In vitro-expansion of induced regulatory T cells and its inhibitory effects on corneal allograft rejection

Background

Researches showed that CD4⁺ CD25⁺ natural regulatory T cells (nTregs) play an important role in maintaining peripheral immune tolerance, while immunotherapy using in vitro-expanded induced regulatory T cells (iTregs) suppresses allograft rejection in multiple organ transplantation.The inducing method of iTregs still needs to be optimized.Furthermore, the effect of iTregs on grafts of keratoplasty is unclear.

Objective

This study was to investigate the inducing and expansion method of iTregs and explore its inhibitory effects on corneal allograft rejection.

Methods

Bone marrow-derived dendritic cells (BMDCs) were isolated from C57BL/6 mice femora and cultured.CD4⁺ CD25⁺ T cells and CD4⁺ CD25^– T cells were isolated from mouse spleen and separated using flow cytometry.The CD4⁺ CD25^– T cells were divided into negative control group (CD4⁺ CD25^– T cells), CD3/28 antibody bead group (CD4⁺ CD25^– T cells+ CD3/28 antibody bead), 2.5 ng/ml transforming growth factor (TGF)-β₁ induced group and 10.0 ng/ml TGF-β₁ induced group.The iTregs was formed after induction of different concentrations of TGF-β₁ and CD3/CD28 antibody bead (1∶1). CD3/CD28 antibody bead (1∶2), interleukin-2 (IL-2) and TGF-β₁ were used to expand iTregs.The phenotype and proliferation of iTregs were assayed by flow cytometry, and the inhibitory effect of iTregs on effector T cells (Teffs) was analyzed by mixed lymphocyte reaction.Allogenic keratoplasty model (C57BL/6→BALB/c) was build, and 0.1 ml iTregs or nTregs suspension or PBS was injected via posterior venous plexus of fellow eyes to assess the graft survival time.The use and care of the mice followed the ARVO statement.

Results

The CD4⁺ CD25⁺ T cell proportions were (6±3)%, (91±4)%, (91±3)% and (86±6)% in the negative control group, CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β₁ induced group and 10.0 ng/ml TGF-β₁ induced group, showing significant increases in the CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β₁ induced group and 10.0 ng/ml TGF-β₁ induced group compared with the negative control group (all at P<0.01). The Foxp3⁺ T cell proportions of the CD3/CD28 antibody bead group, 2.5 ng/ml TGF-β₁ induced group and 10.0 ng/ml TGF-β₁ induced group were (1.18±0.20)%, (8.70±1.80)% and (21.80±3.36)%, showing significant increases in the 2.5 ng/ml TGF-β₁ induced group and 10.0 ng/ml TGF-β₁ induced group compared with the CD3/CD28 antibody bead group (both at P<0.01). Compared with the nTregs, the expression of CD69 was lower, and the expressions of PD-1 and Foxp3 were raised in the iTregs (all at P<0.01). The proliferation of Teffs were decreased when cocultured with iTregs in comparison with nTregs at 1∶1, 1∶2, 1∶4, 1∶8, 1∶16 Tregs/Teffs rations (all at P<0.01). The survival time of mouse corneal grafts was 4 weeks with the permanent tolerance of 50% in the iTregs injected group, which was superior to the 3 weeks survival time and 17% permanent tolerance in the nTregs injected group(P<0.05).

Conclusions

TGF-β₁ can induce CD4⁺ CD25^– T cells to form iTregs, which highly express Foxp3.iTregs show a stronger inhibitory effect on the growth of lymphocytes than nTregs, and therefore suppress the graft rejection after keratoplasty.

CD4-positive T-lymphocytes/cytology; T-lymphocytes, regulatory/drug effects; Corneal transplantation/therapy; Immune tolerance; Disease model, animal; Mice