May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Assessing Allospecific Ignorance to Graft Endothelium Using "Chimeric Keratoplasty"
Author Affiliations & Notes
  • D. R. Saban
    Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • S. K. Chauhan
    Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • Y. Jin
    Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • Q. Zhang
    Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships D.R. Saban, None; S.K. Chauhan, None; Y. Jin, None; Q. Zhang, None.
  • Footnotes
    Support Fight for Sight PD06009 (Saban), NIH/NEI T32-07156 (Saban), NIH/NEI RO1-12963 (Dana)
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 1922. doi:
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    • Get Citation

      D. R. Saban, S. K. Chauhan, Y. Jin, Q. Zhang; Assessing Allospecific Ignorance to Graft Endothelium Using "Chimeric Keratoplasty". Invest. Ophthalmol. Vis. Sci. 2007;48(13):1922.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: Due to its high cellularity, corneal epithelium is considered to be a major contributor in sensitization to alloantigens shared by graft endothelium--the critical target tissue for rejection in penetrating keratoplasty. Therefore, we hypothesize that transplantation of chimeric grafts with 3rd-party epithelium, allodisparate to graft stroma/endothelium and recipient, results in allospecific ignorance to graft endothelium.

Methods:: Harvested donor buttons from adult male C57BL/6 (H-2b), C3H/He (H-2k), or BALB/c (H-2d) mice were treated with 20 mM EDTA for separation of the epithelium from the stroma/endothelium layer. Tissues were thoroughly washed and assembled into full-thickness isografts (H-2d), full-thickness allografts (H-2b), or chimeric grafts (epithelium [H-2k] + stroma/endothelium [H-2b]) followed by orthotopic transplantation on recipient (H-2d) mice (n of 3 per group). Draining lymph nodes were harvested 2 weeks post-transplantation for magnetic T lymphocyte sorting. 2uM CFSE fluorescently labeled sorted T lymphocytes. Cells were washed and co-cultured with naïve APC for recall stimulation measurement to alloantigens of graft endothelium (H-2b), or to syngeneic controls (H-2d). Recall stimulation was measured by multiplex cytokine screening, and quantifying CFSE+ T lymphocyte divisions by FACS.

Results:: Recall stimulation resulted in negligible levels (≤10pg/ml) of polarizing cytokines (i.e., IL-4, 5, 10, IFN-g) in all transplanted recipient groups. However, robust IL-2 levels (≥145pg/ml) were observed in response to recall stimulation in all recipient groups, in contrast to syngeneic stimulation (≤12 pg/ml). Recall stimulation resulted in 3 successive CFSE divisions of CD4+ gated cells. Strongest proliferation was observed in allografted recipients, since the majority of CD4+ cell progeny were found in the last division (20.9%). Recall stimulation in isografted recipients resulted in a 4-fold decrease of progeny cells found in the last division (4.9%). Interestingly, there was a similar decrease of progeny cells in chimeric graft recipients (8.1%).

Conclusions:: While IL-2 levels are comparably strong in all transplanted recipient groups, CD4+ cell proliferation is clearly strongest in allografted recipients. Conversely, proliferation profiles of chimeric grafted recipients are weak and closely resemble those of isografted recipients. Therefore, our model suggests that chimeric grafts do not appreciably sensitize recipient T lymphocytes to graft endothelium. Experiments testing the potential for chimeric keratoplasty in promoting transplantation survival are underway.

Keywords: transplantation • cornea: epithelium • immunomodulation/immunoregulation 
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