Kidney and kidney-pancreas transplantation
An In Vitro System for the Determination of Individualized Immunosuppression

https://doi.org/10.1016/j.transproceed.2008.04.006Get rights and content

Abstract

The risk of complications of immunosuppressive treatment in organ transplantation increases with the aggregate amount of immunosuppressive medication given to the patient. As the doses of immunosuppressive agents required to achieve comparable effects show considerable variability, methods to assess individual sensitivity toward immunosuppressive regimens are urgently needed. The aim of this study was to develop such an in vitro test system. As immunological model for allogeneic transplantation, individual pairs of recipient-derived lymphocytes and of donor-derived B lymphocytes mimicking HLA expression of cells in the transplanted organ were isolated and assessed in mixed-lymphocyte cultures (MLC). Alloreactivity was readily observed and MLC consisted of CD8+ and CD4+ T cells as well as CD56+ natural killer cells. A proliferation assay to measure the response of individual MLC on the immunosuppression by cyclosporine (CsA) was developed. The concentrations of CsA leading to growth reductions by 50% (inhibitory concentration 50, IC50) were found between 110 and 220 ng/mL, which was near the trough whole blood levels for CsA. Accordingly, the IC90 values (660 to 1760 ng/mL) were near the target values for peak whole blood levels. We believe that these data present a simple and potentially useful in vitro technology that allows for the prediction of individual responses to immunosuppressive therapeutic regimens.

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Patients and Methods

As a surrogate for the transplanted organ, donor lymphocytes were transformed by Epstein-Barr virus (EBV) subsequent to isolation. Recipient lymphocytes were isolated from a single blood specimen (10 to 20 mL) obtained after written informed consent was given by the patient. Blood collection was performed after preoperative immunosuppression was given (including 250 mg corticosteroids, 2 g mycophenolate mofetil, 5 mg/kg tacrolimus, and/or 0.2 mL/kg antithymocyte globuline). The study protocol

Results

Blood samples were obtained from 12 patients subsequent to organ transplantation (nine men, three women), mean age 44.3 years (range 27 to 72), having undergone transplantations of a kidney (n = 8), combined pancreas/kidney (n = 2), or a liver (n = 2). Lymphocyte isolation was successfully performed for all 12 blood samples. Seven EBV-transformed B cell lines (B-LCL) were established out of 11 lymphocyte preparations derived from organ donors (58.3%).

Using these cells, eight individual MLC

Discussion

As B cells of donor origin express both class I and class II HLA molecules and therefore resemble transplanted cells, they represent an appropriate target cell for this model of specific allogeneic stimulation. However, the analysis was hampered by the relatively poor yield of EBV transformation that must be improved for future analysis.

Specific alloreactivity could be demonstrated in all MLC pairs analyzed by ELISpot analysis. This method is well suited to measure alloreactivity as has been

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This work was supported by a FORUN grant of the Medical Faculty of the University of Rostock.

M.L. and C.R. contributed equally to this work.

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