The CD1 System in Humanized Mice Figure 4. Analysis of CD1-mediated antigen presentation. Mononuclear cells from spleen or liver were used to stimulate cytokine secretion by human CD1-restricted T cell lines in the presence of specific cognate antigens. Black squares show cytokine secretion detected from incubation of human T cells with spleen or liver cells from engrafted mice in the presence of the glycolipid antigen concentrations indicated on the x-axis; grey squares show cytokine secretion detected from T cells incubated with APCs in the absence of glycolipid antigen; and white squares show the results from T cells incubated alone. The squares represent the mean cytokines values from 2–4 replicate analyses with error bars showing standard deviations. A) CD1a-mediated antigen presentation was tested using a cell line called CD8-2 and the antigen didehydroxymycobactin (DDM). B) CD1b-mediated antigen presentation was tested using a cell line called LDN5 and the antigen glucose monomycolate (GMM). C) CD1c-mediated antigen presentation was tested using a cell line called CD8-1 and the antigen mannosylphosphomycoketide (MPM). D) CD1d-mediated antigen presentation was tested using an iNKT cell line called J24L.17 and the antigen a-galacosylceramide (a-GalCer). Results are representative of 2–3 independent experiments. doi:10.1371/journal.pone.0021701.g004 apparent using the liver APCs (which would also be expected to contain cells expressing murine antigen presenting molecules), however, further studies will be required to determine whether xeno-presentation is a factor. Our ELISpot analyses revealed that there are cells in the spleens of engrafted mice that produce IFN-c in a CD1-dependent manner without requiring addition of foreign glycolipids, suggest- ing the presence of CD1-restricted T cells that respond to endogenous antigens. This is similar to previous observations of CD1-restricted cells from human tissue samples that are not dependent on the addition of foreign antigens [38,39,40,41,42]. Flow cytometric analyses using human CD1d tetramers revealed a T cell population that was specifically stained by a-GalCer loaded tetramer, and analysis of a cell line derived from the tetramer- stained population demonstrated the use of a canonically rearranged TCR. Whereas murine iNKT cells comprise about Figure 5. Analysis of CD1-restricted T cell responses. A) Splenocytes from engrafted mice were combined with human K562 antigen presenting cells transfected with the indicated CD1 molecules, or with the untransfected K562 parental cell line (‘‘UT’’), and the frequencies of IFN-c producing cells were estimated by ELISpot analysis. Each symbol represents the mean number of spots from 3 or 4 replicate analyses of the splenocytes from one engrafted mouse. The data were statistically analyzed using a one-tailed Wilcoxon paired t-test, yielding the p values shown below the x-axis labels for each CD1 transfectant compared to the untransfected parental cell line. B) Results from a similar analysis using peripheral blood mononuclear cells (PBMC) purified from healthy adult human donors. doi:10.1371/journal.pone.0021701.g005 PLoS ONE | www.plosone.org 10 June 2011 | Volume 6 | Issue 6 | e21701