The CD1 System in Humanized Mice Figure 3. Expression of human CD1 molecules in peripheral tissues. A and B) Flow cytometric analysis of splenocytes for CD1a, CD1b,CD1c, and CD1d. The samples were first gated on the human CD45+ subset, and then gated by forward and side scatter to focus on myeloid or lymphoid cells. C) Histological analysis of CD1a expression in spleen, liver, and lung sections from engrafted mice (bottom row) or human tissue samples (top row). CD1a expression is visualized by DAB staining (brown colored cells). The sections were counterstained with hematoxylin, producing blue staining of cell nuclei. All images are shown at 406 magnification. doi:10.1371/journal.pone.0021701.g003 characterized by elevated levels of IFN-c and IL-4 in the serum that correlate with the frequency of iNKT cells [43]. To test the effects of in vivo administration of a-GalCer in this model, hu-NSG mice were injected intraperitoneally with 3 mg of a-GalCer or an equivalent amount of vehicle alone. Blood samples were taken from the mice at 2–4 or 24–48 hours post-injection, and the plasma was analyzed for human IFN-c and IL-4 by ELISA. The median IFN-c concentration of samples from the vehicle treated mice was 95 pg/ml, with about a third of the samples having IFN- c values that were near or below the limit of detection for the ELISA (Figure 8A, left plot). In contrast, only one sample from the 2–4 hour post a-GalCer injection time point was below the limit of detection of the ELISA, and the median IFN-c concentration was 240 pg/ml (Figure 8A, left plot). Although the amount of IFN-c from the a-GalCer treated mice was only 2–3 fold greater than that of the vehicle treated mice, a Mann-Whitney test indicated that the difference is statistically significant (p,0.009). The median IFN-c detected from the 24–48 hours post a-GalCer injection samples was 155 pg/ml, and statistical analyses indicated that this was not significantly elevated compared to the vehicle treated mice (Figure 8A, left plot). By comparison, when we incubated samples of 0.5 million human PBMCs in vitro in 0.2 mL of culture medium containing either a-GalCer or vehicle, we detected a median IFN- c concentration of 50 pg/ml for the vehicle treated and 255 pg/ ml for the a-GalCer treated samples (Figure 8A, right plot). Interestingly, whereas we were not able to detect any increased IL- 4 in the culture supernatants from human PBMCs incubated in vitro with a-GalCer compared to vehicle (Figure 8B, right plot), we observed a significant increase in the amount of IL-4 detected from blood samples of hu-NSG mice taken at 2–4 hours post a- GalCer injection compared to those of vehicle treated mice (Figure 8B, left plot). These results demonstrate that treatment with a-GalCer induces a rapid biological response in vivo in the hu- NSG mice, with systemic cytokine levels apparently becoming elevated in the first few hours after treatment and then receding to baseline levels within 24–48 hours. Analysis of plasma samples from a-GalCer or vehicle treated mice that were taken at 48-hours post-injection showed no difference in levels of the liver transaminases AST and ALT (Figure 8C), suggesting that the liver injury that has been observed in laboratory mice following a- GalCer treatment is much less significant in this model [47]. Discussion Great progress has been made using murine model systems to understand the development and function of CD1d-restricted iNKT cells, however, it remains unclear whether human iNKT cells are identical to their murine counterparts. Additionally, little is known about the development and in vivo function of T cells that are restricted by other CD1 molecules. The analyses presented here demonstrate that immunodeficient mice that are transplanted with human tissues (hematopoietic stem cells, fetal thymus, and fetal liver) develop a functional human CD1 antigen presenting system in the periphery and acquire human iNKT cells with a canonical TCR rearrangement. Additionally, our findings suggest that the development of other human CD1-restricted T cells is supported. Thus, these mice provide a very promising model system for further investigating specific aspects of the development and functions of human CD1-restricted T cells in vivo. In this model system NSG mice are engrafted with human tissues when they are six to eight weeks old. At this age the murine thymus appears to have atrophied and disappeared (presumably because the mice do not produce their own T cells). In any case, we are not able to detect a murine thymus even after engraftment of the human hematopoietic stem cells (data not shown). Therefore, it is not clear whether any of the developing human T cells in this model are selected on murine antigen presenting molecules. However, it is clear that the transplanted human fetal thymic tissue increases in size and persists as a long-lasting thymic organoid. We show here for the first time that there is prominent expression of CD1a, CD1b, CD1c, and CD1d molecules by human T cells in this thymic organoid. In our histological analyses, we observed that there were two distinct CD1a+ cell types in the thymic organoid: abundant small, round lymphocytic cells that appear to be cortical thymocytes, and less abundant, larger cells in the medullary area that may be DCs. An interesting possibility is that these two populations are involved in different aspects of thymic selection of CD1-restricted T cells. Whereas MHC- restricted T cells are positively selected by MHC molecules expressed on thymic epithelial cells in the thymic cortex, murine CD1d-restricted NKT cells have been shown to be positively selected in the thymic cortex by CD4 and CD8 double positive thymocytes [32]. Thus, we would expect that the CD1+ cortical thymocytes present in the human thymic organoid might mediate positive selection of CD1a-, CD1b-, CD1c-, or CD1d-restricted human T cells. Moreover, since negative selection of murine NKT cells appears to be mediated by DCs of hematopoietic origin [33], we would hypothesize that the CD1a+ DC-like cells we observed in the medullary regions of the human thymic organoid might play a role in negative selection. We also found expression of CD1 molecules by human cells in peripheral tissues of engrafted mice. CD1c and CD1d were clearly detectable on myeloid cell types that were present in the spleen (see Figure 3) and in the liver (data not shown), and these CD1 molecules were also expressed by B lymphocytes (Figure 3). Splenocytes expressing CD1a or CD1b appeared infrequent by flow cytometric analysis, but by histological analysis we were able to detect rare cells that stained positively for CD1a in the spleen, as well as somewhat more abundant CD1a+ cells in liver and lung sections. Functional analyses demonstrated that liver mononuclear cells from engrafted mice were able present specific glycolipid antigens to CD1a-, CD1b-, CD1c-, or CD1d-restricted human T cells. Spleen cells were able to present glycolipid antigens to CD1b- and CD1d-restricted T cells, but the CD1a- and CD1c- restricted T cells we tested showed a high response to the splenocytes alone (without added antigen) and it was difficult to see enhanced activation in response to added antigen. Thus, the splenic APCs may stimulate responses by some CD1-resticted T cells that are not dependent on the addition of foreign antigens. It is not clear whether this is due to presentation of endogenous antigens by the relevant CD1 molecules, or whether this is due to some sort of cross-reactivity by the human T cell lines tested here for murine antigen presenting molecules. We favor the first explanation, since the antigen-independent responses were not PLoS ONE | www.plosone.org 8 June 2011 | Volume 6 | Issue 6 | e21701