A Fungal Glycosphingolipid Directly Activates Natural Killer T Cells and Rapidly Induces Airways Disease
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1 A Fungal Glycosphingolipid Directly Activates Natural Killer T Cells and Rapidly Induces Airways Disease Lee A. Albacker, Vinod Chaudhary, Ya-Jen Chang, Hye Young Kim, Ya-Ting Chuang, Muriel Pichavant, Rosemarie H. DeKruyff, Paul B. Savage, and Dale T. Umetsu Supplemental Figure 1 R L % baseline WT splenocyte into Tcra-J18 / A. fumigatus BALB/c A.fumigatus Tcra-J18 / A. fumigatus * Methacholine (mg/ml) Supplemental Figure 1. Reconstitution of inkt cell deficient mice with WT splenocytes rescues AHR. 1 7 WT splenocytes were used to reconstitute Tcra-J18 / recipients with unactivated inkt cells, which were then challenged with intranasal A. fumigatus extract. One representative experiment of two is shown. Significant differences were found between reconstituted Tcra-J18 / mice and Tcra-J18 / mice that did not receive splenocytes (n=5, * p<.5 Two-Way ANOVA, Bonferoni pose-test). Nature Medicine: doi:1.138/nm.3321
2 Supplemental Figure 2 a. b. c. 11, 1, * BALB/c Cd1d1 / Myd88 / Ticam1 / * * D pasp-b (µg ml 1 ) PBS PBS57 sasp-b (µg ml 1 ) D pasp-b sasp-b Lipid Supplemental Figure 2. IFN-γ production by inkt cells after stimulation with Asperamide B. (a) inkt, BMDC cocultures stimulated with pasp-b. (b) inkt, BMDC cocultures stimulated with sasp-b. (a-b) * p <.5, p <.1, One-Way ANOVA, Dunnett s Multiple Comparison Test to DMSO Control. (c) inkt cocultured with the indicated BMDC stimulated witih the indicated lipids ( p <.1, * p <.1, Two-Way ANOVA, Bonferoni post-test). PBS57 Nature Medicine: doi:1.138/nm.3321
3 Supplemental Figure 3 a. Unloaded Tetramer Unloaded Tetramer TCRβ b % of Max % of Max 4 6 % of Max Vβ8.1/2 Vβ Vβ c. inkt cell line % of Max Vβ8.1/2 Vβ Vβ d. Only Only PBS Supplemental Figure 3. Asperamide B load CD1d tetramers stain primary inkt cell lines. (a) Primary inkt cell lines stain with Asperamide B and PBS57 loaded CD1d tetramers. (b) The Vβ usage of the inkt cell line stained with the Asperamide B loaded CD1d tetramers. (c) The Vβ usage of the inkt cell line stained with PBS57 loaded CD1d tetramers was similar to the V β usage of Asperamide B loaded CD1d tetramer stained inkt cells in the line. This suggests that the Asperamide B CD1d tetramer did not bind to a subset of inkt cells. (d) Incubation of inkt cell lines simultaneously with Asperamide B and PBS57 loaded CD1d tetramers (right panel) results in staining only with the PBS57 loaded CD1d tetramers. Nature Medicine: doi:1.138/nm.3321
4 Supplemental Figure 4 a. b. inkt cells (1 2 ) * IL IL-13 Vehicle pasp-b PBS c. Cells (1-3 ) * BALB/c Vehicle BALB/c pasp-b CD1d -/- Vehicle CD1d -/- pasp-b IL-4/13 -/- Vehicle IL-4/13 -/- pasp-b Mac Eos Neut Lymph Cell Type Supplemental Figure 4. Asperamide B induces IL-4 and IL-13 expression in BAL inkt cells (a) Total numbers of lung inkt cells after intranasal administration of vehicle, pasp-b, or PBS57 (* p <.1, student s t-test). (b) Representative IL-4 and IL-13 FACS plots gated on inkt cells. Numbers indicate percentage of inkt cells within that gate. (c) BAL cell count of BALB/c, CD1d1 /, or Il4 / Il13 / treated with vehicle or pasp-b (with Figure 5i-j) (* p <.1, Two-Way ANOVA, Bonferoni post-test).. Nature Medicine: doi:1.138/nm.3321
5 Supplemental Figure 5 Supplemental Figure 5. ST2 deficient mice have normal numbers of inkt and T cell populations. The spleens of WT and ST2 deficient (Il1rl1 / ) mice were stained for TCRβ, PBS57 loaded CD1d tetramers, CD4, and CD8. Panels show gating on all cells for TCR and tetramer staining. Sub-gating on inkt and other T cells shows a normal CD4/CD8 profile for these populations. Nature Medicine: doi:1.138/nm.3321
A Fungal Glycosphingolipid Directly Activates Natural Killer T Cells and Rapidly Induces Airways Disease
A Fungal Glycosphingolipid Directly Activates Natural Killer T Cells and Rapidly Induces Airways Disease The Harvard community has made this article openly available. Please share how this access benefits
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Pair-fed % inkt cells 0.5. EtOH 0.0
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ndln NK Cells (x10 3 ) Days post-infection (A/PR/8) *** *** *** Liver NK Cells (x10 4 ) Days post-infection (MCMV)
A mln NK Cells(x ) 6 1 * ndln NK Cells (x ) ns C Lung NK Cells(x ) 1 1 7 * D LN NK Cells (x ) 1 7 1 7 Days post-infection (A/PR/8) * * E Liver NK Cells (x ) 1 7 Days post-infection (A/PR/8) * * * 1 7 Days
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pplementary Figur Supplementary Figure 1. a.
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doi: 1.138/nature775 4 O.D. (595-655) 3 1 -ζ no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 1 Soluble and plated anti- Abs induce -! signalling. B3Z cells stably expressing!
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