1. Volume 33, Issue 2, Pages 216-228 (August 2010)
Lysosomal α-Galactosidase Controls the Generation of Self Lipid Antigens for Natural Killer T Cells 
Alexandre Darmoise, Susann Teneberg, Lauriane Bouzonville, Roscoe O. Brady, Michael Beck, Stefan H.E. Kaufmann, Florian Winau 
Immunity 
Volume 33, Issue 2, Pages (August 2010)
DOI: /j.immuni
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2. Immunity 2010 33, 216-228DOI: (10.1016/j.immuni.2010.08.003)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 DCs Lacking α-Gal-A Activity Induce iNKT Cell Responses
(A) DCs from wild-type (WT) or Gla−/− mice were pulsed with αGalCer (100 ng/ml) for 16 hr, or were left untreated, before coculture with liver iNKT cells from Vα14-Jα18 transgenic mice. Additionally, DCs were treated with CD1d-blocking antibody (20 μg/ml) or isotype control (iso, 20 μg/ml). Two days after stimulation, antigen-specific proliferation was determined by [3H]-thymidine incorporation as depicted by counts per minute (CPM).
(B) DCs from WT or Gla−/− mice were treated with DGJ (1 mM), or human recombinant α-Gal-A (Replagal, 20 μg/ml), or were left untreated. In parallel, DCs were pulsed with iGb3 (1 μg/ml) for 16 hr. After washing, DCs were cocultured with liver iNKT cells. Moreover, DCs additionally lacking CD1d expression (Cd1d1−/−) were used as APCs. Three days after stimulation, cytokines released in the culture supernatants were determined by ELISA.
(C) DCs from WT, Gla−/−, Gla−/−Cd1d1−/−, or β-Gal-deficient (Glb1−/−) mice were cocultured with the invariant NKT cell hybridoma DN32.D3 or the noninvariant TCB11. Additionally, WT APCs were preincubated with 1 mM DGJ, and Gla−/− DCs were reconstituted with 10 or 20 μg/ml recombinant α-Gal-A. One day after coculture, IL-2 secretion of hybridoma cells was measured by ELISA. (A–C) Graphs show mean ± SD of triplicate cultures.
(D) IB4-mediated blocking of endogenous antigens abrogates iNKT cell activation by α-Gal-A-deficient APCs. Gla−/− and WT DCs were pulsed for 6 hr with iGb3 (400 ng/ml), αGalCer (100 ng/ml), or were left unpulsed. In addition, DCs were preincubated with 0.5–5 μg/ml IB4 for 2 hr or were left untreated. Subsequently, APCs were cocultured with the invariant NKT cell hybridoma DN32.D3 for 1 day, prior to measurement of IL-2 by ELISA. Depicted is the percentage of the remaining IL-2 response of iNKT cells upon IB4 treatment compared to untreated DCs (Unpulsed, 200 pg/ml; iGb3, 277 pg/ml and 638 pg/ml; αGalCer, 2866 pg/ml and 3824 pg/ml, for WT or Gla−/− DCs, respectively). In the right panel, Gla−/− DCs were preincubated with 0.5–2 μg/ml IB4 or were left untreated. Subsequently, APCs were cocultured with primary liver iNKT cells, and secretion of IFN-γ and IL-4 was measured by ELISA 2 days after stimulation. Depicted is the percentage of the remaining cytokine response of liver iNKT cells upon IB4 treatment compared to untreated DCs (758 pg/ml for IFN-γ and 304 pg/ml for IL-4)
(E) WT and Gla−/− DCs were incubated with 100 μg/ml NB-DGJ for the indicated period of time or were left untreated. Subsequently, APCs were cocultured with the invariant NKT cell hybridoma DN32.D3, and IL-2 secretion was measured by ELISA 1 day after stimulation. Depicted is the percentage of the remaining IL-2 response of iNKT cells upon NB-DGJ treatment compared to untreated DCs (60 pg/ml for WT and 235 pg/ml for Gla−/− DCs). Graphs show mean ± SEM of triplicate cultures. (A–E) Results depicted are representative of at least three independent experiments with similar results. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < as analyzed by Student's t test. NS, not significant.
