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Blood, Vol. 112, Issue 5, 2101-2110, September 1, 2008
Absence of donor Th17 leads to augmented Th1 differentiation and exacerbated acute graft-versus-host disease
Blood Yi et al.
112: 2101
Supplemental materials for: Yi et al
Files in this Data Supplement:
- Figure S1. No significant difference between IL-17−/− and WT C57BL/6 mice regarding spleen T cell percentage, surface phenotype, and proliferative function (JPG, 200 KB)
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Spleen cells from 8-week old, male, WT and IL-17−/−C57BL/6 mice were analyzed with multiple color flow cytometry. Sorted CD4+ T and CD25−CD4+ T cells were stimulated with anti-CD3/CD28 and T cell proliferation was measured with 3H-TdR incorporation. Sorted CD25hiCD4+ T cells were tested in the assay of suppression of the CD4+CD25− T proliferation. (A) spleen cells were stained with anti-TCRβ, CD4, CD8, CD44, and CD62L. First, TCRβ vs forward scatter (FS) was shown; second, the gated TCRβ+ cells were shown in CD4 vs CD8; third, the gated CD4+TCRβ+ or CD8+TCRβ+ cells were shown in CD44 vs CD62L. One representative of 4 individual mice in each group is shown. The mean ± SE of total spleen mononuclear cells was 78 ± 3.7 × 106 (WT) vs 77.6 ± 6.3 × 106 (IL-17−/−). The mean ± SE of percentage of TCRβ+ T cells among spleen mononuclear cells was 24.4 ± 0.8 (WT) vs 26.3 ± 0.6 (IL-17−/−). The ratio of CD4+ T /CD8+ T was 1.61 ± 0.04 (WT) vs 1.68 ± 0.03 (IL-17−/−). The percentage of naïve CD62LhiCD44loCD4+ T cells among total CD4+ T cells was 73.3 ± 0.6 vs 73.1 ± 0.8, and the percentage of naïve CD62LhiCD44loCD4+ T cells among total CD8+ T cells was 70.7 ± 1.4 (WT) vs 74.1 ± 1.1 (IL-17−/−). There was no significant difference between WT and IL-17−/− mice regarding above comparison (P>0.1). (B) spleen T cells purified by negatively selection were stained with anti-TCRβ, CD4, and CD8. First, TCRβ vs FS was shown; second, gated TCRβ+ cells were shown in CD4 vs CD8. One representative is shown of 2 replicated experiments, in which the purified T cells were used to induce GVHD as described in Fig. 1D and E. The purity of TCRβ+ cells was 95.9 ± 0.8% (WT) and 96.9 ± 0.3% (IL-17−/−), and the ratio of CD4+ T/CD8+ T is 1.61 ± 0.08 (WT) vs 1.63 ± 0.02 (IL-17−/−) (P>0.5). (C) spleen cells were stained by anti-TCRβ, CD4, CD25, and Foxp3 (intracellular). First, TCRβ vs CD4+ was shown; second, gated CD4+TCRβ+ T cells were shown in Foxp3 vs CD25. One representative of 4 individual mice is shown. The percentage of Foxp3+CD25+CD4+ T cells among CD4+ T cells was 7.8 ± 0.3 (WT) vs 8.0 ± 0.5 (IL-17−/−) (P>0.5). (D) titrated dose of purified CD4+ T cells from WT or IL-17−/− C57BL/6 mice were stimulated with anti-CD3/CD28, and 3H-TdR incorporation was measured. Mean ± SE of cpm of triplicated culture was calculated, and one representative of three replicated experiments is shown. (E) sorted CD25−CD4+ T cells from WT (left panel) or IL-17−/−(right panel) C57BL/6 mice were stimulated with anti-CD3/CD28 with or without presence of CD25+CD4+ T cells from WT or IL-17−/C57BL/6, and 3H-TdR incorporation was measured. Mean ± SE of cpm of triplicated culture was calculated, and one representative of three replicated experiments is shown.
- Figure S2. Sorting of CD44loCD4+ T cells (JPG, 62.9 KB)
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Enriched CD4+ T cells from WT or IL-17−/− C57BL/6 mice were stained with anti-CD44, and sorted into CD44hi and CD44lo subsets. After sorting, a small portion (0.2 × 106) of each subset was stained with CD62L. One representative of 3 replicated experiments is shown.
- Figure S3. Loss of IFN-γ leads to exacerbated GVHD and augmented Th17 differentiation (JPG, 56.6 KB)
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(A and B) Sublethally irradiated BALB/c recipients were transplanted with 2.5 × 106 TCD-BM cells and 2.5 × 106 spleen cells from wild-type (WT) or IFN-γ−/− C57BL/6 donors. The recipients were checked for clinical signs of GVHD and body weight every 5 days and monitored for survival daily. Body weight change and survival curves are shown. There were 12 mice in each group. Three replicated experiments were combined. (C) MLN cells of the recipients given WT or IFN-γ−/− donor cells were stained for H-2b, CD4, and intracellular IL-17, 13 days after HCT. Gated H2b+ cells are shown in IL-17 versus CD4. The percentage of IL-17+ cells is shown beside the gating boxes. One representative of 3 recipients in each group is shown.
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