Amplified Natural Killer Cell Activity and Attenuated Regulatory T-cell Function Are Determinants for Corneal Alloimmunity in Very Young Mice

Abstract

Background: Corneal transplantation outcomes are generally less favorable in young children compared with adults. The purpose of this study was to determine the immunological mechanisms underlying this difference.

Methods: A murine model of allogeneic corneal transplantation was used in the study, and graft survival was determined by evaluating opacity scores for 8 wk. Syngeneic transplantation in the very young host served as a surgical control. The frequencies of total and activated natural killer (NK) cells in cornea posttransplantation were kinetically evaluated using flow cytometry. The regulatory T cell (Treg) frequency and function in naive animals were assessed by flow cytometry and in vitro suppression assays, respectively. Finally, graft survival and immune responses were determined in NK cell-depleted, or adult naive Treg-transferred, young hosts.

Results: Corneal allograft survival in the very young recipients was significantly lower than in adult hosts. The frequencies of total NK cells and their interferon gamma-expressing subset in the cornea were significantly higher in the very young mice posttransplantation. In ungrafted mice, frequencies of Treg in draining lymph nodes as well as their capabilities to suppress NK-cell secretion of interferon gamma were lower in the very young compared with adults. In NK cell-depleted or adult Treg--transferred very young recipients, the allograft survival was significantly improved along with the suppressed NK-cell response.

Conclusions: Our data demonstrate that amplified activity of NK cells, together with lower suppressive function of Treg, contributes to early rejection of corneal allografts in very young graft recipients.

Figure 1.. Very young mice show higher opacity scores and decreased allograft survival after corneal allograft transplantation.

A, Opacity scores were significantly higher in very young (solid circle) vs adult (square) recipients (n = 10 mice/group). Syngeneic corneal transplantation in the very young (solid triangle) serves as a procedure control. Data are presented as mean ± SEM. B, Accelerated graft rejection and significantly reduced survival rate in very young recipients. *P<0.05, **P<0.01, ***P<0.001 as compared to adult group.

Figure 2.. Increased infiltration of NK cells in the corneal graft in very young recipients.

A, The frequencies of CD45⁺CD3⁻CD49b⁺ NK cells in the cornea were significantly higher in very young than in adult mice at day 21 and at day 28 posttransplantation. Data are presented as mean ± SD (n = 3 mice/group). B, Flow cytometry analysis of NK cell frequencies in dLNs of very young vs adult transplant recipients. Before and after transplantation the frequencies of NK cells are not significantly different in very young compared to adult graft recipients. Data are presented as mean ± SD (n = 5 mice/group). C and D, Bar charts of cumulative flow cytometric data showing IFN-γ⁺ NK cell frequencies in very young vs adult transplant recipient mice. Data are presented as mean ± SD (n = 4–6 mice/group). At 7 days following transplantation, very young mice had increased IFN-γ⁺ NK cell frequencies compared to adult graft recipient mice in the cornea (C) and draining lymph nodes (D). *P<0.05, **P<0.01. dLN, draining lymph node; IFN-γ, interferon gamma; NK, natural killer; n.s., not significant.

Figure 3.. Very young mice have decreased levels of Tregs with lower suppression capacities.

A, Representative flow cytometry dot plots showing CD4⁺CD25⁺Foxp3⁺ Tregs in the draining lymph nodes of very young vs. adult graft recipients before transplantation (day 0). B, Bar chart of cumulative flow cytometric data showing Treg frequencies in very young vs. adult (n = 5/group). The frequencies of Tregs were significantly lower in very young mice compared to adult mice. C, Chart showing the capacity of Tregs to suppress T cell proliferation in very young vs adult mice before transplantation. Tregs derived from very young mice had significantly lower suppressive effect on T cell proliferation than adult mice. The protein levels of IL-10 (D) and TGF-β1 (E) in the culture supernatants from the suppression assay were significantly lower in very young mice compared to adult mice. Data are presented as mean ± SD (n = 4/group). **P<0.01, ****P<0.0001. CD, cluster of differentiation; dLN, draining lymph node; IL-10, interleukin 10; TGF-β1, transforming growth factor beta 1; Treg, regulatory T cell.

Figure 4.. Tregs from the very young mice show minimal effect on suppressing NK cell activation.

A, The protein levels of IL-12 in the corneal grafts were significantly higher in very young recipients compared to adult recipients at day 7 after transplantation (n = 4/group). B, The protein levels of IFN-γ secreted by NK cells stimulated with a series of concentrations of IL-12 were significantly higher in very young mice (n = 4/group each concentration). C and D, Tregs from very young mice have lower capacity to suppress IFN-γ expression by NK cells compared to Tregs from adult mice. Bar charts showing (C) the summary of flow cytometry data of IFN-γ⁺ NK cell frequencies (n = 5/group) and (D) ELISA data of protein levels of IFN-γ in the supernatants (n = 4/group). Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. IFN-γ, interferon gamma; IL-12, interleukin 12; NK, natural killer; n.s., not significant; Treg, regulatory T cell.

Figure 5.. Allograft survival rate in NK cell–depleted mice.

A, The protein levels of IFN-γ in the corneal grafts were significantly lower in NK cell–depleted very young recipients compared to nondepleted control recipients at day 21 after transplantation Data are presented as mean ± SD (n = 4 mice/group). B, NK depletion delays graft rejection in very young recipients. C, NK depletion did not affect graft survival in adult recipients. Grey lines in the plots denote NK cells depleted recipient mice, and black lines indicate nondepleted control. n = 8 mice/group for (B) and (C). **P<0.01. IFN-γ, interferon gamma; NK, natural killer.

Figure 6.. Adoptive transfer of Tregs from adult mice into the very young mice suppresses NK cell activity and graft rejection.

A, Representative flow cytometry dot plots showing CD3⁻CD49b⁺ NK cells gated on CD45⁺ cells, and bar chart summarizing the frequencies of NK cells in the cornea (mean ± SD, n = 5/group per time point). B, Representative flow cytometry dot plots showing IFN-γ⁺ NK cells, and bar chart summarizing the frequencies in the cornea (mean ± SD, n = 5/group per time point). C, Representative flow cytometry dot plots showing CD3⁻CD49b⁺ NK cells gated on CD45⁺ cells, and bar chart summarizing the frequencies of NK cells in the dLN (mean ± SD, n = 5/group per time point). D, Representative flow cytometry dot plots showing IFN-γ⁺ NK cells, and bar chart summarizing the frequencies in the dLN (mean ± SD, n = 5/group per time point). E, Corneal grafts were monitored for up to 8 weeks posttransplant, and Kaplan-Meier curves were plotted to evaluate the graft survival. n = 10 mice/group. *P<0.05, **P<0.01, ***P<0.001. CD, cluster of differentiation; dLN, draining lymph node; IFN-γ, interferon gamma; NK, natural killer; Syn, syngeneic control; Treg, regulatory T cell.