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Blood, Vol. 109, Issue 8, 3316-3324, April 15, 2007
The Pten/PI3K pathway governs the homeostasis of V 14iNKT cells
Blood Kishimoto et al.
109: 3316
Supplemental materials for: Kishimoto et al, Vol 109, Issue 8, 3316-3324
Files in this Data Supplement:
- Document S1. Supplemental materials and methods (PDF, 78.1 KB)
- Figure S1. Characterization of Vα 14iNKT development in the absence of Pten (PDF, 258 KB) -
(A) Normal V 14iNKT cell proliferation in the mutant thymus. Ptenflox/flox and LckCrePtenflox/flox mice (7 weeks of age) were intraperitoneally injected twice daily with BrdU for 2 days, and the thymi were harvested. The percentage of BrdU-positive cells in the immature stage 2 V14iNKT population (GalCer-CD1d+TCR +NK1.1− cells) was determined as described in “Supplemental materials and methods.” (B) Decreased mature V14iNKT cells in the mutant periphery. The spleens and livers of 8-week-old Ptenflox/flox and LckCrePtenflox/flox mice were examined by flow cytometry for the percentages of NK1.1+ and NK1.1− V14iNKT cells present. While NK1.1+ cell numbers were significantly reduced in mutant spleen and liver, NK1.1− cells were present at normal levels. Results are expressed as the mean ± SEM for 3 mice per group. **P < .01. (C) Normal expression of CD1d and H-2Kb in mutant thymus and spleen. Levels of CD1d and H-2Kb expression on CD4/CD8 double-positive (DP) thymocytes and thymic and splenic DCs of Ptenflox/flox and LckCrePtenflox/flox mice were analyzed by flow cytometry. Data shown are from 1 mouse per genotype and are representative of 3 animals examined per genotype.
- Figure S2. Increased expression of Ly49A inhibitory receptor (PDF, 120 KB) -
GalCer-CD1d+TCR+NK1.1+ cells from the thymi of 8-week-old Ptenflox/flox and LckCrePtenflox/flox mice were analyzed by flow cytometry to determine expression levels of Ly49A. Data shown are from 1 mouse per genotype and are representative of 3 animals examined per genotype.
- Figure S3. Normal response of Pten-deficient NKT cells to IL-15 (PDF, 91.6 KB) -
NK1.1+TCR+ cells (1 × 104) purified from liver MNCs of 7-week-old Ptenflox/flox and LckCrePtenflox/flox mice were cultured in the absence (none) or presence of IL-15 (25 ng/mL) for 48 hours and pulsed with 3H-thymidine for 12 hours. No significant differences in proliferation were observed; P = .22. Results are expressed as the mean ± SEM and are representative of 3 trials.
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