The CD1 System in Humanized Mice Figure 8. Activation of NKT cells in vivo. A) The plot on the left shows results from engrafted mice that were injected intraperitoneally with 3 mg a-GalCer or with an equivalent amount of vehicle alone. Blood samples were collected at 2–4 and 24–48 hours after injection and analyzed for human IFN-c by ELISA. Each symbol represents the mean of 3 replicates. The P value shown on the plot was calculated using a one-tailed unpaired t test. The plot on the right shows results from human PBMC samples that were incubated in vitro with a-GalCer or vehicle alone. Supernatants were collected after 48 hours and analyzed for IFN-c by ELISA. The P value shown on the right plot was calculated using a one-tailed paired t test. B) Results from the same experiments analyzed for human IL-4 by ELISA. C) Heparinized plasma samples taken at 48-hours post injection were analyzed for enzyme activity of the liver transaminases AST and ALT, which serve as biomarkers of liver injury. The plot shows the enzyme activities detected from samples from mice that were treated with vehicle (open squares) or with a-GalCer (filled squares). There are no significant differences between the groups. doi:10.1371/journal.pone.0021701.g008 adaptive immune systems during immune activation and therefore they may occupy a very powerful functional niche. Although humanized mouse models are not without concerns, for example, batch to batch variations, heterogeneity of engraftment within batches, and the possibility of artifacts due to the chimeric nature of the immune system, our results suggest that the hu-NSG mouse model described here will be an extremely useful tool for studying the thymic development and peripheral function of human CD1- restricted T cells in vivo. Moreover these mice provide a highly novel opportunity to study the role of the CD1 system in vivo during immune responses to human specific pathogens such as Epstein-Barr virus, HIV, and Dengue. Materials and Methods Generation of humanized mice Research involving mice was performed in accordance with a protocol that was approved by the University of Wisconsin’s Animal Care and Use Committee, and in accordance with a protocol approved by the University of Wisconsin’s Institutional Review Board. NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ mice (abbreviat- ed as NOD/SCID/ccnull or ‘‘NSG’’ mice) were obtained from Jackson Laboratory (stock #005557). At 6–8 weeks old, mice were conditioned with sub-lethal (2.5 Gy) whole-body irradiation, and within 4 hours were surgically implanted under the kidney capsule with fragments (approximately 1 mm3) of human fetal thymus and liver (14–22 weeks gestation). Immediately following this surgery, the mice were given an intravenous injection (46105/mouse) of CD34+ cells isolated from autologous fetal liver tissue that was processed and stored in X-vivo 15 serum free culture medium (Cambrex Biosciences Walkersville, MD). The CD34+ cells were prepared by gently disrupting the liver tissue by cutting it into small pieces and repeatedly pipetting up and down, then filtering the resulting cell suspension through a 70 mm mesh, and purifying the mononuclear cells by Ficoll-Paque density gradient separation (GE Healthsciences). CD34+ cells were then isolated by magnetic sorting (Miltenyi Biotec, Auburn, CA). The purity of the injected CD34+ cells was determined by flow cytometric analysis to be at least 80–90%, with less than 0.5% contamination of CD3+ cells. The drinking water for the mice was supplemented with 0.17– 0.25 mg/ml Enrofloxacin (Bayer Healthcare, KS) for 10 days post surgery to prevent infections. Flow cytometric analysis Antibodies used to detect specific human markers were as follows: pan CD45 (clone HI30); CD4 (clone RPA-T4 or OKT4); CD8a (clone RPA-T8); CD14 (clone M5E2); CD19 (clone HIB19); CD11c (clone S-HCL-3); CD123 (clone 9F5); pan HLA class I (clone W6/32); pan HLA class II (clone LN3); CD1a (clone OKT6); CD1b (clone BCD1b3.1); CD1c (clone BDCA-1); CD1d (clone CD1d42); CD3 (clone SPVT-3b); Va24 (clone C15B12); Vb11 (clone C21D2). Negative control antibodies were clone P3 (IgG1) and clone UPC10 (IgG2a). For flow cytometric analysis, tissues (i.e. bone marrow, spleen, liver, blood, thymic organoid) were collected from mice 12–20 weeks after implantation of human cells. Human tissue samples used as controls (i.e. blood and spleen) were collected in accordance with a protocol approved by the University of Wisconsin Minimal Risk IRB. Single cell suspensions were prepared from solid tissues by gentle homoge- nization followed by filtration through a 0.45 mm strainer. Samples were subject to ACK lysis to remove red blood cells, or mononuclear cells were purified by density gradient centrifuga- tion. The samples were blocked with human and murine serum, and then incubated on ice for 30 minutes with 10 mg/ml specific antibodies in FACS buffer (1 mg/ml PBS/BSA) containing 10 mg/ml 49, 69-diamidino-2-phenylindole (DAPI) or propidium iodide (PI). The samples were washed with FACS buffer, then resuspended and analyzed on a Becton Dickenson LSRII or a FACSCalibur flow cytometer. Data were analyzed using Flowjo software (Tree Star, Inc., Ashland, OR). Histologic analysis Tissues were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections from liver, spleen, lung, lymph node and thymic organoid were de-paraffinized and rehydrated with water for hematoxylin and eosin (H&E) staining or immunohistochem- istry. Antigen retrieval was performed using rodent decloaker (Biocare Medical), followed by incubation with a mouse monoclonal against human CD1a (clone CD1a007, Biocare Medical). Antibody labeling was detected and visualized by the Mouse-on-Mouse Horseradish peroxidase (HRP)-Polymer Kit with diamionbenzidine (DAB) development (Biocare Medical). Antigen presentation analyses Spleen and liver tissues were collected from mice 12–20 weeks after implantation of human cells, and single cell suspensions were prepared. The samples were tested for stimulation of human CD1- restricted cell lines in the presence or absence of purified or synthetic lipid antigens. Assays were carried out in 96-well round bottom plates in 200 ml culture medium (RPMI 1640, 10% Bovine Calf Serum, 2 mM L-glutamine, and 100 mg/ml each of penicillin and streptomycin), using 56104/well hu-NSG cells and an equal number of human T cells. After 16 hours incubation at 37uC in a 5% CO2 incubator, culture supernatants were collected and tested for cytokine concentration using commercially available ELISA reagents. Human T cell lines used were as follows: CD8-2, a CD1a-restricted T cell line [48]; LDN5, a CD1b-restricted T cell line [11]; CD8-1, a CD1c-restricted T cell line [48]; and J24L.17, J3N.5, or Jc2.4, human CD1d-restricted NKT cell clones, [36,49]. Human T cell lines were cultured in T cell medium (RPMI 1640 medium, 2 mM L-glutamine, 100 mg/ml each of penicillin and streptomycin, 10% FBS, 5% bovine calf serum, 5% human AB serum from Gemini Bio-Products, and 400 U/ml recombinant human IL-2 from Chiron), with periodic re-stimulation by irradiated allogeneic feeder cells and PHA. Lipid antigens For stimulation of the CD1a-restricted CD8-2 line, a fraction enriched for the lipopeptide didehydroxymycobactin (DDM) was PLoS ONE | www.plosone.org 14 June 2011 | Volume 6 | Issue 6 | e21701