Abstract
Purpose :
To evaluate the immunosuppressive effect of corneal myofibroblasts on effector T cells in corneal transplantation.
Methods :
Allogeneic corneal transplantation (penetrating keratoplasty, PK) was performed in eight to ten week old male BALB/c mice. MK/T-1 corneal fibroblasts were cultured with transforming growth factor-beta-1 (TGF-β) for 48 hours to induce differentiation into myofibroblasts. Allo-primed CD4+CD25- effector T cells (Teff) were isolated from the cervical draining lymph nodes of PK mice 14 days post-operatively. 5x105 Teff were co-cultured with either corneal myofibroblasts or MK/T-1 corneal fibroblasts (control) for 24 hours in the presence of anti-CD3 and anti-CD28 in 24 well culture plates. Teff cell activation was evaluated by assessing the expression level of CD69 using flow cytometry. The functional phenotype of Teff cells was assessed by measuring the intracellular cytokine staining level of IFN-γ by flow cytometry as well. To evaluate the effect of corneal myofibroblasts on T cell proliferation, naïve CD4+CD25- T cells were co-cultured with either corneal myofibroblasts or corneal fibroblasts for 72 hours and proliferation was assessed by carboxyfluorescein succinimidyl ester (CFSE) staining.
Results :
We confirmed the differentiation of corneal fibroblasts to myofibroblasts by cell morphology and increased expression of α-smooth muscle actin (α-SMA). When Teff were co-cultured with corneal myofibroblasts, the frequency of CD69+ Teff cells was 0.16±0.05% as compared to 2.50±0.3% when Teff were co-cultured with corneal fibroblasts (p=0.011). CD69 expression by Teff cells was determined by evaluating CD69 MFI levels. The mean CD69 MFI on Teff co-cultured with corneal myofibroblasts was 1153.4±134.2 as compared to 2005.6±155.2 when Teff were co-cultured with corneal fibroblasts (p=0.039). In addition, the frequency of IFN-γ+ Teff was 2.34±0.8% in the corneal fibroblast co-culture group and 0.80±0.4% in the corneal myofibroblast co-culture group (p=0.0076). Finally, corneal myofibroblasts significantly suppressed T cell proliferation as compared to fibroblasts (41.84±1.7% vs. 70.70±2.3% proliferative cells, p<0.0001).
Conclusions :
In addition to their well-established role in fibrosis and extracellular matrix production, corneal myofibroblasts may also serve as immunoregulatory cells capable of suppressing T cell activation, function, and proliferation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.