in 100 µl T-buffer using a single 20 ms, 2,200 V pulse. Transfected BM cells were then cultured with GM-CSF to facilitate BMDC differentiation, as described in the T cell activation (inhibitors) section.The pCAG-Cre plasmid was supplied by C. Cepko (Harvard University, Boston, MA) through the Addgene depository (Addgene plasmid 13775). Cells expressing CRE were selected and purified based on PHA-L staining. In brief, MGat2flox BMDCs previously transfected with pCAG-Cre were labeled with 20 µg/ml biotin-yl ated PHA-L (Vector Laboratories) and anti-biotin magnetic microbeads then passed through a magnetic MACS LS column (Miltenyi Biotec). The non- binding cells (PHA-L negative) found in the flow through (MGatn2eg) were analyzed by RT-PCR (see the RT-PCR section), and flow cytometry for purity using biotin-PHA-L and a fluorescent streptavidin-APC conjugate (BioLegend) and compared with MGat2wt cells (MGat2pos). For T cell assays using MGat2pos and MGat2neg BMDCs, CD4+ T cells were isolated from the spleens of C57BL/6 or OT-II mice (The Jackson Laboratory) by positive selection using CD4+ magnetic beads (Miltenyi Biotec). CD4+ T cells were added to the BMDC cultures at a 10:1 ratio (i.e., 10 T cells per BMDC) in the presence or absence of 50 µg/ml OVA or PSA.Activation was assessed on day 3 using IFN- production by sandwich ELISA according to the manu- facturer’s protocol (BioLegend). Western blots. Protein extracts were prepared from BM cells 18 h after transfection with the CRE recombinase expression construct pCAG-Cre (see above). In brief, cells were lysed for 30 min in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, and Complete Mini protease inhibitor cocktail (Roche). Protein samples were normalized using the microBCA Protein Assay kit (Thermo Fisher Scientific) before separation by SDS-PAGE and transfer to an Immobilon-P membrane (Millipore). Membranes were probed with either rabbit anti-CRE (Immuno-Biological Laboratories), rabbit anti-myc (Cell Signaling Technology), or mouse anti–-actin (clone 2F1-1; BioLegend) antibodies, followed by HRP-conjugated secondary antibodies.Visualization was performed on film using ECL Plus (GE Healthcare) or ECL (Thermo Fisher Scientific). MGat2 RT-PCR. RNA was isolated from both BMDC populations using the PerfectPure RNA Cell and Tissue kit (5 Prime) to verify the presence or absence of MGat2 transcription. Using equal amounts of RNA, mRNA reverse transcription and PCR were performed using the SuperScript III First-Strand Synthesis System for RT-PCR kit following the manufacturer’s instructions (Invitrogen). Primers used for PCR (IDT) were as follows:MGat2, forward 5-TTGCTGATAGACTCGCTTCG-3 and reverse 5-GCTGAACGGAA- AGAACACTTG-3; -actin, forward 5-TGAGAGGGAAATCGTGC- GTGA-3 and reverse 5-CACTATTGGCAACGAGCGGTTC-3. Animal studies. C57BL/6 mice were bred and maintained in a specific pathogen–free environment at CaseWestern Reserve University and were treated under IACUC-approved guidelines in accordance with approved protocols. All injections were given i.p.Age-matched C57BL/6 mice were treated with 250 µg KF (dissolved in PBS at 1 mg/ml) or PBS alone for three consecutive days.Where indicated, some mice received a cell transfer (i.p.) of 5 × 106 WT APCs on day 4, 2 h before additional treatments. On day 4, all mice received an additional dose of KF or PBS, including a sensitizing dose consisting of 100 µg purified PSA or 100 µg OVA protein (Sigma-Aldrich) with a 1:3-1:5 dilution of sterilized cecal contents (Cobb et al., 2004) in a final volume of 200 µl PBS.At the indicated time point after immunization, spleens were harvested to assay antigen-specific CD4+ T cell recall responses. In brief, CD+4T cells were purified by positive selection using CD4 magnetic labeling beads per the manufacturer instructions (Miltenyi Biotec). APCs were isolated from splenocyte populations after depletion of T cells using CD90.2 magnetic labeling beads per the manufacturer’s instru-c tions (Miltenyi Biotec). 2.5 × 105 CD4+ T cells and 2.5 × 105 APCs were added per well in triplicate with or without 50 µg/ml antigen in a final volume of 200 µl RPMI 1640 media (Invitrogen) supplemented with 10% FBS, pen/ strep, l-glutamine, andME. Supernatants were assayed for the presence of IFN- by ELISA according to the manufacturer’s protocol (eBioscience). Statistical ana-l yses of the resulting data were performed using an unpaired Student’st test. 1052 Online supplemental material. Table S1 shows the Consortium for Functional Glycomics Glycan Array Information List of all glycans rep-re sented on the glycan-binding array. Fig. S1 shows BMDC control anal-y ses. Online supplemental material is available at http://www.jem.org/cgi/ content/full/jem.20100508/DC1. The authors wish to thank Colleen Lewis, Lili Zhang, and Luke Bury for technical assistance. We also thank Lori Kreisman for critical evaluation of this manuscript, Richard Cummings for helpful discussions and experimental advice, and Derek Abbott for technical advice, Western blot antibodies, and the use of his Neon transfection system instrument. We thank Dr. Roy Marriuza for supplying Escherichia coli–expressed HLA-DR protein. The glycan-binding array was performed by Core H of the Consortium for Functional Glycomics, which is funded by grant GM062116 through the National Institute of General Medical Sciences. This work was funded by grants OD004225 and GM082916 to B.A. Cobb. The authors claim no competing financial interests. Submitted: 12 March 2010 Accepted: 17 March 2011 REFERENCES Altmann, F., E. Staudacher, I.B.Wilson, and L. März. 1999. 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