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T. Ritter, M. Morcos, M. Maenz; A Comprehensive Flow-Cytometric Analysis of Serum, Draining Lymph Nodes and Graft Infiltrating Lymphocytes During the Rejection Phase in a Fully Allogeneic Rat Cornea Transplantation Model. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1559.
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© ARVO (1962-2015); The Authors (2016-present)
To foster the notion that cervical lymph nodes (LNs) are prime locations for the immunologic reaction towards corneal allografts, we studied the cellular and humoral immune response after penetrating keratoplasty.
Full thickness donor corneas (PVG-RT1c) were placed on Brown Norway (BN-RT1n) recipients and fixed with 10-0 interrupted sutures. Using multicolour flow cytometry (FACS), we quantified and compared the composition of ipsi- and contra-lateral LNs as well as a distal LN together with serum samples from rats receiving allo- or syngeneic corneas. Additionally, graft infiltrating lymphocytes (GIL) were isolated and measured by FACS at day 7 or the onset of rejection.
On average we find elevated numbers of activated CD4+ cells as shown by increased expression of CD25 and CD134. Additionally we found sharply decreased levels of CD45RC (Th2 indicator) on CD4+ T-cells from ipsi-lateral LN which correlates with a pronounced expansion of B-cells. In concordance with an increase in B-cell numbers we often detected copious amounts of allo-antibodies in the serum of rejecting animals, in particular IgM, IgG1 and IgG2a but no IgA. FACS analysis of lymphocytes isolated from collagenase digested rejected corneas revealed the following five distinct subpopulations: CD4+ T-cells, CD8+ cytotoxic T-cells, CD161mid large granular lymphocytes (LGL/possibly monocytes), CD3+ CD8+ CD161mid NK-T-cells and CD161high CD3- NK cells. Surprisingly, the latter two cell populations comprised almost 50% of all GIL. At an earlier time point (day 7, before clinical signs of rejection) only CD161mid MHC2neg LGLs are present in transplanted corneas irrespective of the donor MHC status.
Our results demonstrate that despite its immune privileged status allogeneic corneal grafts mount a full fledged Th1 and Th2 response. The presence of NK-T-cells and NK-cells in rejecting corneas shows the synergy between innate and adaptive immunity during allograft destruction.
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