BackgroundIn healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions.MethodsObjective of this work was the optimization and technical validation of an IFN-γ ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated® immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay.ResultsOptimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 × 104 and 2 × 105 PBMC per well upon stimulation with T-activated® IE-1 (R2 = 0.97) and pp65 (R2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated® IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3+CD4+ (Th), CD3+CD8+ (CTL), CD3−CD56+ (NK) and CD3+CD56+ (NKT-like) cells. Accordingly, the optimized IFN-γ ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive.ConclusionThe combined use of T-activated® IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-γ ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0195-y) contains supplementary material, which is available to authorized users.

Antigen-specific stimulation of T helper (Th) cells initiates signaling cascades that ultimately result in the activation of the transcription factors NF-jB, NFAT, and AP-1 which regulate, together with other factors, many T-cell functions such as cytokine production, proliferation, and differentiation. Ordered assembly and different phosphorylation events, along with subcellular translocation of the CARMA1/Bcl-10/MALT1 complex, determine NF-jB activation after T-cell receptor (TCR) triggering. We now provide evidence that inhibition of the Ser/Thr phosphatase calcineurin (CaN) prevents dephosphorylation of Bcl-10. CaN, in constant interaction with the Bcl-10/MALT1 complex, is able to dephosphorylate Bcl-10. The CaN inhibitor cyclosporine A (CsA) converts a transient phosphorylation of Bcl-10 Ser138 during the immediate early phase of T-cell activation into a persistent state. Thus, subsequent processes such as IKKb phosphorylation, IjBa degradation, p65 nuclear translocation, and DNA binding are diminished. Consistently, CsA treatment does not affect the phosphorylation pattern of the upstream kinase PKCh. Together, our findings demonstrate that CaN functions as a critical signaling molecule during Th cell activation, regulating Bcl-10 phosphorylation and thereby NF-jB activation.Keywords: Calcineurin . Cell activation . T cell . TCR signaling . Transcription factors Supporting Information available online IntroductionAntigen-specific stimulation of T helper (Th) cells induces activation of the transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) via a complex signaling network. The cooperative action of these and other transcription factors results in gene expression profiles leading to the activation of Th cells, production of growth factors, in particular cytokines, and proliferation [1][2][3]. T-cell receptor (TCR) engagement triggers phosphorylation of membrane-associated tyrosine kinases that activate PLCg, which in turn generates the second messengers IP 3 and DAG. The IP 3 -induced Ca 21 influx triggers the Ser/Thr phosphatase calcineurin (CaN) which dephosphorylates NFAT. This dephosphorylation step is crucial for the nuclear translocation of NFAT and binding to promoters of target genes. The second messenger, DAG, activates the NF-kB signaling cascade by binding to the protein kinase C-y (PKCy) leading to a change in PKCy conformation into an open, active state. The precise nature of these changes is not entirely clear, but phosphorylation at different sites, subcellular translocation, and assembly of assorted molecules contributes to this process (reviewed in [4,5] [6,7]. The importance of Bcl-10 for NF-kB activation is underlined by studies in Bcl-10 knockout mice showing that Bcl-10 is essential for NF-kB-driven cytokine production, cell proliferation, and B-as well as T-cell development [8,9]. Furthermore, Bcl-10 overexpression in MALT B-cell lymphomas is characterized by constant NF-kB activation [10,11]. Bcl-1...

BackgroundUncontrolled cytomegalovirus (CMV) replication in immunocompromised solid-organ transplant recipients is a clinically relevant issue and an indication of impaired CMV-specific cell-mediated immunity (CMI). Primary aim of this study was to assess the suitability of the immune monitoring tool T-Track® CMV to determine CMV-reactive CMI in a cohort of hemodialysis patients representative of patients eligible for renal transplantation. Positive and negative agreement of T-Track® CMV with CMV serology was examined in 124 hemodialysis patients, of whom 67 (54%) revealed a positive CMV serostatus. Secondary aim of the study was to evaluate T-Track® CMV performance against two unrelated CMV-specific CMI monitoring assays, QuantiFERON®-CMV and a cocktail of six class I iTAg™ MHC Tetramers.ResultsPositive T-Track® CMV results were obtained in 90% (60/67) of CMV-seropositive hemodialysis patients. In comparison, 73% (45/62) and 77% (40/52) positive agreement with CMV serology was achieved using QuantiFERON®-CMV and iTAg™ MHC Tetramer. Positive T-Track® CMV responses in CMV-seropositive patients were dominated by pp65-reactive cells (58/67 [87%]), while IE-1-responsive cells contributed to an improved (87% to 90%) positive agreement of T-Track® CMV with CMV serology. Interestingly, T-Track® CMV, QuantiFERON®-CMV and iTAg™ MHC Tetramers showed 79% (45/57), 87% (48/55) and 93% (42/45) negative agreement with serology, respectively, and a strong inter-assay variability. Notably, T-Track® CMV was able to detect IE-1-reactive cells in blood samples of patients with a negative CMV serology, suggesting either a previous exposure to CMV that yielded a cellular but no humoral immune response, or TCR cross-reactivity with foreign antigens, both suggesting a possible protective immunity against CMV in these patients.ConclusionT-Track® CMV is a highly sensitive assay, enabling the functional assessment of CMV-responsive cells in hemodialysis patients prior to renal transplantation. T-Track® CMV thus represents a valuable immune monitoring tool to identify candidate transplant recipients potentially at increased risk for CMV-related clinical complications.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-017-0194-z) contains supplementary material, which is available to authorized users.

The enteric bacterium Escherichia blattae has been analyzed for the presence of cobalamin (B₁₂) biosynthesis and B₁₂-dependent pathways. Biochemical studies revealed that E. blattae synthesizes B₁₂ de novo aerobically and anaerobically. