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

Figure 2

iTregs expanded in vitro by mtDCs retained Foxp3 expression and efficiently inhibited CD4⁺ T cell proliferation. (a) Schematic overview of the induction/expansion strategy for iTregs. The two expansion cycles were established as described in the Materials and Methods. The differentiation of iTregs was first induced with an anti-CD3/CD28 mAb, TGF-β, and IL-2 for 5 days in vitro. Then, iTreg_mtDC or iTreg_mDC were generated by expanding iTregs for 4 days using mtDCs or mDCs, respectively. The expression of the transcription factor Foxp3 and CD25 in cells was determined in each cycle using flow cytometry. Data are representative of three independent experiments. (b) Expansion of different types of iTregs was determined by counting the cells, and Foxp3 expression levels were determined by flow cytometry. All data are presented as means ± SEM (), and , , and for the comparisons of the indicated groups using the unpaired t-test. (c) The ability of iTregs to inhibit the proliferation of CD4⁺ T cells was assessed using the proliferation assay. In this assay, CD4⁺ T cells (responders) were isolated from splenocytes of D1 mice, stained with CFSE, and stimulated with an anti-CD3/CD28 mAb and IL-2. 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. Progressive dilution of CFSE in responder cells was used as a readout of proliferation in the presence or absence of different types of iTregs. The results are presented as means ± SEM (). and compared with the indicated groups using the unpaired t-test.

(a)

(b)

(c)