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. 2008 May 12;205(5):1037–1048. doi: 10.1084/jem.20072528

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© 2008 Fang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jem.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 5. — DN TNFR25 transgene blocks TNFR25 signaling and interferes with Th2 cytokine production upon secondary stimulation. (A) Expression of DN-TNFR25-tg on lymph node cells. Lymph node cells were gated on CD4, CD8, CD11c, B220, DX5, or NK1.1/CD3; TNFR25 expression is displayed in the histogram. Black curves, isotype controls; red curves, DN TNFR25-tg lymph node cells; green curves, nontransgenic lymph node cells from littermates; all cells are in resting, nonactivated state. (B) DN TNFR25-tg blocks cytokine co-stimulation by agonistic anti-TNFR25 (4C12). Purified WT and DN TNFR25-tg (DN) CD4 cells were stimulated for 3 d with anti-CD3 with or without the agonistic anti-TNFR25 antibody 4C12 (5 μg/ml). The supernatants were analyzed for cytokines by ELISA. (C) DN TNFR25-tg CD4 T cells do not exert Th2 cytokine production in secondary activation. WT and DN TNFR25-tg CD4 T cells were purified by negative selection and activated with 2 μg/ml immobilized anti-CD3 and 1 μg/ml soluble anti-CD28 for 3 d under Th cell neutral conditions (no cytokines were added). Cells were harvested, washed, replated, and restimulated with 1 μg/ml immobilized anti-CD3 for 2 d. The supernatants were collected for cytokine ELISA assay. All experiments were performed more than three times with reproducible results. Error bars represent the mean ± the SEM. ***, P < 0.001.