(BCD) cFLIP mRNA expression in blood-derived CD4(+) and CD8 (+) T cells and B cells from blood donors and patients with EOMG, MG(?) thymomas, and TAMG(+) thymomas (***value for chi-square testtolerance once they are released from thymomas is unclear. in a separate window Figure 3 Intrapersonal comparison of cFLIP mRNA expression in thymoma (T) and the adjacent, non-neoplastic residual thymus (RT): Midodrine hydrochloride six pairs each of TAMG(+) and MG(?) type AB thymomas and the respective RT from the same patients studied by qRT-PCR (** 0.0001]). (BCD) cFLIP mRNA expression in blood-derived CD4(+) and CD8 (+) T cells and B cells from blood donors and patients with EOMG, MG(?) thymomas, and TAMG(+) thymomas (***value for chi-square testtolerance once they are released from thymomas is unclear. The failure of thymomas to generate FoxP3(+) regulatory T cells36 is a likely candidate mechanism that may become relevant only Midodrine hydrochloride when AChR-directed, potentially autoreactive effector T cells are released from a given thymoma (i.e. in the TAMG(+) setting). In addition, overexpression of cFLIP can block the FAS/FASLG-dependent death of TH1 and TH17 cell. This escape mechanism from peripheral tolerance is operative in several autoimmune diseases and mouse models,37 including experimental autoimmune myasthenia gravis.33 Therefore, the increased resistance of cFLIPhigh PBMCs of thymoma patients to FASLG-induced apoptosis (Fig.?(Fig.6),6), and the overcoming of this resistance PDGFD by pharmacological cFLIP downregulation (Fig.?(Fig.8A8A and B) strongly suggest that cFLIP overexpression in PBMCs of thymoma patients plays a role in the escape of these potentially autoreactive T cells from peripheral tolerance. This hypothesis is not in conflict with the observation that PBMCs of both TAMG(+) and MG(?) thymoma patients are resistant to FASLG-induced apoptosis (Fig.?(Fig.6).6). The resistance of PBMCs in MG(?) thymoma patients is likely due to the high number of thymoma-derived CD8+ T cells in the blood17,18,22 that we now find to have increased levels of cFLIP (Fig.?(Fig.4).4). While export only of CD8+ T cells from thymomas is insufficient to elicit TAMG,17 CD8+ T cells exported from MG(?) thymomas may not be innocent, since autoimmune diseases other than TAMG are very common in both MG(?) and TAMG(+) thymoma patients.34 The mechanism underlying the overexpression of cFLIP in intratumorous thymocytes in TAMG is unknown. Furthermore, the observed association between CTLA4 genotypes and cFLIP expression levels in TAMG(+) thymomas remain enigmatic since no direct linking of CTLA4 signaling to modulation of cFLIP expression has been reported.38 By contrast, the nuclear accumulation of p65/NF- em /em B in PBMCs of TAMG(+) patients in?vivo, the downregulation of cFLIP by pharmacological NF- em /em B inhibition in PBMCs from thymoma patients, and the sensitization of cFLIPhigh PBMCs by NF- em /em B inhibition to FAS-mediated apoptosis imply that increased NF- em /em B signaling is a driver of cFLIP overexpression in PBMCs. Since both cFLIP and nuclear NF- em /em B expression decline in PBMCs after thymoma resection, it is tempting to speculate that NF- em /em B signaling is initiated in thymocytes inside the thymoma. Notwithstanding this open question, the impact of NF- em /em B on cFLIP expression in PBMCs justifies the consideration of pharmacological NF- em /em B inhibition as therapeutic strategy for TAMG. Furthermore, the elucidation of the currently unknown activators of NF- em /em B in PBMCs of TAMG(+) patients may offer additional therapeutic perspectives. The decline of cFLIP expression in PBMCs after thymoma removal resembles the postoperative drop of recent thymic emigrants as defined by T-cell receptor excision circles (TRECs).18,39 The fact that cFLIP expression was highest in CD4+CD45RA+ blood T cells at the time of surgery and dropped thereafter hints to their derivation from the thymoma and shows that the cFLIPhigh phenotype is tumor dependent. Whether the disappearance of cFLIPhigh PBMCs after thymoma surgery reflects absence of adequate stimuli in the extratumorous microenvironments or homeostatic elimination of cFLIPhigh cells from the repertoire through lack of appropriate niches22 cannot be decided. Midodrine hydrochloride Nevertheless, the normalization of cFLIP levels after thymoma removal warrants testing cFLIP overexpression in PBMCs as adjunct tool for the diagnosis of primary and recurrent thymomas, and for the delineation of thymic hyperplasia in EOMG patients, in whom cFLIP levels were adequate for age (Fig.?(Fig.4A).4A). This differential diagnosis may be further facilitated in the future by measuring the expression of another anti-apoptotic protein, survivin, that was recently described as being overexpressed in PBMCs in EOMG.40 In summary, the current findings suggest that thymoma-dependent cFLIP.