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Blood, Vol. 110, Issue 4, 1199-1206, August 15, 2007
Role of CD28 in fatal autoimmune disorder in scurfy mice
Blood Singh et al.
110: 1199
Supplemental materials for Singh et al, Vol. 110, Issue 4, 1199-1206
Mouse breeding protocol
foxp3sf/+ females were crossed with cd28−/− males to obtain foxp3sf/Ycd28+/− and foxp3sf/+cd28+/− mice. foxp3sf/+cd28+/− mice were backcrossed with cd28−/− males to obtain foxp3sf/Ycd28−/− males. From this mating we screened 4 litters and obtained 3 cd28−/−scurfy males, which were mated with cd28−/− foxp3sf/+ females to derive cd28−/− scurfy males and females. Mice carrying the scurfy mutation or CD28 gene disruption were identified by polymerase chain reaction (PCR) using primers and conditions suggested by Jackson laboratory.
Files in this Data Supplement:
- Figure S1. Visual comparison of thymus and lymph node from 3 week old mice with following genetic backgrounds (JPG, 69.9 KB)
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WT: C57BL/6, CD28(−): cd28−/− mice with C57BL/6 background, CD28(−)scurfy: cd28−/− foxp3sf mice, scurfy: cd28+/+ foxp3sf mice. The magnification of the image is shown by the scale bar in the lower right corner.
- Figure S2. IL-10 and IFN-γ production by CD62L+CD44− and CD62L−CD44+CD4 T cells in response to anti-CD3 stimulation in the presence or absence of anti-CD28 antibody (JPG, 68.1 KB)
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Supernatants from figure 4 were tested for IL-10 and IFN- by ELISA. The error bars represent the SD of the duplicate samples.
- Figure S3. Cytokine production by cd28−/− and cd28+/+ scurfy mouse CD8 and NKT cells (JPG, 55.3 KB)
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Mo-Flo sorted CD8+DX5−TCR+ (CD8) and DX5+TCR+ (NKT) T cells (2 × 104 cells/well in flat bottomed 96 well plate) were activated with 1µg/mL of anti-CD3 coated plates in the absence (open bars) or presence (closed bars) of anti-CD28 antibody (0.5 µg/mL). Forty-eight hours later, a portion of culture supernatant was harvested for analysis of cytokines indicated on the right side of panels. IL-4 and IL-10 were not detectable in samples from CD8 T cells and not shown. The error bars represent the SD of the duplicate samples.
- Figure S4. Histology of lung and liver from cd28−/− and cd28+/+ scurfy mice (JPG, 77.7 KB)
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Paraformaldehyde fixed (A) lung and (B) liver tissues were sliced for 5 µm and were analyzed by hematoxylin and eosin (H&E) staining (×600, numerical aperture 1.4). Mononuclear cell infiltration was observed in cd28+/+ CD28(+) but not in cd28−/−CD28(−) scurfy mice. Tissues were fixed in 10% buffered formalin and embedded in paraffin. Hematoxylin and eosin (H&E)–stained sections (5 µM) were mounted with Cytoseal™60 (Richard Allan Scientific, Kalamazoo, MI) and analyzed by light microscopy. Images were acquired at room temperature using an Olympus Provis (Model #AX70TRF, Japan) microscope equipped with a Spot Camera, (Model #1.3.0, Diagnostic Instruments, Sterling Heights, MI) and Spot advance software (Version 4.0.9, Diagnostic Instruments). All the images were adjusted for uniform color and brightness using Adobe Photoshop (version 7.0, San Jose, CA). Original magnification ×200/0.5 NA dry objective (Figure 4a ) or ×600/1.4 NA oil objective (Figure 4b).
- Figure S5. Histology of lung from CTLA4-Ig injected scurfy mice (JPG, 325 KB)
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Representative images from lungs of PBS-injected 22 day old scurfy mice (PBS), of CTLA4-Ig–injected scurfy 22 day old (day22) and 48 day old (day48) mice are shown. Tissues were processed and images were acquired as in Figure S4 (original magnification ×200.
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