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

Figure 2

Stability of human CD8⁺Tregs without induction factors or in an inflammatory microenvironment in vitro. (a) Foxp3 expression when induction factors were removed in vitro. Compared with normal induction/expansion conditions, the level of Foxp3 expression in induced human CD8⁺Tregs when induction factors (TGF-β1 and RAPA) were removed or induction factors were removed with only 1/2 or 1/10 of normal expansion factors. (b) Stability of induced human CD8⁺Treg cells in two different inflammatory conditions in vitro. Two inflammatory microenvironments were modeled with different inflammatory cytokine mixtures (Infla-A: IL-2 10 IU/mL, IL-1β 10 ng/mL, IL-6 4 ng/mL, and TGF-β1 5 ng/mL; Infla-B: IL-2 10 IU/mL, IL-21 25 ng/mL, IL-23 25 ng/mL, and TGF-β1 5 ng/mL). Then, CD8⁺Tregs were cultured in these two different inflammatory microenvironments, and IL-2, Foxp3, and IFN-γ expression of human CD8⁺Tregs was evaluated on days 3, 6, and 9 by FACS. (c) Compared with human natural CD4⁺Tregs, IFN-γ and IL-17A secretion in the supernatant on day 6 was investigated by CBA. Human natural CD4⁺CD25⁺Treg cells were purified from PBMCs and expanded with anti-CD3CD8/CD28 beads plus RAPA in vitro. After 9 days of expansion, CD4 Treg cells were harvested and washed. Expended CD8⁺Tregs and CD4⁺Tregs were cultured in the above two inflammation conditions (Infla-A and Infla-B) for 6 days. The supernatant was collected, and IL-17A and IFN-γ secretion was evaluated with CBA. Each mean represents at least 3 individual samples; the bars indicate the ; , , and .

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