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Hey1, Hey2 and HeyL are downstream effectors of the Notch signalling pathway. Hey genes play decisive roles during embryonic development for example in cardiovascular development. However, the precise transcriptional programmes and genes, which are affected by each single Hey gene, are still poorly understood. One drawback for the analysis of Hey1, Hey2 or HeyL single gene function is that these genes are co-expressed in many tissues and share a high degree of functional redundancy. Thus, it was necessary to establish a system, which is either devoid of Hey expression, or just comprises one single Hey gene family member. For this, Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- as well as Hey-triple- knock out (KO)-ES cells (embryonic stem cells) were generated in this work, because ES cells and their differentiation as EBs (embryoid bodies) represent a valuable tool for the in vitro analysis of embryonic developmental processes. After the establishment of Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells, it could be seen by ALP staining and pluripotency marker expression that loss of Hey expression did not affect ES cell pluripotency features. Thus, these ES cells represent bona fide ES cells and could be further used for the differentiation as EBs. Here, differences in gene expression between Hey1(fl/fl)/Hey2(-/-)/HeyL(-/-)- and Hey-triple- KO-ES cells (after the loss of Hey1) could be observed in realtime-RT-PCR analysis for the endodermal marker AFP as well as for neural and myogenic markers in d10 EBs. However, the establishment of inducible Hey1, Hey2 or HeyL ES cell lines will be essential to confirm these findings and to search for novel Hey target genes. To get further insight into the mode of Hey action, the analysis of Hey interaction partners is necessary. One such binding partner, the Bre protein, has previously been found in a yeast-two-hybrid screen. Bre has been described to be a member of two distinct complexes (i.e. the nuclear BRCA1-A complex with a function in DNA damage response and the cytoplasmic BRISC complex), to directly interact with the TNF-receptor and Fas and to interfere with apoptotic signalling. The Hey-Bre interaction could be further corroborated in this work; yet, it was not possible to narrow down the interaction site of Bre with Hey1. It rather seems that non-overlapping parts of the Bre protein may bind to Hey. This interaction may be direct– pointing to more than one interaction site inside the Bre protein – or via a common binding partner such as the endogenous Bre protein itself. Besides the interaction studies, functional assays were performed for a more detailed characterisation of Hey1 and Bre interaction. Here, it could be shown that Hey1 over-expression did not have any influence on Bre sub-cellular localisation. Interestingly, it could be demonstrated that Bre positively interfered with Hey1 repressive function in luciferase assays at three of four promoters analysed. Moreover, interaction with Bre seems to lead to a stabilisation of Hey1. As Bre has been described to modulate the E3-ligase activity intrinsic to the BRCC complex it was analysed whether Bre over-expression results in an ubiquitination of Hey1. Yet, this could not be observed in the present work. Furthermore, an interaction of Bre with ubiquitinated proteins could not be demonstrated in an ubiquitin binding assay. To obtain a better insight into Bre function, Bre LacZ gene trap-ES cells and animals were generated. However, realtime-RT-analyses revealed that these cells and mice did not show a loss of Bre expression on mRNA level indicating that insertion mutagenesis did not occur as expected. However, embryos derived from these mice could nevertheless be used for the detection of tissues with Bre expression by β-galactosidase staining. Bre deficiency on mRNA levels was only achieved after the deletion of the floxed exon 3 resulting in the generation of Bre del-mice. Bre del-mice were fertile and without any obvious phenotype and they were used for the generation of Bre del- and wt-MEFs (murine embryonic fibroblasts). Characterisation of these cells showed that proliferation was not affected after loss of Bre (neither under normal nor under stress conditions). However, loss of Bre notably resulted in a reduction in the BRCA1 DNA damage response, in a slightly increased sensitivity towards apoptosis induction by FasL treatment and in an increase in the K63-poly-ubiquitin content in Bre del-cytoplasmic fractions, probably linked to a change in the BRISC de-ubiquitinase activity. Even though these results have the same tendencies as observed in former studies, the effects in the present work are less striking. Further studies as well as intercrossing of Bre del- to Hey KO-animals will be necessary to further understand the functional relevance of Hey and Bre interaction.
Cytomegalovirus (CMV) infection is a major cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). Measuring CMV-specific cellular immunity may improve the risk stratification and management of patients. IFN-γ ELISpot assays, based on the stimulation of peripheral blood mononuclear cells with CMV pp65 and IE-1 proteins or peptides, have been validated in clinical settings. However, it remains unclear to which extend the T-cell response to synthetic peptides reflect that mediated by full-length proteins processed by antigen-presenting cells. We compared the stimulating ability of pp65 and IE-1 proteins and corresponding overlapping peptides in 16 HSCT recipients using a standardized IFN-γ ELISpot assay. Paired qualitative test results showed an overall 74.4% concordance. Discordant results were mainly due to low-response tests, with one exception. One patient with early CMV reactivation and graft-versus-host disease, sustained CMV DNAemia and high CD8\(^+\) counts showed successive negative protein-based ELISpot results but a high and sustained response to IE-1 peptides. Our results suggest that the response to exogenous proteins, which involves their uptake and processing by antigen-presenting cells, more closely reflects the physiological response to CMV infection, while the response to exogenous peptides may lead to artificial in vitro T-cell responses, especially in strongly immunosuppressed patients.
Impaired cytomegalovirus (CMV)-specific cell-mediated immunity (CMV-CMI) is a major cause of CMV reactivation and associated complications in solid-organ transplantation. Reliably assessing CMV-CMI is desirable to individually adjust antiviral and immunosuppressive therapy. This study aimed to evaluate the suitability of T-Track® CMV, a novel IFN-γ ELISpot assay based on the stimulation of peripheral blood mononuclear cells with pp65 and IE-I CMV proteins, to monitor CMV-CMI following kidney transplantation. A prospective longitudinal multicenter study was conducted in 86 intermediate-risk renal transplant recipients. CMV-CMI, CMV viral load, and clinical complications were monitored over 6 months post-transplantation. Ninety-five percent and 88–92% ELISpot assays were positive pre- and post-transplantation, respectively. CMV-specific response was reduced following immunosuppressive treatment and increased in patients with graft rejection, indicating the ability of the ELISpot assay to monitor patients' immunosuppressive state. Interestingly, median pp65-specific response was ninefold higher in patients with self-clearing viral load compared to antivirally treated patients prior to first viral load detection (P < 0.001), suggesting that reactivity to pp65 represents a potential immunocompetence marker. Altogether, T-Track® CMV is a highly sensitive IFN-γ ELISpot assay, suitable for the immunomonitoring of CMV-seropositive renal transplant recipients, and with a potential use for the risk assessment of CMV-related clinical complications (ClinicalTrials.gov Identifier: NCT02083042).