Decay accelerating factor is essential for successful corneal engraftment

Abstract

In contrast to immune restrictions that pertain for solid organ transplants, the tolerogenic milieu of the eye permits successful corneal transplantation without systemic immunosuppression, even across a fully MHC disparate barrier. Here we show that recipient and donor expression of decay accelerating factor (DAF or CD55), a cell surface C3/C5 convertase regulator recently shown to modulate T-cell responses, is essential to sustain successful corneal engraftment. Whereas wild-type (WT) corneas transplanted into multiple minor histocompatibility antigen (mH), or HY disparate WT recipients were accepted, DAF's absence on either the donor cornea or in the recipient bed induced rapid rejection. Donor or recipient DAF deficiency led to expansion of donor-reactive IFN-gamma producing CD4(+) and CD8(+) T cells, as well as inhibited antigen-induced IL-10 and TGF-beta, together demonstrating that DAF deficiency precludes immune tolerance. In addition to demonstrating a requisite role for DAF in conferring ocular immune privilege, these results raise the possibility that augmenting DAF levels on donor corneal endothelium and/or the recipient bed could have therapeutic value for transplants that clinically are at high risk for rejection.

FIGURE 1

A) F5 ♂ Daf1+/+ corneas were transplanted into F5 ♂ Daf1+/+ (◆, n=10) or ♂ F5 Daf1−/− (▲, n=9) recipients and F5 ♂ Daf1−/− corneas were transplanted into F5 ♂ Daf1+/+ (■, n=6) recipients. For the Daf1+/+ → Daf1+/+ group, the non-failure was 100% (1.0) at day 7, 80% at day 21, and >70 % at day 28. P values are given in the text. B–D) Corneas analyzed by H&E staining on day 28 from ♂ Daf1+/+ to ♂ Daf1+/+ transplants compared to those from ♂ Daf1+/+ to Daf1−/− and from ♂ Daf1−/− to ♂ Daf1+/+ transplants. *While some sections showed pigmented cells, others did not and there was no correlation with nonclarity.

FIGURE 2

A) F5 ♂ Daf1+/+ corneas were transplanted into F5 ♀ Daf1+/+ (◆, n=9) or F5 Daf1−/− (▲, n=6) recipients and F5 ♂ Daf1−/− (■, n=7) corneas were transplanted into F5 ♀ Daf1+/+ recipients. P values are given in the text. B) F12 ♂ Daf1−/− corneas were transplanted into F12 ♀ Daf1+/+ (solid line, X, n=2) recipients, and F12 Daf1+/+ ♂ (dotted line, △, n=2) were transplanted into F12 Daf1−/− ♀ recipients. As controls F12 ♂ Daf1+/+ corneas were transplanted into F12 ♂ Daf1+/+ (solid line, ◆, n=6) recipients, and F12 ♂ Daf1+/+ (dotted line, ▲, n=6) corneas were transplanted into F12 Daf1+/+ ♀ recipients. C) F12 ♂ Daf1−/− (◆, n=2) corneas were transplanted into F12 ♀ Daf1−/− recipients. As controls, F12 ♀ Daf1−/− (■, n=5) or F12 ♂ Daf1−/− (▲, n=3) corneas were transplanted into F12 ♂ Daf1−/− recipients. P values are given in the text.

FIGURE 3

C3 deposition in 21 day ♂ Daf1+/+ corneas transplanted into ♀ Daf1−/− or Daf1+/+ recipients (100x magnification). Isotype control stained sections are shown for comparison. Lower left insets in top panels are positive staining controls of sections of livers from ova transgenic mice sensitized in vivo with anti-ova antibody.

FIGURE 4

A) CD4+ and CD8+ T cell IFN-γ responses of spleen cells harvested on day 28 from F5 ♀ Daf1+/+ and Daf1−/− recipients of F5 ♂ Daf1+/+ corneas and from F5 ♀ Daf1+/+ recipients of ♂ Daf1+/+ or ♂ Daf1−/− corneas. B) CD4+ and CD8+ T cell IFN-γ responses of spleen cells harvested on day 35 from F12 ♂ Daf1+/+ corneas to F12 ♀ Daf1+/+ or F12 ♀ Daf1−/− recipients and F12 ♂ Daf1−/− corneas to F12 ♀ Daf1+/+ recipients. C) Anti-Dby and anti-Uty Ig isotype levels were assayed in 21 day plasma from ♀ Daf1+/+ recipients of ♂ Daf1+/+ or Daf1−/− corneas.

FIGURE 5

♂ Daf1+/+ corneas were transplanted into ♀ Daf1−/− or Daf1+/+ recipients. Day 35 recipient splenocytes were incubated for 3 days with HYDby or HYUty after which IL-10 and TGF-β levels in supernatants were assayed by specific ELISAs.