The PCR based analysis of TRECs in
The PCR-based analysis of TRECs in general is laborious, technically challenging and comparison of results obtained in different laboratories is difficult as different normalization standards exist. Moreover the analysis is restricted to analysis of cell populations (Douek et al., 1998; Jamieson et al., 1999; Sodora et al., 2000; Ye and Kirschner, 2002). In contrast, flow cytometric determination of naïve CD4 T cell homeostasis by measurement of frequencies and absolute counts of CD31+ thymicnaive or CD31- centralnaïve CD4+ T T-5224 determination represents a sensitive, robust and rather simple method requiring only minute amounts of blood (Kohler et al., 2005b). This method has reproducibly been demonstrated to correlate with thymic function in health (Duszczyszyn et al., 2006; Junge et al., 2007; Kilpatrick et al., 2008; Kimmig et al., 2002b; Kohler et al., 2005a) and disease (Bofill et al., 2006; Duszczyszyn et al., 2006; Isgro et al., 2005; Thiel et al., 2008) and can thereby be regarded as reliable tool to investigate thymic function (Kohler and Thiel, 2009). In this study we wanted to confirm the reported increased naïve CD4+ T cell generation by MG-associated thymoma (Buckley et al., 2001), that has been detected by determination of TREC levels, by our method. Here, in line with the previously reported abnormal thymopoesis in thymoma patients (Buckley et al., 2001), we have detected a significantly increased frequency of CD31+ thymicnaive CD4+ T cells in peripheral blood in correlation with thymic pathology. This difference is even more striking after exclusion of thymoma subtypes A and A/B where histologically by definition only a low content of lymphatic cells is present (Marx et al., 2015). With respect to this one might expect a drastically lower export of lymphocytes to the periphery as compared to other, more lymphatic, subtypes. Our findings not only confirm previous results but also imply that by peripheral blood analysis of CD31+ thymicnaive CD4+ T cells a differentiation into thymomas with rather epithelial or lymphatic subtype could be possible (Marx et al., 2015). Unfortunately we did not have enough numbers of thymoma patients with different subtypes to prove this statistically. As the thymoma group included patients with and without coexisting MG, we can not draw any conclusion whether aberrant naïve CD4+ T cell generation might be specific for MG. We could not identify differences when comparing MG patients with thymoma and patients with thymic hyperplasia. This can be explained by the fact that, as reflected in our cohort, in which the oldest patient with thymic hyperplasia in our study was only 45 years, patients with thymic hyperplasia are usually of younger age (Tsinzerling et al., 2007) when high frequencies of CD31+ thymicnaive CD4+ T cells are also detected in healthy donors (Kimmig et al., 2002a; Kohler and Thiel, 2009; Kohler et al., 2005b). Therefore, due to the high level of physiological CD31+ thymicnaive CD4+ T cells in this age group, it is more difficult to detect aberrant thymopoesis. In contrast thymectomy in our samples has been performed in adult patients some of them well over the age of 50 years, thus at an age in which thymic function is already low. In this setting the sensitivity for the detection of a further decrease of thymopoesis is rather low and this might explain that in our study we could not show a significant impact of thymectomy on naïve CD4+ T cell subsets. In conclusion, we have been able to confirm altered naïve CD4+ T cell homeostasis in MG patients in a cohort selected for the absence of potentially modulating factors like immunosuppressive therapy or thymoma. Additionally we have been able for the first time to detect aberrant thymopoesis in thymoma patients in the peripheral blood on the cellular level. Yet, further studies are needed in order to determine, whether this tool can be used as follow-up method for thymoma patients in order to analyse for signs of thymoma recurrence.