Abstracts (complete list) - Wissenschaft Online

Bernhard Fleischer, Juliane Ladhoff, Elke Effenberger, Cornelia Doebis, Hans-Dieter
Volk, Martina Seifert
Characterisation of NK cell attacks towards MHC class I
deficient rat aortic endothelial cells in vitro
Background: MHC (major histocompatibility) class I deficient rat aortic endothelial cells
(RAEC) have been generated in the past by expression of an anti-MHC class I-intrabody.
The MHC-I-down regulation resulted in a reduced susceptibility of RAEC to humoral and
CTL-mediated immune attacks. Those cells are interesting tools for generating vascular
allografts. However, due to their MHC class I deficiency RAEC may become a target of
Natural Killer (NK) cell lysis.
Methods: Rat NK cells of the recipient rat strain were isolated from spleen by density
gradient centrifugation combined with nylon wool adherence and MACS-separation
technology using an anti-NKR-P1 antibody. Characterization of isolated NK cells was
performed flow cytometrically. Rat NK cells were cultured for 2 days in IL-2 containing
medium and then used as killer cells in a Calcein-based cytotoxicity assay. NK cell lysis
was tested with syngeneic, allogeneic and allogeneic but MHC class I deficient cells. YAC-
1 tumor cell line was used as a positive control for NK cell killing.
Results: Rat NK cells could be isolated with a purity of 70-80 %, defined by the coexpression
of ratNKR-P1 and ratNKp46 and the absence of CD3/T cell receptor (TCR)
expression. In cytotoxicity assays wild type RAEC of syngeneic as well as allogeneic
origin were killed to a low degree (10-40 %) by NK cells, whereas the YAC-1 cells were
almost completely lysed. MHC class I deficient RAEC displayed comparable low specific
lysis rates similar to that of wild type cells.
Conclusion: Down regulation of surface MHC class I on RAEC does not increase
susceptibility to NK cell mediated lysis. This immunological resistance of MHC I deficient
RAEC would allow their use as an essential component for covering the luminal surface
of vascular allografts.