The CD1 System in Humanized Mice Figure 1. Immune reconstitution of engrafted mice. A) Schematic representation of the protocol used to engraft mice with human cells and tissues. B) A photograph taken from the abdominal side shows an engrafted human thymic organoid growing on the lateral superior surface of a murine left kidney. Histological analysis of the thymic tissue shows a lobular organization with cortical and medullary areas. The medullary area shown at 206 magnification on the right includes several bodies that closely resemble the Hassall’s corpuscles found in human thymus. C) Blood samples from engrafted mice were taken at the indicated times and analyzed by flow cytometry to determine the percentage of the total blood leukocytes expressing human CD45. D) Flow cytometric analysis of cells isolated from spleen (top panels) and liver (bottom panels) of engrafted mice. The samples were first gated on the human CD45+ subset, and then gated by forward and side scatter to focus on lymphocytic cells (left panels) or myeloid cells (right panels). doi:10.1371/journal.pone.0021701.g001 areas appeared negative for CD1a (Figure 2B). However, there were some larger cells present in the medullary areas that showed intense staining for CD1a, and these tended to be localized near the edges of the Hassall’s corpuscles (Figure 2B). These results suggest that that there are two distinct types of CD1+ cells within the engrafted human thymic organoid: developing thymocytes localizing mainly to the cortical areas, and larger cells in the medullary areas that may be dendritic cells (DCs). Based on this analysis, we would expect that human CD1-restricted T cells can be both positively and negatively selected in the engrafted thymic organoid. Peripheral expression of human CD1 molecules We next evaluated the expression of human CD1 molecules in peripheral tissues of engrafted mice. Human CD1a, CD1b, and CD1c molecules are most prominently expressed on myeloid DC lineages, although a fraction of B lymphocytes are typically positive for CD1c. Spleen cells from hu-NSG mice were analyzed by flow cytometry for co-expression of CD1 molecules and lineage markers characteristic of myeloid antigen presenting cells and B cells. Human cells within the myeloid gate that were positive for CD11c (a marker of DCs) showed only very little positive staining for CD1a or CD1b, but a substantial fraction were typically positive for CD1c (Figure 3A). CD1c expression was observed on both CD14+ (i.e. monocytes and macrophages) and CD142 myeloid cells (e.g. DCs). Additionally, a substantial fraction of the CD19+ lymphocytes typically stained positively for CD1c (Figure 3A). CD1c expression on B cells appeared limited to those that co-expressed CD20, a phenotype that is characteristic of mature B cells. Human CD1d molecules are constitutively expressed at low cell surface levels by monocytes, macrophages, myeloid DCs, and B lymphocytes [34]. Consistent with this, human CD14+ cells in the engrafted mice appeared uniformly positive for CD1d, and a small fraction of the CD142 myeloid cells (possibly myeloid DCs) also expressed high levels of CD1d (Figure 3B). Moreover, B cells in the engrafted mice appeared nearly uniformly positive for CD1d, with very low cell surface expression levels (Figure 3B). These patterns of CD1a, b, c, and d expression closely resemble what we have observed in similar flow cytometric analyses of mononuclear cells freshly isolated from human blood (data not shown). To further assess the presence of CD1+ APCs in peripheral tissues of engrafted mice, we performed immunohistochemical staining for CD1a on sections of spleen, liver, lung, and skin. Some of the hu-NSG spleen sections analyzed showed isolated cells that stained positively for CD1a, and similarly rare positive staining for CD1a was observed in adult human spleen sections (Figure 3C). Multiple cells staining positively for CD1a could be observed in liver sections from the hu-NSG mice and in sections of adult human liver, suggesting that CD1a+ APCs might be more abundant in this tissue (Figure 3C). We also observed CD1a+ cells in lung sections analyzed from the hu-NSG mice, although they appeared less abundant than those in adult human lung sections (Figure 3C). We were not able to identify CD1a+ cells in skin sections from the hu-NSG mice (data not shown), although CD1a+ Langerhans cells are typically prominently expressed in human skin. Together, these results indicate that CD1+ APCs are present at major sites of immunological activity of engrafted mice, including spleen, liver, and lung, although they may be largely lacking in the skin. Presentation of glycolipid antigens To investigate CD1-mediated antigen presentation by APCs from hu-NSG mice, we isolated cells from spleen or liver and tested their ability to stimulate antigen-dependent cytokine secretion by previously established and characterized human CD1-restricted T cell lines. To test for antigen presentation by CD1a, we used transfected JRT.3 cells expressing a TCR (CD8-2) that has been found to specifically recognize didehydroxymyco- bactin (DDM) presented by CD1a [14]. We observed marked human IFN-c production when CD8-2 transfected JRT.3 cells were incubated with spleen cells from an engrafted mouse, however, the cytokine response was not increased by addition of the DDM antigen (Figure 4A, left panel). In contrast, liver mononuclear cells stimulated only modest IFN-c production from the transfected JRT.3 cells in the absence of DDM, and addition of the antigen resulted in enhanced cytokine secretion (Figure 4A, right panel). Thus, APCs from the liver of hu-NSG mice appeared to be able to perform CD1a-mediated antigen presenting functions, but it was not clear whether splenic APCs shared this ability. A CD1b-restricted human T cell line called LDN5, which specifically recognizes the mycobacterial lipid glucose monomy- colate (GMM) [11], was used to evaluate CD1b-mediated antigen presenting function. Co-incubation of LDN5 cells with splenocytes or liver cells from hu-NSG mice resulted in some IFN-c production in the absence of added antigen, but there were clear dose-dependent increases in IFN-c secretion in response to GMM (Figure 4B). Notably, the form of GMM used for these studies contains a long (80 carbon) acyl chain, and has been shown to require internalization into endocytic compartments for loading into CD1b molecules before it can be presented at the cell surface [35]. Thus, these results suggest both spleen and liver contained cells capable of internalizing and presenting a glycolipid antigen via CD1b. CD1c-mediated antigen presentation was tested using the CD8- 1 human T cell line, which recognizes a microbial lipid called mannosylphosphomycoketide (MPM) and also responds to structurally related mammalian lipids called mannosylphosphodo- lichols [7]. Similar to the results for CD1a-mediated antigen presentation, we found that co-incubation of the CD8-1 cells with hu-NSG splenocytes resulted in marked IFN-c secretion, and there was little evidence of enhanced responses in the presence of added MPM (Figure 4C, left panel). However, when CD8-1 cells were incubated with liver mononuclear cells from hu-NSG mice there was only modest cytokine production in the absence of added antigen, and clear dose-dependent increases were observed in response to added MPM (Figure 4C, right panel). From these results, it seems that APCs in hu-NSG liver can present added glycolipid antigens via CD1c. However, it is not clear whether the PLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e21701