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 20× 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).
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 40× magnification, using an antibody against human CD1a, with DAB chromogenic development (brown color) and hematoxylin counterstaining of nuclei (blue color).
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 40× magnification.
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 α-galacosylceramide (α-GalCer). Results are representative of 2–3 independent experiments.
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-γ 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.
Figure 6.
A) Flow cytometric analysis of splenocytes from an engrafted mouse using fluorescently labeled human CD1d tetramer (y-axis) and an antibody against human CD3 (x-axis). The left plot shows staining using vehicle treated CD1d tetramer, and the right plot shows α-GalCer loaded CD1d tetramer. B) Percentages of CD1d tetramer-positive cells detected from splenocytes of 10 engrafted mice, compared to PBMCs from 15 healthy adult human donors. The medians for each data group are as follows: spleen cells from hu-NSG mice, tetramer+vehicle = 0.0039%, tetramer+α-GalCer = 0.048%; human PBMCs, CD1d tetramer+vehicle = 0.0041%, CD1d tetramer+α-GalCer = 0.0745%. C) Analysis of the splenocytes and liver mononuclear cells from 4 engrafted mice for human cells stained by CD3 and α-GalCer loaded CD1d tetramer.
Figure 7.
CD1d tetramer-positive and -negative T cells were flow cytometrically sorted from an engrafted mouse and expanded in vitro. A) Flow cytometric analysis of the expanded cells (tetramer-positive top row, tetramer-negative bottom row). Filled histograms show staining with fluorescently labeled anti-CD3, α-GalCer loaded human CD1d tetramer, anti-Vα24, and anti-Vβ11 antibodies, respectively. Open histograms show staining with vehicle treated CD1d tetramer or isotype-matched negative control antibodies. B) Cytokine secretion by the tetramer-positive and -negative cell lines in response to CD1d-transfected or untransfected (UT) APCs in the presence of α-GalCer. Open bars indicate assays that were performed in the presence of an anti-CD1d blocking antibody, and filled bars show assays performed in the presence of an isotype matched negative control antibody. C) Predicted amino acid sequences of the junctional regions of the TCRα and β chains from hu-NSG tetramer-positive T cells compared to those of NKT cell clones previously derived from the human tissues shown in parentheses. Dashes indicate identity with the consensus sequences shown at the bottom.
Figure 8.
Activation of NKT cells in vivo.
A) The plot on the left shows results from engrafted mice that were injected intraperitoneally with 3 µg α-GalCer or with an equivalent amount of vehicle alone. Blood samples were collected at 2–4 and 24–48 hours after injection and analyzed for human IFN-γ by ELISA. Each symbol represents the mean of 3 replicates. The P value shown on the plot was calculated using a one-tailed unpaired t test. The plot on the right shows results from human PBMC samples that were incubated in vitro with α-GalCer or vehicle alone. Supernatants were collected after 48 hours and analyzed for IFN-γ by ELISA. The P value shown on the right plot was calculated using a one-tailed paired t test. B) Results from the same experiments analyzed for human IL-4 by ELISA. C) Heparinized plasma samples taken at 48-hours post injection were analyzed for enzyme activity of the liver transaminases AST and ALT, which serve as biomarkers of liver injury. The plot shows the enzyme activities detected from samples from mice that were treated with vehicle (open squares) or with α-GalCer (filled squares). There are no significant differences between the groups.