The CD1 System in Humanized Mice Figure 2. Expression of human CD1 molecules in the engrafted thymic organoid. A) Flow cytometric analysis of human CD3+ cells from the thymic organoid. Top row: staining with antibodies specific for the indicated CD1 molecules is shown by the filled histograms, open histograms show staining by isotype-matched negative control antibodies. Bottom row: contour plots showing CD4 and CD8 staining of the CD1-positive populations. B) Immunohistological analysis of the engrafted thymic organoid at 406 magnification, using an antibody against human CD1a, with DAB chromogenic development (brown color) and hematoxylin counterstaining of nuclei (blue color). doi:10.1371/journal.pone.0021701.g002 comparatively abundant CD1c+ cells we detected in our flow cytometric analyses of hu-NSG spleen samples (see Figure 3A) are also able to present an exogenous glycolipid. Finally, we used a human invariant NKT cell clone to investigate CD1d-mediated antigen presentation. Human iNKT cells respond to the synthetic glycolipid a-GalCer as a strong TCR agonist [36], but they typically also show detectable cytokine production in response to CD1d+ APCs in the absence of added antigens, which is thought to be due to recognition of endogenous CD1d ligands such as lyso-phosphatidylcholine [29]. Similar to the autoreactive responses of human iNKT cells to human CD1d+ APCs that we have observed previously, there was clearly detectable cytokine production when human iNKT cell clones were co-incubated with either spleen or liver cells from hu-NSG mice in the absence of added antigens (Figure 4D). The addition of a-GalCer stimulated increased cytokine production from co- cultures of hu-NSG spleen or liver cells with human iNKT cell clones (Figure 4D), indicating it was presented by CD1d+ APCs. Notably, the cytokine production was blocked by addition of an antibody that is specific for human CD1d and blocks iNKT cell responses to this antigen presenting molecule, but does not block their responses to murine CD1d (Figures S1 and S2). Together these results suggest that the main route of a-GalCer presentation was through human CD1d molecules. However, it important to note that murine CD1d is not ablated in these mice, and therefore it is possible that CD1d-restricted responses could also be mediated by murine APCs in this system. We also observed enhanced cytokine secretion from the addition of GalGalCer to the co-cultures of human iNKT cells with hu-NSG liver mononuclear cells (Figure S3). GalGalCer is a form of a-GalCer that requires intracellular processing in order to be recognized by iNKT cells [37], and thus this finding indicates that hu-NSG APCs are able to internalize an exogenous glycolipid and trim off excess sugars before presenting it via CD1d. Detection and analysis of CD1-restricted T cells Previous analyses have indicated that a significant fraction of human CD1a-, CD1b-, and CD1c-restricted T cells resemble iNKT cells in that they are able to become activated in a CD1- dependent manner in the absence of foreign antigens [38,39,40,41,42]. To test for the presence of such T cells in hu- NSG mice, we performed IFN-c ELISpot analyses using CD1- transfected K562 myelo-monocytic cells as APCs. Hu-NSG splenocytes were incubated in ELISpot wells with CD1a-, CD1b-, CD1c-, or CD1d-transfected K562 cells, or with untransfected (CD1-negative) K562 cells. K562 cells were used for these analyses because they are negative for MHC class I and II molecules, and therefore these APCs would be unlikely to stimulate an allo-response by MHC-restricted T cells [42]. The untransfected K562 cells stimulated IFN-c spot numbers that were close to background levels (i.e. splenocytes without any added APCs). In contrast, the CD1a-, CD1b-, and CD1d-transfected K562 cells consistently stimulated greater numbers of spots (Figure 5A). In some cases, the CD1c-transfected K562 cells also stimulated elevated numbers of spots, but this was not sufficiently consistent to attain statistical significance (Figure 5A). Peripheral blood mononuclear cells isolated from healthy adult human donors showed a similar pattern of ELISpot results in response to the CD1-transfected K562 cells (Figure 5B). Moreover, we observed similar responses to the CD1-transfected K562 cells by liver mononuclear cells from engrafted mice (data not shown). Thus, the hu-NSG mice appear to possess IFN-c producing lymphocytes that respond to CD1 molecules loaded with cellular ligands. In our analyses of CD1-mediated antigen presentation by splenocytes from hu-NSG mice, we did not observe significantly increased IFN-c secretion by splenocytes that were treated with a- GalCer compared to untreated splenocytes (Figure S4), raising the question of whether iNKT cells are present in the engrafted mice. Therefore, to specifically investigate the frequency of CD1d- restricted iNKT cells, we used CD1d tetramers loaded with a- GalCer. By flow cytometric analysis we observed T cells that stained specifically with a-GalCer loaded CD1d tetramer, whereas there was little or no staining using a vehicle treated control tetramer (Figure 6A). The frequency of CD1d tetramer+ T cells varied substantially among the hu-NSG mice tested, but the mean and the range appeared similar to that observed in PBMC samples of healthy human donors (Figure 6B). Notably, whereas commonly used laboratory strains of mice have substantially elevated frequencies of iNKT cells in the liver compared to the spleen [43], the hu-NSG mice typically showed similar frequencies of tetramer positive cells in the spleen and liver (Figure 6C). This lack of iNKT cell enrichment in the liver is similar to what has been observed in previous analyses of primary human liver samples [44,45,46]. To investigate the TCR usage of CD1d-restricted NKT cells from hu-NSG mice, we sorted T cells stained by the a-GalCer loaded CD1d tetramer and expanded them in vitro. CD1d tetramer-positive and -negative T cells were sorted separately, and expanded by exposure to PHA and irradiated allogeneic human feeder cells in the presence of recombinant IL-2. Flow cytometric analysis of the expanded cells indicated that the CD1d tetramer-positive cells used Va24 and Vb11 TCR chains, whereas the tetramer-negative T cell line sorted in parallel showed little or no staining for these TCR chains (Figure 7A). The CD1d tetramer-positive cells secreted cytokines in response to CD1d- transfected APCs that were pulsed with a-GalCer, but did not respond to the untransfected parental cell line, and the cytokine secretion was blocked by an anti-CD1d antibody (Figure 7B). DNA sequence analysis confirmed that the TCRa chain of the CD1d tetramer-positive cells was Va24, and demonstrated that it was rearranged to Ja18 using the same canonical rearrangement found in human iNKT cells (Figure 7C, top). Analysis of the TCRb chain identified a single dominant sequence consisting of Vb11 rearranged to Jb2.7, with a unique sequence at the V-J junctional region that is suggestive of N-region diversification (Figure 7C, bottom). Thus, the hu-NSG mice possess human CD1d-restricted T cells that utilize the canonical TCRa chain paired with Vb11, and that recognize a-GalCer presented by CD1d. Activation of NKT cells in vivo Injection of a-GalCer into common strains of laboratory mice results in rapidly detectable immunological activation that is PLoS ONE | www.plosone.org 6 June 2011 | Volume 6 | Issue 6 | e21701