Higher Corneal Allograft Rejection Rates in the Very Young Are Associated with a Heightened NK cell Response in the Setting of Regulatory T cell Dysfunction

Abstract

Purpose : The success rates of corneal allografts are substantially lower in children than in adults. However, the immunologic mechanisms underlying higher failure rates in very young graft recipients are not known. The aim of this study was to investigate how the balance between natural killer (NK) cells and regulatory T cells (Tregs) affects the fate of corneal allografts in very young and adult mice.

Methods : Allogeneic corneal transplantation was performed in 3.5 week-old (very young) and 10 week-old (adult) BALB/c mice using C57BL/6 donors (n=10). Frequencies of interferon gamma (IFNγ)-positive NK cells (CD45⁺CD3^-CD49b⁺) in the cornea and draining lymph nodes were assessed at day 7 after transplantation using flow cytometry. Frequencies of draining lymph node CD4⁺CD25⁺Foxp3⁺ Tregs and their suppressive function were assessed in naïve very young and adult mice. To evaluate the effect of Tregs on NK cell function in vitro, NK cells and Tregs isolated from lymph node and spleen of naïve very young and adult mice were co-cultured in the presence of IL-12, and IFNγ protein levels were assessed using ELISA.

Results : The frequencies of IFNγ-positive NK cells in very young mice were significantly higher than adult mice in the cornea (20.21% vs. 10.83%; P=0.0059) and draining lymph nodes (17.67% vs. 12.40%; P=0.0042) on day 7 post-transplantation. The frequencies of Tregs were significantly lower in the very young mice compared to adult mice (9.05% vs. 13.10%, p<0.0001). Additionally, Tregs derived from very young mice had significantly lower suppressive effect on T cell proliferation than adult mice (45.93% vs. 62.46%; p<0.0001). Furthermore, our co-culture data demonstrated that Tregs from very young mice had lower capacity in suppressing IFNγ secretion by activated NK cells compared to Tregs from adult mice (2.07% vs. 18.0%; p=0.039).

Conclusions : These data suggest that higher frequencies of activated NK cells coupled with reduced frequencies and suppressive function of Tregs contribute to higher rejection rates in very young graft recipients.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.