Adoptive Cell Therapy of Induced Regulatory T Cells Expanded by Tolerogenic Dendritic Cells on Murine Autoimmune Arthritis

Figure 5

iTreg_mtDC exerted a more potent immunosuppressive effect on antigen-specific CD4⁺ T cell proliferation and suppressed Th17 cell differentiation more effectively. (a) The ability of iTreg_mtDC to inhibit the proliferation of antigen-specific CD4⁺ T cells was assessed using the proliferation assay. In this assay, CD4⁺ T cells (responders) were isolated from splenocytes of CIA mice (on day 25 after the primary immunization), stained with CFSE, and stimulated with CII-loaded mDCs (stimulators) derived from the BM of normal D1 mice. Then, different ratios of iTregs, iTreg_mDC, or iTreg_mtDC (suppressor cells) were added to the responder cultures. Four days later, the cells were harvested and analyzed by flow cytometry. The results are shown as means ± SEM (). and for the comparisons of the indicated groups using the unpaired t-test. (b) The suppression of Th17 cell differentiation by coculture with iTreg_mtDC was assessed using the standard Th17 cell differentiation system. As described in the Materials and Methods, naive CD4⁺ T cells were isolated from the splenocytes of CIA mice, stained with CFSE, and stimulated with an anti-CD3/CD28 mAb, IL-1β, IL-6, and TGF-β1. Then, a 1 : 1 ratio of iTregs, iTreg_mDC, or iTreg_mtDC was added to this differentiation system. After 4 days of cocultivation, the percentages of CFSE⁺ CD4⁺ IL-17A⁺ cells in the different groups were determined by flow cytometry as the percentage of induced Th17 cells. Data are reported as means ± SEM (), and , , and represent the comparisons of the indicated groups using unpaired t-test. (c) After 4 days of cocultivation, supernatants were collected from the groups mentioned in (b). The concentration of soluble IL-17A in the coculture supernatant was determined using CBA. Data are reported as means ± SEM (), and , , and represent the comparisons of the indicated groups using the unpaired t-test.

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