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. 2007 Jan 22;204(1):33–39. doi: 10.1084/jem.20061531

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Copyright © 2007, The Rockefeller University Press

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Figure 2. — CD62L⁻ T reg cells from infliximab-treated RA patients are more potent suppressors than their CD62L⁺ counterparts and mediate their suppressive effects through IL-10 and TGF-β. CD4⁺CD25⁻, CD4⁺CD25^hiCD62L⁺, and CD4⁺CD25^hiCD62L⁻ were FACS (MoFlo) sorted from the PBMCs of healthy controls, active RA patients, and infliximab-treated RA patients. In all experiments, cells were stimulated with 2 μg/ml of soluble anti-CD3/CD28. (A) CD4⁺CD25⁻ T cells were cocultured with either CD62L⁺ or CD62L⁻ T reg cells (2:1 ratio shown) for 5 d, with [³H]thymidine added in the last 18 h of culture. Mean triplicate values shown from six patients. (B) CD4⁺CD25⁻ T cells were cultured alone or cocultured at a 2:1 ratio with either CD62L⁺ or CD62L⁻ T reg cells for 48 h. Cells were intracellularly stained for TNF-α and IFN-γ. Data shown expressed as mean ± SE of six patients and six healthy controls, and are represented as the percentage inhibition of cytokine production compared with CD4⁺CD25⁻ T cells alone. The means for percent IFN-γ⁺/TNF-α⁺ cells are as follows: healthy CD4⁺CD25⁻ 3.2/3.4, CD4⁺CD25⁻/CD62L⁺ T reg cells 0.7/0.9, CD4⁺CD25⁻/CD62L⁻ T reg cells 1.9/2.1; active RA CD4⁺CD25⁻ 5.2/13.2, CD4⁺CD25⁻/CD62L⁺ T reg cells 3.6/9.0, CD4⁺CD25⁻/CD62L⁻ T reg cells 3.1/7.8; post-infliximab CD4⁺CD25⁻ 4.2/8.6, CD4⁺CD25⁻/CD62L⁺ T reg cells 3.8/6.3; and CD4⁺CD25⁻/CD62L⁻ T reg cells 1.1/2.2. (C) CD4⁺CD25⁻ T cells from healthy and infliximab-treated RA patients were cultured alone or with CD62L⁻ T reg cells (2:1 ratio) for 48 h with anti-CD3/CD28 alone or in the presence of 2 μg/ml anti–TGF-β1, 0.5 μg/ml anti–IL-10, or anti–TGF-β1 and anti–IL-10 together, and stained for TNF-α and IFN-γ. Data depicted represent mean ± SE of six patients and healthy controls. (D) CD4⁺CD25⁻ T cells were cocultured, either directly or separated by a transwell membrane, with CD62L⁻ T reg cells from RA patients after infliximab, and stimulated with 2 μg/ml anti-CD3/CD28 for 48 h. Cells were intracellularly stained for TNF-α and IFN-γ, and the results are depicted as the percentage inhibition of cytokine production compared with CD4⁺CD25⁻ T cells alone. Data represent mean ± SE of four patients.