Supplementary Materials1. postcapillary venules we report that both PSGL-1 and CD43, but not CD44, function as E-selectin ligands for Th17 cells. Moreover, our results indicate that CD43 functions as a major E-selectin ligand for Th17 cells independent of PSGL-1 and uniquely participates in Th17 cell recruitment to the dermal air pouch model and to the spinal cord in Experimental Autoimmune Encephalomyelitis (EAE), contrary to Th1 cells. Using competitive rolling assays and confocal intravital microscopy, we provide compelling evidence that CD43 mediates Th17 cell rolling to the activated vascular endothelium in an E-selectin dependent manner. Further examination of triple knock out (TKO) CD43?/?PSGL-1?/?CD44?/? mice suggest that there are most likely no additional glycoprotein ligands that function as E-selectin ligands in Th17 cells. Our data position CD43 as the major E-selectin ligand responsible for Th17 cell rolling on activated vasculature and recruitment during inflammation and autoimmunity. Materials and Methods Reagents Recombinant mouse IL-23, E-selectin and P-selectin Fc-chimeras were from R&D Systems (Minneapolis, MN). Recombinant mouse IL-12, IL-2, IL-6, TNF-, recombinant human TGF-, and the following antibodies to mouse cytokines and DIAPH1 adhesion molecules: ANA-12 IL-4 (clone 11B11), IFN (clone XMG 1.2), IL-2 (clone JES6-1A12), CD4 (clone GK 1.5), CD3 (clone145-2C11), CD28 (clone 37.51), IL-17A (clone 2C11-18H10.1), CD43 activation-associated glycoform (clone 1B11), and CD44 (clone IM7) are all from Biolegend (San Diego, CA). Anti- PSGL-1 and mouse TNF- were purchased from BD-Pharmingen (San Jose, CA), and carrier free CCL20 from Peprotech (Rocky Hill, NJ). PMA and ionomycin were from SIGMA (St. Louis, MO). Secondary Abs coupled to alkaline phosphatase were from Promega (Madison, WI). Vibrant CFSE and Alexa 680 were from Life Technologies (Carlsbad, CA). Myelin Oligodendrocyte glycoprotein was purchased from Anaspec (Fremont, CA) and Pertussis Toxin was purchased from List Biological Laboratories (Campbell, CA). Anti-E-selectin (clone 9A9) antibody was generously provided by Dr. F. William Luscinskas (Brigham and Women’s Hospital, Boston, MA) and IgG control was from Biolegend (San Diego, CA). Mice All mice used were bred in the pathogen free facility at Tufts University School of Medicine, Ziskind Building, in accordance with the guidelines of the committee of Animal research at Tufts University School of Medicine, Tufts Medical Center and the NIH Animal research guidelines. C57Bl/6 (WT) mice were purchased from Jackson Laboratory (Bar Harbor, Maine) or used as littermates from heterozygous crosses. Double knock out (DKO) PSGL-1?/?CD44?/? and PSGL-1?/?CD43?/? mice were obtained from Dr. Rodger McEver (OMRF, Oklahoma City, OK). CD43 (CD43?/?) and PSGL-1 (PSGL-1?/?) were generated ANA-12 from inter-crosses of PSGL-1?/?CD43?/? DKO and PSGL-1?/?CD44?/? DKO mice with C57Bl/6 (WT) mice. Triple knock out (TKO) PSGL-1?/?CD43?/?CD44?/? were generated by crossing ANA-12 PSGL-1?/?CD43?/? DKO mice with PSGL-1?/?CD44?/? DKO mice. CD44?/? mice were purchased from Jackson Laboratories. Mice were sacrificed at 7-12 weeks of age for harvest of na?ve ANA-12 T cells, or used between 8-10 weeks of age for air pouch and intravital microscopy experiments. The genotypes were determined by PCR, and null mutations were also confirmed by FACS analysis of spleen cells. All deficient mice in this study were viable and fertile as previously described (13,14,23). Preparation of effector T cells CD4+ cells were isolated from spleen and lymph node cell suspensions of WT or genetically deficient mice using positive selection by immunomagnetic beads (Invitrogen, Carlsbad, CA). Th1 cells were derived from the na?ve T cells by anti-CD3 and anti-CD28 stimulation in the presence of IL-12 and IFN-, as previously described (8). To achieve Th17 differentiation, na?ve T cell were stimulated with anti-CD3 in the presence of human TGF- (3ng/ml), mouse IL-6 (30ng/ml), ANA-12 mouse IL-23 (20ng/ml), plus anti-IFN- (10ug/ml), anti-IL-4 (10g ml), and anti-IL-2 (10g/ml) mAb. On day 3, Th1 and Th17 cultures were.