A Prospective Analysis of the Natural Course of Donor Chimerism Including the Natural Killer Cell Fraction After Liver Transplantation

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After solid organ transplantation, circulating hematopoietic cells of the donor can be found in the circulation of the recipient, so-called donor chimerism (1). Donor chimerism has gained attention primarily for two reasons: first, because of the importance of circulating donor cells in the development of graft versus host disease (GVHD), and second, because of the potential role of these donor cells in the development of tolerance (2). Previous studies on donor chimerism vary in their prospective or retrospective nature, methods used, clinical correlations, and also in the cell populations that were studied. We aimed to prospectively study the natural course of donor chimerism after liver transplantation in whole blood, T cells, and natural killer (NK) cells. The NK cells are of particular interest because of the recently described potential tolerogenic properties (3). We prospectively collected blood samples of 21 liver transplant recipients in the first 3 months after transplantation. Blood samples were collected for analysis every week during admission after transplantation, and thereafter at every visit to the outpatient clinic up to 3 months after transplantation. Clinical and demographic data were obtained. Microchimerism was defined as the presence of circulating donor DNA in the studied cell population up to a maximum of 1%. Macrochimerism was defined as the presence of more than 1% of circulating donor DNA in the studied cell population. DNA was isolated from whole blood and from T- or NK-cell fractions using QIAamp blood kit (QIAGEN, Venlo, The Netherlands). NK- and T-cell fractions were obtained by a negative selection using RosetteSep kits (Stemcell Technologies, Grenoble, France). Patient and organ donor were typed for seven short tandem repeat markers. The percentage of donor DNA was calculated using the fluorescence intensity of the peaks. Lymphocyte subsets (total lymphocyte number, CD3+ T cells, and CD3-CD56+CD16+ NK cells) were measured as absolute numbers using the Becton Dickinson MultiTest TruCount method with four-color MultiTest reagent CD3/16+56/45/19 (Becton Dickinson, Rutherford, NJ). All patients survived during the first 3 months. A total of 137 samples were obtained from the 21 patients. Of the 137 samples analyzed, chimerism was detected in whole blood, T cells, and NK cell fraction in 34 (25%), 35 (26%), and 69 (50%) samples, respectively. In six patients (29%), at no time after transplantation donor DNA was detected in the peripheral blood. In 15 patients (71%), at least one sample showed donor DNA. Of the 15 patients with donor DNA, 5 showed microchimerism (≤1% donor DNA) and 10 showed macrochimerism (>1% donor DNA). In 14 of the 15 patients, the highest amount of donor DNA was found in the first month after transplantation. Patterns of donor chimerism in T cells and NK cells are shown in Figure 1. We could not find a relationship between the development of chimerism and any of the clinical parameters studied, including recipient gender, age of recipient or donor, use of a donation after cardiac death donor liver, duration of intensive care unit stay, the number of units of red blood cells transfused perioperatively, and normalization of liver tests. There were no episodes of GVHD or rejection.FIGURE 1.: Graphical representation of the maximum percentage of donor DNA during the first, second, and third month after transplantation. (A) Represents results from T cells and (B) from NK cells.From these findings, we can conclude that donor chimerism is a frequent event after liver transplantation, especially occurring during the first month after transplantation. Probably, the most important findings of our study are related to NK-cell chimerism. We have shown that compared with whole blood and T-cell fraction, chimerism is most prevalent in the population of NK cells (CD3-CD56+CD16+). In the first month after transplantation, 14 of 21 patients (67%) tested positive for NK-cell chimerism. Few studies have looked specifically at NK-cell chimerism, and the number of patients in these studies is small (3–5). This study is the largest to date on this particular subject. In a recent and elegant study, Moroso et al. (3) have shown that chimeric NK cells after liver transplantation have unique properties including a high state of activation, abundant presence of cytolytic enzymes, and a specific pattern of interleukin expression. This may make the NK-cell pool particularly suitable to inhibit the recipient immune response and in this way add to graft tolerance. In conclusion, we found donor chimerism in 71% of our liver transplant recipients. Chimerism was most frequently found in the first month after transplantation. Donor cells were more frequently seen in the NK cell subset than in whole blood or T-cell fraction. In this prospective study, we were not able to find a relationship between donor chimerism and previously suggested risk factors for GVHD or clinical outcome. Robert C. Verdonk1 Elizabeth B. Haagsma1 Theo Jongsma2 Robert J. Porte3 Caroline Roozendaal2 Aad P. van den Berg1 Bouke G. Hepkema2 1 Department of Gastroenterology and Hepatology University Medical Center Groningen University of Groningen Groningen, The Netherlands 2 Department of Laboratory Medicine University Medical Center Groningen University of Groningen Groningen, The Netherlands 3 Department of Surgery Section of Hepatobiliary Surgery and Liver Transplantation University Medical Center Groningen University of Groningen Groningen, The Netherlands

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