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Blood, Vol. 114, Issue 3, 667-676, July 16, 2009
NKT cells mediate pulmonary inflammation and dysfunction in murine sickle cell disease through production of IFN- and CXCR3 chemokines
Blood Wallace et al.
114: 667
Supplemental materials for: Wallace et al
Immunostaining of cells for flow cytometric analysis. Purified murine cells were washed twice (1% BSA in PBS) and erythrocytes were lysed (Biolegend). Cells were washed and resuspended at 1 × 106 cells/ml in 100 µl of staining buffer (1% BSA, 0.1% sodium azide in PBS). Non-specific Fcγ receptor binding of labeled antibodies was blocked by incubation with CD16/32 (clone 93) for 10 min prior to primary antibody staining. iNKT cells were stained first with the anti-mouse αGalCer-analog (PBS57) loaded CD1d tetramer (controls were stained with unloaded CD1d tetramer) for 30 min at room temperature in the dark. Other leukocytes were then incubated for 30 min at 4°C with various cell surface markers and a fixable LIVE/DEAD® stain was used for viability (Invitrogen). Cells were then washed, fixed, and permeabilized for intracellular staining. Alternatively, 100 µl of whole human blood was stained. iNKT cells and other markers were stained as described above. However, immediately after non-bound cell surface markers and the fixable LIVE/DEAD® stain were washed off, red blood cells were lysed. Cells were then washed, fixed, and permeabilized for intracellular staining. Fluorescence intensity was measured with a CyAn™ ADP LX 9 Color analyzer (DakoCytomation) with 405 (450/50, 530/40 emitter filters), 488 (530/40, 575/25, 613/20, 680/30, 750LP emitter filters), and 642 (665/20, 750LP emitter filters) nm excitation lasers. Data analyses were performed with FlowJo software (Tree Star, Inc.). Gates to determine mouse leukocyte populations were created based upon fluorescence minus one staining and isotype staining of single (low pulse width), live (Aqua−), and CD45-PerCP+ (BD; 30-F11) cells. Murine, neutrophils were identified as anti–neutrophil-FITC+ (Serotec; 7/4), CD11b-APC-AF750+ (EBioscience; M1/70), and Ly-6G/GR1-Pacific Blue+ (EBioscience; RB6–8C5). Murine NK cells were identified as NKp46-FITC+ (R&D; polyclonal) and CD3ε-Pacific Blue− (EBioscience; 500A2). Murine iNKT cells were identified as CD1d-tetramer-AF647+ (NIH tetramer facility) and CD3ε-Pacific Blue+. Murine CD4 T cells were identified as CD1d-tetramer-AF647−, CD3ε-Pacific Blue+, and CD4-PEAF610+ (Caltag; L3T4). Murine CD8 T cells were identified as CD1d-tetramer-AF647−, CD3ε-Pacific Blue+, and CD8α-APCAF750+ (EBioscience; 53–6.7) (Fig. S1). Additional surface and intracellular activation markers used: CD69-PE (EBioscience; H1.2F3), IFN-γ-PE (EBioscience; XMG1.2), CXCR3-PE (R&D; 220803). Human iNKT cells were identified as: single (low pulse width), live (Aqua−), and CD45-APCAF750+ (EBioscience; RAB-6B2), CD3-PEAF610+ (Caltag; S4.1), and CD1d-tetramer-APC+ (NIH tetramer facility) (Fig. S2). Additional surface and intracellular activation markers used: CD69-PE (EBioscience; FN50), IFN-γ (EBioscience; 4S.B3), CXCR3-FITC (R&D; 49801).
Files in this Data Supplement:
- Figure S1. Murine flow cytometric gating strategy (JPG, 138 KB)
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Murine pulmonary cells were identified as single cells (low pulse width), live (Aqua−) and CD45-PerCP+ (BD; 30–F11). Murine, neutrophils were identified as anti–neutrophil-FITC+ (Serotec; 7/4), CD11b-APC-AF750+ (EBioscience; M1/70), and Ly-6G/GR1-Pacific Blue+ (EBioscience; RB6–8C5). Murine NK cells were identified as NKp46-FITC+ (R&D; polyclonal) and CD3ε-Pacific Blue− (EBioscience; 500A2). Murine iNKT cells were identified as CD1d-tetramer-AF647+ (NIH tetramer facility) and CD3ε-Pacific Blue+. Murine CD4 T cells were identified as CD1d-tetramer-AF647−, CD3ε-Pacific Blue+, and CD4-PEAF610+ (Caltag; L3T4). Murine CD8 T cells were identified as CD1d-tetramer-AF647−, CD3ε-Pacific Blue+, and CD8α-APCAF750+ (EBioscience; 53–6.7). SSC: side-scatter; FSC: forward-scatter.
- Figure S2. Human flow cytometric gating strategy (JPG, 104 KB)
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Human iNKT cells were identified as: single cells (low pulse width), live (Aqua−), and CD45-APCAF750+ (EBioscience; RAB–6B2), CD3-PEAF610+ (Caltag; S4.1), and CD1d-tetramer-APC+ (NIH tetramer facility). SSC: side-scatter; FSC: forward-scatter.
- Figure S3. Liver and spleen from NY1DD mice have increased and activated iNKT cells at baseline that are hyper-responsive to hypoxia-reoxygenation (JPG, 172 KB)
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(A) Representative flow cytometry plots of iNKT cells (CD3+ and CD1d-tetramer+). (B) iNKT cells were identified from the live, CD45+ lymphocyte gate as CD1d-tetramer+ CD3+ cells. Compared to C57BL/6 mice, NY1DD mouse liver and spleen have a higher number of resident iNKT cells and 3 hrs of hypoxia followed by 4 hrs of reoxygenation increased this effect. (B,C) iNKT cells from NY1DD mice are more activated as defined by higher percent of surface CD69 and intracellular IFN-γ that is amplified by H/R. Data were analyzed by one-way ANOVA with Neuman-Keuls post-testing. * P<0.05 was considered significant. SSC: side-scatter; H/R: hypoxia-reoxygenation.
- Figure S4. The activation marker CD69 and intracellular IFN-γ are increased on pulmonary lymphocytes from NY1DD mice and are transiently decreased after iNKT cell inhibition with anti-CD1d (JPG, 121 KB)
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Representative flow cytometry plots of CD69 and IFN-γ expression on pulmonary lymphocytes. NY1DD mice have increased CD69 surface expression and intracellular IFN-γ levels on pulmonary lymphocytes that decrease 1 day, but not 5 days after treatment with anti-CD1d. Data were analyzed by one-way ANOVA with Neuman-Keuls post-testing. * P<0.05 was considered significant. SSC: side-scatter; H/R: hypoxia-reoxygenation.
- Figure S5. Hypothetical mechanism of iNKT cell activation and downstream effects (JPG, 25.4 KB)
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Chronic IRI in SCD may create an altered self-lipid that can be presented via CD1d on APCs to iNKT cells. This presentation may activate the iNKT cells to release IFN-γ and IL-2. IFN-γ triggers the release of IFN-γ inducible chemokines (CXCL9 and CXCL10) from resident epithelial (EpiC) or endothelial cell (EC). IL-2 has been previously shown to upregulate CXCR3 expression on lymphocyte effector cells. Taken together, the release of CXCL9/CXCL10 and the upregulation of CXCR3 could potentially enhance the trafficking of lymphocyte effector cells to the lung. IRI: ischemia-reperfusion injury.
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