1. Determination of cytotoxic T lymphocyte activity against
human spleen cells and
estimation of microchimerism
for tolerance measurement
M. Schenk, P. Kaur Gill, A. Königsrainer
Department of General, Visceral and Transplant Surgery,
University Hospital, Tuebingen
Objective
Clinical organ transplantation between genetically disparate individuals presently requires non-specific immunosuppressive agents to prevent rejection. However, the use of immunosuppressive drugs caused side effects and does not always guarantee success of the graft. Therefore, there is a need to establish methods of “donor specific transplantation tolerance” and that could not be achieved directly in patients. So, aim was to establish mixed lymphocytes culture (MLC) and fluorescence activated cell sorting (FACS) cytotoxicity assay and a very sensitive assay for microchimerism based on cytospin preparation of peripheral blood lymphocytes.
Materials and Methods
In this study, the spleen cells and PBMC from different patients were stimulated with different concentration of Con-A (2-6 µg) up to 6 days. Than, spleen cells were labelled (5x105 cells/ml in PBS buffer) with 3 µM of Dio18(3) for 45 min at 37°C, 5% CO2. Dio18 dye remains stably incorporated in the membrane for long periods of time (20 days). The MLC were established by culturing different ratios (1:1, 2:1, 4:1) of unstained PBMC (effectors lymphocytes) with an cells/ml irradiated (30 Gy) spleens cells in RPMI medium supplemented with 50 µg/ml Gentamycin, 10% FCS, 200 mM Glutamin, 10 mM Hespes pH 7.2 and 2 µg Con-A into 96 well flat bottom plates. After different time intervals, the cells suspension were harvested and by adding PI to samples, FACS analysis was carried out on a FACScan cytometer. By excitation at 488 nm with an argon laser, the emission of two fluorochromes was recorded through specific band pass filter 520 nm for Dio18(3) (FL1) and > 425 for PI (FL3). Cytospin preparations of Bw4/Bw6-different donor-recipient combinations were stained with the corresponding antibody.
Results
Maximum stimulation of spleen cells was observed with 2 µg Con-A after 24 h. Whereas, the stimulation of peripheral blood lymphocytes (PBMC) was also with 2 µg Con-A, but after 8 days of activation of PBMC from different patients, generated markedly higher levels of cytotoxicity against targets cells. Maximum reduction of spleen cells (2-3%) in the MLC was observed after 6 days of incubation at 37°C, 5% CO2. The degree of decrease of spleen cells was 95% using PBMCs from healthy persons and from third-party immunosuppressed patient 20%. With PBMC from the recipient no decrease was observed.
Conclusions
In conclusion, the Dio18(3) assay is a quantitative methods for the detection of cell- mediated cytotoxicity and providing a greater insight into the mechanisms of cell-mediated cytolysis by different effector populations. Cytospin preparations provide reproducible results in mircochimerism rates even below 1%. A clinical study using both methods on liver transplant recipients is on the way can make this large change a little bit smaller and shows more or less helpful items in the personal transplant preparation.