The CD1 System in Humanized Mice Figure 7. Analysis of hu-NSG NKT cells. CD1d tetramer-positive and -negative T cells were flow cytometrically sorted from an engrafted mouse and expanded in vitro. A) Flow cytometric analysis of the expanded cells (tetramer-positive top row, tetramer-negative bottom row). Filled histograms show staining with fluorescently labeled anti-CD3, a-GalCer loaded human CD1d tetramer, anti-Va24, and anti-Vb11 antibodies, respectively. Open histograms show staining with vehicle treated CD1d tetramer or isotype-matched negative control antibodies. B) Cytokine secretion by the tetramer-positive and -negative cell lines in response to CD1d-transfected or untransfected (UT) APCs in the presence of a-GalCer. Open bars indicate assays that were performed in the presence of an anti-CD1d blocking antibody, and filled bars show assays performed in the presence of an isotype matched negative control antibody. C) Predicted amino acid sequences of the junctional regions of the TCRa and b chains from hu-NSG tetramer-positive T cells compared to those of NKT cell clones previously derived from the human tissues shown in parentheses. Dashes indicate identity with the consensus sequences shown at the bottom. doi:10.1371/journal.pone.0021701.g007 1–5% of splenic T cells and 10–30% in the liver, the iNKT cell subset generally comprised about 0.1% of the total human T cells in the spleens of the engrafted mice, and the frequency did not appear to be enriched in the liver. The iNKT cell frequencies we observed in the hu-NSG mice and their lack of enrichment in the liver are consistent with iNKT cell frequencies observed in previous analyses of human tissues [22,44,45,46], and this therefore appears to constitute an important difference between humans and commonly used strains of laboratory mice. Given the differences in iNKT cell frequency between the hu-NSG mice and laboratory strains, it is perhaps not surprising that injection of a- GalCer resulted in only modest increases in IFN-c and IL-4 levels in the blood of engrafted mice, and did not appear to produce elevated levels of transaminase enzymes that are indicative of damage to the liver. Nevertheless, our data indicate that there is a rapid cytokine response to a-GalCer in vivo in the engrafted mice, which suggests that these mice will be a valuable tool for investigating the impact of glycolipid antigens in a human immune context. The CD1 system has a number of key advantages from the perspective of developing new methods of therapeutic immune modulation: the antigens utilized by the CD1 system are highly conserved molecules that may reduce the chances of pathogen escape through mutation, also there is very little allelic polymorphism in the human CD1 system, and finally, CD1- restricted T cells seem to temporally bridge the innate and PLoS ONE | www.plosone.org 12 June 2011 | Volume 6 | Issue 6 | e21701