purified from M. tuberculosis [14]. For stimulation of the CD1b- restricted LDN5 line, a long chain form of glucose monomycolate (GMM C80) was purified from M. phlei [11]. For stimulation of the CD1c-restricted CD8-1 line, a synthetic preparation of mannosyl- b1-phosphomycoketide (MPM C32) was used [50]. For in vitro analyses of CD1d-restricted NKT cells, a-GalCer was prepared as described [51]. CD1a-, CD1b-, and CD1c-restricted T cell lipid antigens were stored in organic solvents at 220uC, and prior to use were transferred to glass vials and dried down under nitrogen gas, then resuspended in culture medium and sonicated in a heated water bath. a-GalCer was dissolved in DMSO at 100 mg/ ml, and this solution was sonicated then diluted into culture medium prior to use. For in vivo injection, a-GalCer was synthesized by coupling 2,3,4,6-tetra-(O-trimethylsilyl)-D-galacto- syl iodide with a sphingosine acceptor containing azide function- ality, and the resulting compound was modified by azide- reduction, N-acylation and deprotection of benzyl-protected hydroxyl groups. The a-GalCer was dissolved in a vehicle solution containing 0.5% Tween 20, 5.7% sucrose, and 0.75% histidine. ELISpot analyses of CD1-restricted T cell frequency Mononuclear cells were prepared from hu-NSG spleen or liver, or from human peripheral blood. Human blood was collected with written consent from volunteer donors, in accordance with a protocol approved by the University of Wisconsin’s Minimal Risk Institutional Review Board. The samples were incubated with untransfected human K562 myelomonocytic cells [52] or with K562 cells that were transfected with human CD1a, CD1b, CD1c, or CD1d, at a 1:1 ratio (200,000 cells per well total) in serum-free medium (CELLect medium, MP Biomedicals, Inc.) in 96-well PVDF membrane plates (Whatman) coated with anti-human IFN- c mAb (clone NIB42 from Biolegend). The cells were incubated for 48 hr at 37uC and 5% CO2. Secreted IFN-c was detected using biotinylated anti-human IFN-c mAb (clone M701B from Thermo Scientific), and revealed by development with streptavi- din-alkaline phosphatase and BCIP/NBT chromogenic substrate. Spots were quantitated using AID 5.0 software. Generation of CD1d-restricted T cell line Hu-NSG spleen was sterilely harvested and mononuclear cells were purified by density gradient centrifugation. The cells were then stained with sterile fluorescently labeled antibodies against human CD3 and CD19, and with human CD1d tetramer loaded with a-GalCer. Between 50 and 75 tetramer-positive human T cells were flow cytometrically sorted into a well of a 96-well tissue culture plate and cultured with irradiated allogeneic human PBMCs in RPMI 1640 supplemented with 2 mM L-glutamine, 100 mg/ml penicillin and streptomycin, 10% fetal bovine serum (Hyclone), 5% bovine calf serum (Hyclone), 3% human AB serum (Atlanta Biologicals), PHA (Sigma) and 400 U/ml recombinant human IL-2 (Chiron). References 1. Brigl M, Brenner MB (2004) CD1: Antigen Presentation and T Cell Function. Annu Rev Immunol 22: 817–890. 2. Martin LH, Calabi F, Milstein C (1986) Isolation of CD1 genes: a family of major histocompatibility complex-related differentiation antigens. PNAS 83: 9154–9158. 3. Gumperz JE (2006) The ins and outs of CD1 molecules: bringing lipids under immunological surveillance. Traffic 7: 2–13. 4. Sieling PA, Jullien D, Dahlem M, Tedder TF, Rea TH, et al. (1999) CD1 expression by dendritic cells in human leprosy lesions: correlation with effective host immunity. J Immunol 162: 1851–1858. The CD1 System in Humanized Mice TCR sequence analysis Total RNA was prepared from approximately 1–56106 CD1d- restricted T cells using Trizol (Invitrogen), and 1 mg was used for preparation of cDNA using commercially available reagents (Roche). DNA sequences spanning the junctional regions of the TCRa and b chains were amplified by PCR using forward primers that sit down in the Va24 or Vb11 gene segments (59- AAGATACTGGGAGAGGTCCTGTTTC-39 or 59-CCAGGA- ATGGAACTACACCTCATC-39, respectively), and reverse primers that sit down in the TCRa or b constant domains (59- GAATAATGCTGTTGTTGAAGGCG-39 or 59-TTGACAGC- GGAAGTGGTTGC-39, respectively). The resulting PCR prod- ucts were purified by spin column or agarose gel extraction (QIAquick from QIAGEN), and were sequenced using the same primers that were used for amplification. Supporting Information Figure S1 Cytokine production from previously established human iNKT cell clones (Jc25., Jc2.8, JJ2.7) in response to spleen cells from an engrafted mouse treated with a-GalCer is blocked by addition of the CD1d42 anti-human CD1d monoclonal antibody, but not by an isotype-matched negative control antibody. (TIF) Figure S2 The CD1d42 antibody blocks iNKT cell responses to human but not murine CD1d. Untransfected P815 cells (no CD1d), P815 cells transfected with murine CD1d, and P815 cells transfected with human CD1d were treated with a-GalCer and incubated with a human iNKT cell clone (Jc2.5) in the presence of the anti-CD1d antibody CD1d42 (black bars) or an isotype control mAb (grey bars). (TIF) Figure S3 CD1d-mediated antigen presentation by liver cells from engrafted mice was tested using the human iNKT cell clone J24L.17 and a glycolipid called GalGalCer, which requires glycosidic cleavage of the terminal galactose sugar in order to be recognized by iNKT cells. (TIF) Figure S4 Splenocytes from an engrafted mouse show no significant responses to antigens used for evaluation of CD1- mediated antigen presentation to human T cell lines. (TIF) Acknowledgments We thank Dr. Gurdyal S. Besra (University of Birmingham) for kindly providing the a-GalCer lipid preparation that was used for in vitro analyses. Author Contributions Conceived and designed the experiments: JLL XC DR WJB JEG. Performed the experiments: JLL XC YZ DR DAR SH SG. Analyzed the data: JLL XC JEG. Contributed reagents/materials/analysis tools: T-YC RJA GFP DBM. Wrote the paper: JEG. 5. Sieling PA, Ochoa MT, Jullien D, Leslie DS, Sabet S, et al. (2000) Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions. J Immunol 164: 4790–4796. 6. Moody DB, Guy MR, Grant E, Cheng TY, Brenner MB, et al. (2000) CD1b- mediated T cell recognition of a glycolipid antigen generated from mycobac- terial lipid and host carbohydrate during infection. J Exp Med 192: 965–976. 7. Moody DB, Ulrichs T, Muhlecker W, Young DC, Gurcha SS, et al. (2000) CD1c-mediated T-cell recognition of isoprenoid glycolipids in Mycobacterium tuberculosis infection. Nature 404: 884–888. PLoS ONE | www.plosone.org 15 June 2011 | Volume 6 | Issue 6 | e21701