Cell culture and reagents
Under the approval of the Institutional Animal Care and Use Committee of Seoul National University Biomedical Research Institute, spleens and corneas were collected from 8-week-old Fut1 KO mice (C57BL/6 background, B6.129-Fut1tm1Sdo/J; Jackson laboratory, Bar Harbor, ME) and C57BL/6 mice (Jackson laboratory), respectively. The confirmation ofFut1 gene knockdown in Fut1 KO mice was performed following the genotyping protocols provided by Jackson laboratory (10).
Splenocytes were prepared by mashing the spleen, and Fut1 gene knockdown was further confirmed in the cells using real-time RT-PCR (Fig. S1 ). CD4 cells were isolated from splenocytes using mouse CD4 microbeads (CD4 (L3T4) MicroBeads, Miltenyi Biotec, Bergisch Gladbach, Germany). CD4 cells were then cocultured with splenocytes at a ratio of 1 : 1 on plates coated with 5 µg/mL mouse anti-CD3 and anti-CD28 mAbs (BD pharmingen, San Jose, CA) in RPMI1640 media (Welgene, Daegu, Korea) containing 10% fetal bovine serum (Gibco/Thermo Fisher Scientific, Waltham, MA) for 5 d.
Recombinant mouse thrombospondin-1 (THBS1) protein (5 µg/mL) (R&D Systems, Minneapolis MN) was added to some cocultures.