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

Figure 1

Phenotypes, cytokines, and suppressive ability of induced/expanded human CD8⁺Tregs in vitro. (a) The induction and expansion of human CD8⁺Tregs. A total of CD8⁺T cells derived from human PBMCs were sorted and induced with TGF-β1 and RAPA plus anti-CD3/CD28 expansion beads and IL-2 for 9 days. hCD8⁺ T cells were derived from the same CD8⁺ T cells using expansion beads and IL-2, but without TGF-β1 or RAPA. Expended CD8⁺ T and CD8⁺Treg cells were collected and counted with a hemocytometer under a microscope, and cell numbers and expansion folds were calculated. (b) Phenotypes of induced human CD8⁺Tregs analyzed by FACS. Human CD8⁺Tregs and T cells were collected and stained with anti-human CD28, CD103, and PD-1-PE. Foxp3 was also measured after the cells were fixed, permeabilized, and stained with anti-human Foxp3-PE. (c) Cytokine expression in induced human CD8⁺Tregs. On day 9, human CD8⁺Tregs were harvested, washed twice with PBS, and stimulated with PMA, ION, and MON for 4 hours; then, intracellular cytokines, such as IL-2, IL-10, IL-17, and IFN-γ, were evaluated by FACS. Meanwhile, human CD8⁺Tregs and T cells were treated with the above method, but without MON stimulation, for 24 hours, and TGF-β1 secretion in the supernatant was evaluated by ELISA. (d) Inhibition of human CD8⁺Tregs on autologous and allogeneic human CD4⁺CD25⁻ effector T cells. Briefly, human CD4⁺CD25⁻ effector T cells (Teffs) labeled with CFSE were stimulated with anti-human CD3/CD28 expansion beads and IL-2 and cocultured with human CD8⁺ T cells or human CD8⁺Tregs for 3 days (, 1 : 4, 1 : 16, and 1 : 64). For autogenic inhibition, purified CD4⁺CD25⁻ effector T cells and expanded CD8⁺T and CD8⁺Tregs were derived from the same donor, while CD4⁺CD25⁻ effector T cells were purified from different donors with CD8⁺ T and CD8⁺Treg cells in allogeneic inhibition assay. CFSE dilutions were investigated to reflect CD4⁺Teff proliferation. Inhibition ability was calculated by (control Teff proliferation − Teff with Treg proliferation)/(control Teff proliferation) × 100%. Each mean represents at least 3 individual samples; the bars indicate the ; , , and .

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