A r t i c l e A subset of these antigen-pulsed BMDCs were also surface and absence of CS and KF as before. On day 4, the cells were stained with FITC-conjugated MHCII mAb to measure stimulated for 12 h with either 50 µg/ml PSA or 15 nM Pam- increases in plasma membrane–localized MHCII concom-i 3Cysk4 and culture supernatants assayed for IL-8 by ELISA tant with DC maturation (Fig. 5 D). For all antigen combina- (Fig. 5 G).We found robust IL-8 production in response to tions, we found equal increases in surface MHCII localization both stimuli and no change in IL-8 production in CS- or over no antigen controls, thus neither CS nor KF leads to a KF-treated cells. defect in DC maturation. In fact, KF treatment appears to Collectively, these data demonstrate that alterations in have increased the MHCII surface concentration above no- N-glycan formation in APCs does not affect conventional inhibitor controls. protein or peptide antigen–mediated T cell activation, but sig- The remaining antigen-pulsed BMDCs were cultured nificantly reduces T cell activation by three different GlyAgs. with fresh CD4+ T cells from either WT C57BL/6 (PSA, Sp1, and capsule assays) or OT-II (DQ-OVA and OVAp assays) Native complex N-glycans are required for GlyAg-mediated mice to determine the capacity of the BMDCs loaded with responses in vivo antigen to induce T cell activation.At day 3 for DQ-OVA and Reduction of MHCII binding and presentation of GlyAg OVAp assays (Fig. 5 E) or day 4 for the GlyAg assays (Fig. 5 F), negatively effects in vitro T cell recognition and activation.To culture supernatants were assessed for IFN- production by determine the in vivo effect of the reduction of native com- ELISA.We found no significant difference in OT-II T cell ac- plex N-glycans in C57BL/6 mice on GlyAg-mediated r-e tivation between treatment groups when stimulated with in- sponses, animals were injected with 250 µg of KF daily for 1, tact protein antigen DQ-OVA or preprocessed OVAp-pulsed 2, or 3 d. On day 4, the animals were sacrificed and the bulk BMDCs, yet all three GlyAg-mediated T cell activation assays splenocytes were assessed by lectin (PHA-L) staining and flow were significantly reduced in assays containing BMDCs cytometry to test for drug effectiveness (Fig. 6 A). We found grown in CS or KF. that by day 3, essentially all native complex N-glycans were Previous findings have reported that GlyAg-mediated missing from the surface of all splenocytes. T cell activation requires signaling through TLR2 (Wang et al., To assess the effect of the 3-d KF treatment regimen on 2006). To rule out TLR2 as the cause of the lack of T cell GlyAg-driven immune responses, we challenged the KF and activation mediated by GlyAg-pulsed and CS- or KF-grown PBS vehicle–treated (UNT) mice with PSA.After 6 d,the spleens BMDCs, we cultured TLR2+ HEK293 for 3 d in the presence were removed and the cells stimulated with PMA/ionomycin Figure 6. Native complex N-glycans are required for PSA-driven immune responses in vivo. (A) WT C57BL/6 animals (n = 8) were treated daily for 1, 2, or 3 d with 250 µg of KF, and then sacrificed to harvest splenocytes. Cells were stained for native complex N-glycans using PHA-L. Representative histogram shown. (B) WT mice (n = 3 per group) were treated with KF or PBS for 3 d, as in A. Animals were then sensitized with 100 µg PSA. Splenocytes were harvested 6 d later, stained for IFN- and CD4, and analyzed by flow cytometry. (C) KF- or PBS-pretreated mice (n = 6) were sensitized with 100 µg OVA and sterile cecal content adjuvant. 4 d later, CD4+ T cells were isolated and restimulated ex vivo with normally glycosylated APCs and OVA (n = 6). (D) Animals (n = 6) were treated as in C, only with 100 µg of PSA rather than OVA. The PSA-specific recall (n = 18) was measured after ex vivo restimula- tion with normally glycosylated APCs and PSA. (E) Animals (n = 6) were treated as in D, only with the additional adoptive transfer of 5 × 106 fresh nor- mally glycosylated APCs at the time of PSA injection (n = 12). For panels C–E, data were normalized to the antigen-sensitized animals without KF. All bar graphs show the mean ± SEM. JEM VOL. 208, May 9, 2011 1047