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Blood, Vol. 108, Issue 1, 253-261, July 1, 2006
TNF downmodulates the function of human CD4+CD25hi T-regulatory cells
Blood Valencia et al.
108: 253
Supplemental materials for: Valencia et al
Files in this Data Supplement:
- Figure S1. TNFRII crosslinking reverses the suppressive function and downregulates FoxP3 expression by TGFβ1-induced regulatory cells (TGFβTR). (JPG, 38.1 KB)
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A. Purified CD4+CD25— T cells were stimulated with plate bound anti-CD3 at 1µg/ml with mature DC at a 1:20 ratio in the presence of recombinant human TGF 1 for 7 days. After this period, CD4+CD25+ T cells were magnetically purified and used in the in vitro assay of Treg function. Freshly sorted CD4+CD25— T cells or TGFTR alone or mixed together at a ratio of 1:1 were stimulated with platebound anti-CD3 with or without purified anti-TNFRII at (0.2µg/well) coated in a 96 well microtiter plate. After 72hrs, 3H thymidine incorporation was determined. The figure represents the mean data (± SEM) from 7 different experiments. Statistical analysis of the data was carried out using the Mann-Whitney test. The significance of differences between the groups is indicated by the brackets. B. Intracellular FoxP3 staining was carried out with TGFTR after overnight incubation with IL-2 alone or the combination of IL-2 and TNF (50ng/ml). Data are representative of 3 different experiments. Numbers indicate the percentage of positive cells and those in parentheses the mean fluorescence intensity of the stained cells. The horizontal bracket indicates staining with the isotype matched control mAb.
- Figure S2 (JPG, 51.3 KB)
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A. Concentration dependent loss of suppressive function of CD4+CD25hi Treg by TNF. CD4+CD25- T cells (5 × 104/well) or CD4+CD25hi Treg (5 × 104/well) alone or mixed together at a ratio of 1:1 were stimulated with platebound anti-CD3 (1µg/well). Recombinant TNF was added at the beginning of the culture at increasing concentrations as indicated. After 72hrs, 3H thymidine incorporation was determined. Data are the mean ± SD of 3 independent experiments. Statistical analysis was carried out using the two-tailed paired Student’s test. B. TNFRII crosslinking reverses the CD4+CD25hi Treg mediated suppression of the proliferation of CD4+CD25— effectors. Previously activated T cells that had upregulated surface TNFRII expression were used for in vitro regulatory assays as described in Materials and Methods. 96 well microtiter plates were coated with anti-TNFRII mAb at increasing concentrations as indicated along with anti-CD3. CD4+CD25— T cells (5 × 104/well) or CD4+CD25hi Treg (5 × 104/well) alone or mixed together at a ratio of 1:1 were stimulated with platebound anti-CD3 (1µg/well), with or without anti-TNFRII. After 72hrs, 3H thymidine incorporation was determined. Data are the mean ± SD of 3 independent experiments. Statistical analysis was carried out using the two-tailed paired Student’s test.
- Figure S3. CD4+CD25hi Treg from active RA patients fail to suppress normal donor CD4+ effectors (JPG, 38.5 KB)
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CD4+CD25— responder (5 × 104/well) from normal individuals and CD4+CD25hi Treg (5 × 104/well) from active RA patients were cultured with platebound anti-CD3 (1µg/well) either alone or at a 1:1 ratio. After 72hrs, 3H thymidine incorporation was determined. Results are the mean ± SD of 3 separate experiments. No statistical analysis was performed.
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