Where designated, naïve CD45RO⁻/CD4⁺ T cell cultures were generated by CD45RO positive selection of bulk CD4⁺ T cells with Macs beads (Miltenyi Biotec, San Diego, CA) resulting in CD45RO⁻/CD4⁺ T cells at >96% purity. Purity of CD45RO⁻/CD4⁺ T cell cultures was validated in control experiments that included anti CD3 and retinoic acid and no cellular proliferation was observed (Supplementary Figure 8) indicating the contamination by antigen presenting cells did not contribute to proliferative responses. CD8⁺ T cells were depleted in a similar manner, by positive bead selection (Stem Cell Technologies, Vancouver, Canada) to 97% purity.