for intracellular cytokine analysis. We found a significantly MGat2 ablation reduces native complex N-glycans higher percentage of CD4+ T cells producing IFN- in ani- and eliminates GlyAg-mediated T cell activation mals expressing native complex N-glycans compared with Loss of the GlcNAc glycosyltransferase II enzyme (encoded animals treated with KF (Fig. 6 B). by the MGat2 gene) in an animal model of the type IIa con- To verify that this increase in CD4+IFN+ T cell number genital disorders of glycosylation (CDG-IIa) results in several reflects PSA-specific cells, we performed recall experiments osteogenic, hematologic, and gastrointestinal tract abnormal-i from UNT and KF-treated animals sensitized with PSA or ties (Wang et al., 2001). The mice lackingMGat2 also die OVA protein. Animals were injected with KF or PBS vehicle early in postnatal development and lack native branched for three consecutive days. On the fourth day, the animals complex N-glycans. To determine if genetic-based loss of were challenged with OVA protein or PSA and allowed to native complex N-glycans in the APC would also result in respond for 4–6 d. The CD4+ T cells were then harvested GlyAg T cell activation defects, BM was isolated from mice from each animal and restimulated ex vivo with APCs from that carry the MGat2 gene flanked by LoxP sites but do not UNT naive animals (i.e., normally glycosylated cells) and either express the CRE recombinase. BM cells from either WT or PSA or OVA to measure the level of immunological recall. MGat2flox animals were either mock electroporated or electr-o We found that with OVA, the number of responding T cells porated with an expression construct for myc-tagged CRE during sensitization in KF-treated animals increased over recombinase (pCRE) to excise the floxed MGat2 gene, and animals sensitized without KF (Fig. 6 C). In contrast, KF treat- then cultured as usual with granulocyte-macrophage colony- ment during sensitization with PSA resulted in a decreased stimulating factor (GM-CSF) to derive dendritic cells.The T cell response upon recall with PSA compared with UNT four resulting populations of BMDCs were analyzed by PHA-L animals expressing native complex N-glycans (Fig. 6 D).These staining to confirm reduction in native complex N-glycans data show that KF inhibited PSA-specific T cell expansion (Fig. 7 A). Approximately 40% of the MGat2flox BMDCs during in vivo sensitization.To determine if this inhibition is electroporated with pCRE lacked significant PHA-L stain- caused by presentation defects within the resident APCs, ing, whereas the mock transfected MGat2flox BMDCs and KF-treated or UNT animals were sensitized with PSA along both WT populations were uniformly PHA-L positive. with adoptively transferred normally glycosylated APCs i-so Unstained controls are shown in Fig. S1 A. NP-40 lysates from lated from naive mice. Inclusion of normal APCs during PSA these four populations were analyzed using Western blots and sensitization of KF-treated animals allowed for PSA-specific probed with either anti-CRE (Fig. 7 B) or anti-myc (Fig. S1 B) T cell responses in vivo to reach normal levels, as shown by antibodies to demonstrate CRE expression. Both WT and the increased PSA-specific recall response (Fig. 6 E). MGat2flox cells show CRE expression when electroporated Figure 7. Deletion of MGat2 results in deficient PSA, but not OVA-mediated T cell activation. (A) BM cells were harvested from MGat2wt (WT) or MGat2flox animals, electroporated with a CRE expression construct (pCRE) or mock electroporated without DNA, and cultured with GM-CSF for DC dif- ferentiation. Cells were stained with PHA-L to determine the effect of MGat2 excision. Representative histograms are shown. (B) Mock and pCRE electro- porated BM cells were extracted and lysates were analyzed by anti-CRE Western blot using -actin as a loading control. Representative blot shown. (C) PHA-L pCRE electroporated MGat2flox BMDCs were isolated from PHA-L+ cells by negative selection using PHA-L and magnetic beads and compared with PHA-L+ pCRE-electroporated WT cells. Flow cytometry was used to measure the purity and glycophenotype of the populations, whereas RT-PCR on mRNA isolated from the PHA-Lpos and PHA-Lneg BMDCs was performed to demonstrate the excision of MGat2 in PHA-Lneg BMDCs (MGat2neg) but not PHA-Lpos BMDCs (MGat2pos). Representative histograms and gel shown. (D) Flow cytometry was used to measure BMDC differentiation (CD11c) and MHCII expression (MHCII) in both PHA-Lpos and PHA-Lneg BMDC populations. Representative histograms shown. (E) MGat2pos and MGat2neg BMDCs were cultured with either OT-II CD4+ T cells and OVA (50 µg/ml) or WT CD4+ T cells and PSA (50 µg/ml). T cell activation was determined by IFN- ELISA of the culture supernatants. (n = 3 independent experiments). 1048 MHC class II glycosylation in presentation of glycoantigens | Ryan et al.