Lentiviral Knockdown of PSGL-1 in T Cells

Lentiviral vectors carrying shRNAs against PSGL-1 were purchased from Sigma-Aldrich. Virion particles were assembled by cotransfecting HEK293T cells with pHCMV-G, pCMV-ΔR8.2, and lentiviral vectors. Supernatant was collected and used to transduce Jurkat T cells. Cells were selected in puromycin. PSGL-1 knockdown was confirmed by surface staining with an anti–PSGL-1 antibody (KPL-1; BD Pharmingen). Lentiviral vector-mediated ShRNA knockdown of PSGL-1 in primary CD4 T cells was performed as described previously (46 (link)). In brief, blood resting CD4 T cells were purified by negative depletion, transiently stimulated with anti-CD3/CD28 beads, and then transduced with the lentiviral vectors carrying shRNAs against PSGL-1. Additional details of shRNA knockdown and analysis are provided in SI Appendix, Materials and Methods.