Myeloid-derived suppressor cells promote allograft survival by suppressing Treg dysfunction in high-risk corneal transplantation

Abstract

Purpose : The purpose of this study is to determine the rescue effect of MDSC on IL-6-mediated Treg dysregulation and to further evaluate whether subconjunctival injection of MDSC could prolong corneal graft survival in a murine model of high-risk corneal transplantation.

Methods : Syngeneic Treg (2.5x10⁵) were co-cultured either with MDSC or with IL-10 knockdown (KD) MDSC (via IL-10 siRNA [100nM] transfection) at a ratio of 1:1 in the absence or presence of IL-6 (40ng/ml) for 48 hours. The co-cultured Treg were then isolated and subsequently incubated with in vitro-differentiated Th1 cells to assess their suppressive function on Th1 proliferation. Finally, in a high-risk mouse corneal transplantation model, 5x10⁴ MDSC were delivered through subconjunctival route. Expression of FoxP3 and IL-10 by Treg, and expression of T-bet and IFN-γ by Th1 cells, were analyzed by flow cytometry, immunoblotting, and real-time PCR. Functions of Treg in suppressing Th1 proliferation were assessed using CFSE assay.

Results : Addition of MDSC to IL-6-treated Treg cultures significantly inhibited the loss of FoxP3 (31.9±1.0% vs 13.1±0.5%, p<0.0001) and IL-10 (86.1±3.3% vs 11.1±1.8%, p<0.0001) expression by Treg induced with IL-6. However, IL-10 KD MDSC exhibited significantly lower capacity to inhibit IL-6-induced Treg loss of FoxP3 (20.9±2.8%, p=0.0061) and IL-10 (64.6±2.5%, p=0.0019). In addition, Treg pre-cocultured with both MDSC and IL-6 had a higher capacity to suppress Th1 expression of T-bet (1.8±0.2% vs 6.16±0.2%, p<0.0001) and IFN-γ (1.64±0.3% vs 4.33±0.1%, p=0.0001) as compared to those Treg pre-treated with IL-6 alone. Subconjuntivally injected MDSC prolonged graft survival (n=9/10 vs n=0/10 in control saline group) in high-risk corneal transplantation. Mice injected with MDSC showed higher expression of FoxP3 (25.67±1.7% vs 12.67±1.2%, p=0.001) by Treg, and lower expression of T-bet (5.78±0.03% vs 7.74±0.2%, p=0.0002) by Th1 as compared to those obtained from the saline injected mice. Treg from the mice injected with MDSC showed significantly higher capacity of suppressing proliferation of in vitro-differentiated Th1 cells (27.45±4.3% vs 46.34±1.5%, p=0.0042) than those from saline-injected mice.

Conclusions : Our data demonstrate that ex vivo generated MDSC are effective in preventing IL-6-induced impairment of Treg function, and their use can promote higher graft survival.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.