Immunity  , DOI: ( /j.immuni )
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4. Figure 2 Absence of α-Gal-A Mediates iNKT Cell Activation In Vivo
(A) α-Gal-A-deficient DCs activate iNKT cells in vivo. Wild-type (WT) mice received intravenously (i.v.) 1 × 106 WT DCs, pulsed with iGb3 (1 μg/ml) for 16 hr as indicated. Alternatively, mice received 1 × 106 DCs generated from Gla−/− or Gla−/−Cd1d1−/− mice. As controls, recipient animals received PBS or untreated WT DCs. After 18 hr, liver mononuclear cells were analyzed by flow cytometry. The percentages of iNKT cells defined as αGalCer-loaded CD1d-tetramer+ TCRβ+ cells among B220− lymphocytes are depicted in the dot plots. The right panel shows the differences in liver iNKT cell percentages in recipient mice stimulated with the different types of DCs when compared to PBS-treated control animals. For the bar chart, the data from five independent experiments were pooled, including three mice per group. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < as analyzed by one-way ANOVA with variance comparison between all groups followed by a Bonferroni post-test.
(B) Surface expression of CD25 and CD69 by iNKT cells was analyzed upon transfer of Gla−/−, Gla−/−Cd1d1−/−, or iGb3-pulsed WT DCs, as described in (A).
(C) Deficiency of α-Gal-A promotes iNKT cell expansion in vivo. Thymocytes from Vα14-Jα18 transgenic mice were subjected to CD8+ T cell depletion, labeled with CFSE, and injected intravenously (i.v.) into sublethally irradiated WT, Cd1d1−/−, Gla−/−, or Gla−/−Cd1d1−/− mice. Nine days after adoptive transfer, iNKT cells prepared from livers of recipient mice were identified by flow cytometric staining with anti-TCRβ and CD1d-tetramers as shown in the dot plots. In parallel, cotransferred conventional T cells were detected as TCRβ+, CD1d-tetramer− liver lymphocytes. Histograms of the left panel demonstrate the proliferation profiles of CFSE-labeled transferred iNKT cells, including their mean fluorescence intensity (MFI). The right panel depicts CFSE dilution of conventional T cells as controls. (B and C) Shown is one representative experiment of three performed with similar results.
Immunity  , DOI: ( /j.immuni )
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5. Figure 3
Homeostasis of iNKT Cells Is Impaired in the Periphery of α-Gal-A-Deficient Hosts
(A) Specific loss of peripheral iNKT cells is a hallmark of Gla−/− mice. Dot plots show percentages of αGalCer-loaded CD1d-tetramer+ CD44+ iNKT cells among liver mononuclear cells, splenocytes, and thymocytes from WT or Gla−/− mice measured by FACS. The bar charts demonstrate the percentages or absolute numbers of iNKT cells in liver, spleen, or thymus of α-Gal-A-deficient compared to WT mice.
(B) WT and Gla−/− mice received BrdU (0.8 mg/ml) in the drinking water. Five days later, mice were sacrificed and BrdU incorporation by immature (CD44− NK1.1−), semimature (CD44+ NK1.1−), and mature (CD44+ NK1.1+) thymic iNKT cells was analyzed after intracellular staining with a BrdU antibody. iNKT cells were identified as αGalCer-loaded CD1d-tetramer+ TCRβ+ cells. Additionally, the generation of conventional T cells was analyzed in α-Gal-A-deficient and WT thymi by detection of CD1d-tetramer−, NK1.1−, HSA+ lymphocytes. Depicted are the percentages of BrdU-incorporating cells.
(C) Following the same approach as described in (B), liver and splenic iNKT cells in Gla−/− and WT mice were analyzed for BrdU incorporation, including the detection of conventional T cells and NK cells as controls. (A–C) Results are representative of at least three independent experiments with similar results. Error bars depicted in graphs represent mean values ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < as analyzed by Student's t test. NS, not significant.
