Efficient Therapeutic Function and Mechanisms of Human Polyclonal CD8⁺CD103⁺Foxp3⁺ Regulatory T Cells on Collagen-Induced Arthritis in Mice

Figure 6

The therapeutic function of human CD8⁺Tregs on CD4⁺CD25⁻ effector T cells and mature DCs of CIA mice in an in vitro cell coculture assay. (a) Suppression ability of human CD8⁺Tregs on CD4⁺CD25⁻ effector T cells of CIA mice. Mouse CD4⁺CD25⁻T cells were purified from splenic cells from CIA mice on day 35 after the first immunization. CFSE-labeled mCD4⁺CD25⁻ effector T cells (Teffs) were cocultured with or without hCD8⁺Tregs at different ratios (, 1 : 2, 1 : 4, and 1 : 16) under the stimulation of anti-mouse CD3/CD28 expansion beads () and CII (100 ng/mL) for 3 days. A CFSE dilution was investigated to reflect mCD4 proliferation. hCD8⁺Treg inhibitory ability was calculated as (control Teff proliferation − Teff with Treg proliferation)/(control Teff proliferation) × 100%. (b) Associated apoptosis/death induction of CD8⁺Tregs on CD4⁺CD25⁻ effector T cells of CIA mice. Mouse CD4⁺CD25⁻ effector T cells were cocultured with or without CD8⁺Treg cells for 72 hours. Annexin V-FITC and propidium iodide (PI) were used to label the cells, which were evaluated by FACS. The Annexin V⁺PI⁺ cells were considered to indicate apoptotic/dead cells. Cells from 3 individual mice were tested, and a representative graph is shown on the left. (c) The influence of human CD8⁺Tregs on CD4⁺CD25⁻ T cell differentiation in mice. At the ratio of Treg : Teff of 1 : 1, mouse CD4⁺CD25⁻ T cells were selected, and the levels of Foxp3, IL-17A, and IFN-γ were evaluated to investigate the influence of human CD8⁺Tregs on CIA mouse CD4⁺CD25⁻ T cell differentiation. (d) CD80 and CD86 expression in mature mouse DCs. DCs from CIA mouse bone marrow were sorted, matured using LPS (100 ng/mL), and cocultured with or without human CD8⁺Tregs for 24 hours. Then, CD80 and CD86 expression in mDCs was evaluated. The results are expressed as of at least three independent experiments; , , and , compared with the control group by unpaired -tests.

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