Abstract
Purpose :
CD103+dendritic cells (DCs) are key inducers of tolerance and are proposed to promote the conversion of naïve T cells into Foxp3+regulatory T cells (Tregs), a mechanism mediated by αvβ8 integrin. The role of CD103+DCs in inducing tolerance in corneal transplantation has not been investigated. Herein, we evaluated the frequencies and phenotype of CD103+DCs after transplantation, and investigated their role in inducing Tregs.
Methods :
Allogeneic corneal transplantation was performed using C57BL/6J mice as donors and BALB/c mice as recipients. Two weeks after transplantation, cornea, conjunctivae and ipsilateral draining lymph nodes (DLNs) were collected. Frequencies of CD103+DCs and their expression of MHC II and αvβ8 were evaluated by flow cytometry. Expression of Itgb8, Irf8, Itgae, Clec9aand Aldh2genes, involved in CD103+DC function, was evaluated by RT-PCR. Induced CD103+DCs (iCD103+) were generated in vitroby incubating murine bone marrow cells with FLT3-L (100 ng/ml) and GM-CSF (2 ng/ml) for 16 days. iCD103+DCs were stimulated with LPS (10 ng/ml) and allogeneic corneal lysate, then cultured 5 days with naïve CD4+CD25-T cells. Anti-β8 antibody (20 μg/ml) was added to the co-culture to block αvβ8. Frequencies of CD4+Foxp3+Tregs were determined using flow cytometry.
Results :
Frequencies of CD103+DCs were increased in the cornea (15.94±0.25%, p<0.001), conjunctivae (11.6%±1.32, p=0.32) and DLN (25%±3.35, p=0.013) of transplanted mice compared to naïve controls. Expressions of MHC II and αvβ8 by CD103+cells were upregulated in the cornea (2-fold [p=0.025] and 40-fold [p<0.001]), conjunctivae (4-fold [p=0.008] and 4-fold [p<0.001]) and DLN (2-fold [p=0.017] and 3.5-fold [p<0.001]) of transplanted mice compared to naïve controls. mRNA expression of Itgb8, Irf8, Itgae, Clec9a and Aldh2 in the cornea, conjunctivae and DLN was significantly upregulated in transplanted mice (p<0.001). Foxp3+Tregs frequencies were higher in CD4+CD25-T cells cultured with stimulated iCD103+ cells (19±0.65%, p<0.001) compared to non-stimulated (7.19±0.65%) or CD4+CD25-T cells alone (<2%). Addition of anti-β8 blocking antibody suppressed Treg promoting effect (<2%, p<0.001).
Conclusions :
These results suggest that upregulated expression of αvβ8 integrin by CD103+DCs could promote immune tolerance in corneal transplantation by inducing the conversion of CD4+T cells into Foxp3+regulatory T cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.