Immunity  , DOI: ( /j.immuni )
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6. Figure 4
Peripheral iNKT Cells Show Signs of Chronic Exposure to Self Antigens in Gla−/− Mice
(A) WT and Gla−/− mice received i.v. a fluorescent poly-caspase substrate (Flivo). After 45 min, mice were sacrificed, and substrate fluorescence was measured in αGalCer-loaded CD1d-tetramer+ TCRβ+ iNKT cells.
(B) Dot plots show the percentages of liver, spleen, and thymus iNKT cells expressing surface Ly49C/I in WT and Gla−/− mice. iNKT cells were identified as in (A).
(C) Peripheral iNKT cells in Gla−/− mice are tolerant. WT and Gla−/− mice received i.v. αGalCer in PBS containing 0.5% polysorbate 20 or PBS/0.5% polysorbate 20 alone (vehicle). After 3 hr, mice were sacrificed and liver mononuclear cells were isolated and stained with αGalCer-loaded CD1d-tetramers and TCRβ mAb. Cells were fixed and permeabilized prior to intracellular IFN-γ staining analyzed by flow cytometry. (B and C) Numbers in dot plots represent percentages of Ly49C/I+ or IFN-γ+ iNKT cells in the respective quadrants.
(D) Serum concentrations of IFN-γ in WT and Gla−/− mice were determined by ELISA 16 hr following i.p. injection of αGalCer in vehicle or vehicle alone. Error bars depicted in graphs represent mean values ± SEM (A–D). Results are representative of at least three independent experiments with similar results. At least three mice were used per group.
Immunity  , DOI: ( /j.immuni )
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7. Figure 5
DCs Lacking α-Gal-A Amplify Antimicrobial Responses by iNKT Cells
DCs from WT, Myd88−/−, Gla−/−, and Gla−/−Cd1d1−/− mice were incubated for 16 hr with L. monocytogenes, LPS (1 μg/ml), CpG ODN (2 μg/ml), or were left untreated, prior to coculture with liver iNKT cells. After 72 hr, IFN-γ release into the culture supernatants was quantified by ELISA. Results are representative of at least three independent experiments with similar results. Error bars depicted in column graphs represent mean values ± SEM of triplicate cultures. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < as analyzed by Student's t test. NS, not significant.
Immunity  , DOI: ( /j.immuni )
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8. Figure 6
TLR Stimulation Mediated through MyD88 Inhibits α-Gal-A Activity in DCs
WT and Myd88−/− DCs were incubated with L. monocytogenes (MOI 5), exposed to LPS (1 μg/ml), or polyI:C (10 μg/ml) for the indicated period of time. After extensive washing, DC pellets were disrupted, and α-Gal-A activity was measured in lysates using a fluorogenic substrate specific for α-Gal-A. Activity of α-Gal-A is indicated as percentage of remaining enzyme activity as compared to WT, untreated DCs. Results are representative of at least five independent experiments with similar results.
Immunity  , DOI: ( /j.immuni )
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9. Figure 7
α-Gal-A Is a Central Regulator of TLR-Induced Lysosomal GSL Accumulation
(A) DCs from WT and Gla−/− mice were incubated with Alexa 488-coupled STxB (5 μg/ml) for 30 min at 37°C. After extensive washing, DCs were stained with CD11c mAb, washed, and analyzed by FACS. Numbers in dot plots represent percentages of STxB+ DCs in the respective quadrants.
(B) WT and MyD88-deficient DCs were incubated with Alexa 488-coupled STxB (5 μg/ml) for 30 min, before LPS (1 μg/ml), polyI:C (10 μg/ml), CpG ODN (2 μg/ml), or beads were added for 12 hr. In parallel, DCs were treated with bafilomycin A1 (50 nM) for 1 hr prior to incubation with STxB. After extensive washing, DCs were analyzed by flow cytometry. Histograms represent STxB fluorescence measured in untreated compared to treated CD11c+ DCs. (A and B) Shown is one representative experiment out of three performed with similar results.
Immunity  , DOI: ( /j.immuni )
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