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Blood, Vol. 112, Issue 9, 3704-3712, November 1, 2008
IL-7 and IL-15 differentially regulate CD8+ T-cell subsets during contraction of the immune response
Blood Rubinstein et al.
112: 3704
Supplemental materials for: Rubinstein et al
Files in this Data Supplement:
- Figure S1. IL-2, IL-7, and IL-15 cytokine complexes are biologically active and induce the proliferation of CD8+ memory-phenotype T cells (JPG, 101 KB)
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(A) Mice were adoptively transferred with CFSE-labeled T cells enriched from Thy1.1+ IL-7 transgenic mice as previously described.1 Mice were treated every 48 hours for one week with cytokine complexes as indicated. On day 7, spleens were harvested and CFSE dilution among donor CD8+CD44hi T cells was assessed and is indicated by the blue line. The red shaded region shows the CFSE intensity of donor CD8+CD44hi T cells from mice treated with vehicle alone. Note, it is not clear whether IL-15 complexes with human IL-15 are inherently more active than with murine IL-15, or whether this is related to different manufacturing procedures. (B) Spleen sizes from “A” indicate cellular expansion. The cell count (in millions) is shown for each spleen. For this and the remaining portions of this figure, IL-15 complexes were generated with murine IL-15. (C) The distribution of different cell populations within the spleen was assessed by FACS analysis. B cells were identified as B220+IgM+. Pre- and pro-B cells were identified as B220+IgM−. NK cells were identified as NK1.1+DX5+. Other cell populations were identified as described based on expression of Gr1, CD11b, and CD8. (D) Bone marrow harvested from mice in (C) was stained for B220 and IgM. All results are representative of at least two similar experiments.
- Figure S2. IL-7 and IL-15 complexes augment CD8+ T-cell memory (JPG, 58.3 KB)
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(A) Splenocytes were harvested on day 55 of the experiment outlined in Figure 1A and the percentage and (B) number of OT-I CD8+ T cells was assessed in the spleen. (C) A similar experiment as in (A) except that the percentage of OT-I CD8+ T cells was assessed in the PBL on day 68. (D) As in (C) except the percentage of KLRG1hiCD127lo among OT-I CD8+ T cells was determined. For A–D, the triangles indicate individual mice and the bar indicates the mean. (E) Mice were infected with VSV-OVA and treated with or without IL-15 complexes (n=2/group) every 48 hours for two weeks starting on day 1 following infection. Shown are the percentages of OT-I CD8+ donor T cells among PBL. The error bars indicate the standard deviation.
- Figure S3. Expression of cell surface markers on donor OT-I CD8+ T cells on day 17 after cytokine treatment (JPG, 56 KB)
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Mice were treated with IL-7 or IL-15 complexes as described in Figure 1A. On day 17 post-infection, splenocytes were harvested and analyzed by flow cytometry. Shown is the percentage of OT-I CD8+ T cells staining positive for the indicated markers. The small triangles indicate individual mice and the bar indicates the mean from mice pooled from three experiments. **, p <0.01, and *, p <0.05, indicate statistical analysis by Student’s t test.
- Figure S4. KLRG1hi and KLRG1lo CD8+ T cells produce IFNγ irrespective of treatment with cytokine complexes (JPG, 51.6 KB)
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Mice were treated as described in Figure 1A. On day 15 post-infection, splenocytes were stimulated for 5 hours with 0.1mg/ml OVA-peptide and IFNγ was assessed with reagents and according to instructions outlined in the Cytofix/Cytoperm Fixation/ Permeabilization Solution Kit with Golgistop (554715, BD Bioscience). Results are representative of 2 mice per condition. All data are representative of two independent experiments. Similar results were also observed after treatment with IL-2 cytokine complexes.
- Figure S5. Expression of cytokine receptor subunits on different subsets of CD8+ T cells during T-cell contraction (JPG, 59.3 KB)
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OT-I CD8+ T cells (105) were adoptively transferred into B6 mice followed by infection with VSV-OVA. On day 10 post infection, spleens were harvested and stained for surface expression of the markers indicated. Donor and host CD8+ T cells were distinguished based on expression of CD45.1.
REFERENCES
1. Rubinstein MP, Kovar M, Purton JF, et al. Converting IL-15 to a superagonist by binding to soluble IL-15R{alpha}. Proc Natl Acad Sci U S A. 2006;103:9166–9171.
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