[10,11,12,13], as well as lipopeptides such as the didehydrox- ymycobactins [14]. CD1-restricted T cells that recognized phosphatidylethanolamine derived from pollen appeared to be present in increased frequencies in allergic individuals [15], suggesting that the CD1 system may also contribute to allergic responses. Additionally, the CD1 system may play a role in human autoimmune diseases, since CD1b-restricted T cells recognizing self glycolipids such as gangliosides and sulfatide were isolated from multiple sclerosis patients [16], and autoreactive CD1c- restricted T cells from lupus patients were found to promote IgG secretion by B cells [17]. Thus, these data suggest that CD1a-, CD1b-, and CD1c-restricted T cells are involved in a variety of human diseases. However, progress in understanding the role of these T cells in immune responses in vivo has been limited by the lack of a good animal model. CD1 genes have been detected in all mammalian species analyzed to date, and orthologues have even been identified in birds [18,19]. However, not all of the CD1 isoforms are expressed in all species, and in some cases CD1 genes have been duplicated, resulting in several variants of the same isoform. Thus, while humans possess one of each of the five CD1 isoforms, this is not the rule. For example, mice and rats have lost the CD1A, B, C, and E genes and have duplicated the CD1D gene, while guinea pigs express multiple variants of CD1B and C, and rabbits and sheep have lost CD1C [18]. Because of these species differences, small animal models that mimic the pattern of CD1 expression found in humans have been lacking, and most in vivo analyses have focused on CD1d, which is the isoform that is present in mice. The CD1d isoform is responsible for selecting a specialized T lymphocyte population called invariant Natural Killer T (iNKT) cells [1]. Murine and human iNKT cells utilize homologous TCRs, show a striking ability to recognize the same microbial glycolipid antigens, and have similar functional properties, including the ability to rapidly produce both Th1 and Th2 cytokines. Analyses of the in vivo functions of NKT cells using murine model systems have demonstrated that this subset has a potent ability to modulate immune function, and can markedly impact the outcome of anti-microbial, anti-viral, and anti-tumor responses, as well as ameliorating or preventing the progression of autoimmune diseases. These observations have generated consid- erable enthusiasm for the possibility that glycolipid antigens that stimulate iNKT cells could be used as therapeutic agents to treat human diseases [20,21]. However, there are also important differences between humans and mice in regards to the iNKT cell compartment. One of the major differences is that human iNKT cells appear to be present at approximately 100-fold lower frequencies than murine iNKT cells [22,23], and therefore it is not clear that human iNKT cells will have as potent immunological impacts as those observed in laboratory mice. Another important factor is that murine and human CD1d molecules show differences in intracellular traffick- ing, which may result in antigen presentation differences [3]. This may be reflected in the differences between murine and human iNKT cells in their requirements for activation by endogenous antigens. Whereas the activation of murine iNKT cells by endogenous ligands requires CD1d molecules to traffic through the endosomal system [24,25], we have found that human iNKT cells appear to be equivalently activated by wild-type CD1d molecules that undergo endosomal trafficking and mutant CD1d molecules that do not [26]. Moreover, current data suggest that humans lack a glycolipid called isoglobotrihexosylceramide (iGb3) that is thought to endogenously activate murine NKT cells [27,28]. On the other hand, many human NKT cells recognize an endogenous mammalian lipid called lyso-phosphatidylcholine The CD1 System in Humanized Mice (LPC) [29], but it is not yet clear whether murine iNKT cells recognize this antigen. Thus, although mice clearly provide an extremely valuable in vivo model of iNKT cell function, the successful development of glycolipids as therapeutic agents that activate iNKT cells to modulate human immune responses in specific ways may require a system that permits in vivo analysis of the human CD1d pathway. Results Human hematopoietic cell engraftment To generate ‘humanized’ mice we followed an approach that has been described previously [30], in which 6–8 week old NOD/ Prkdcscid/ccnull (NSG) mice are sub-lethally irradiated, then fragments of autologous human fetal liver and fetal thymus are implanted next to each other under the kidney capsule, and concurrently, CD34+ hematopoietic stem cells purified from the fetal liver are injected intravenously (Figure 1A). The human CD34+ cells colonize the murine bone marrow and undergo hematopoiesis, giving rise to a variety of human leukocyte populations that occupy peripheral lymphoid sites such as the spleen, liver, and lymph nodes. The fragments of human fetal thymic tissue, which are about 1 mm3 when they are implanted, expand in size and develop into a vascularized organoid that shows a lobular organization typical of thymus (Figure 1B). In contrast, the human fetal liver fragment becomes undetectable by 8–10 weeks post-engraftment. We have found that the frequency of human hematopoietic cells (i.e. human CD45+) in the blood of the mice increases steadily for about the first 10–12 weeks after engraftment, and thereafter the levels remain comparatively constant [31]. Although there is mouse-to-mouse variation in the degree of human chimerism, blood samples from our humanized mice typically contained 20– 70% (mean 53%) human CD45+ cells by 12–14 weeks post- engraftment (Figure 1C). Analysis of human cells isolated from spleen or livers of mice 12 or more weeks post-engraftment consistently demonstrated the presence of T cells (CD3+), B cells (CD19+), macrophages or monocytic cells (CD14+), and CD11c+ cells that were negative for CD14 (Figure 1D). Thus, we were able to obtain successful reconstitution of multiple lineages of human hematopoietic cells within NSG mice. Analysis of the human thymic organoid Murine CD1d-restricted NKT cells have been found to be positively selected by CD1d molecules that are expressed on other thymocytes (i.e. developing T cells), and to be negatively selected by thymic dendritic cells [32,33]. Therefore, we examined the thymic organoids of engrafted (‘‘hu-NSG’’) mice for evidence that the development of human CD1-restricted T cells might be supported. Histological analysis of thymic organoid sections stained with hematoxylin and eosin showed densely packed cortical zones surrounding somewhat less densely packed medul- lary areas (Figure 1B). Towards the center of the medullary areas, eosinophilic concentric circle-like structures were visible that closely resemble the Hassall’s corpuscles found in human thymus (Figure 1B). Flow cytometric analysis of cells isolated from the human thymic organoid revealed clear expression of human CD1a, CD1b, CD1c, and CD1d on most CD3+ cells (Figure 2A). The CD1+ T cells were mainly double positive for the co-receptors CD4 and CD8 (Figure 2A), as is characteristic of cortical thymocytes. Immunohistochemical analysis of the thymic organoid for human CD1a expression revealed positive staining on small round lymphocytic cells packing the outer cortical areas, while most of the small round lymphocytic cells in the inner medullary PLoS ONE | www.plosone.org 2 June 2011 | Volume 6 | Issue 6 | e21701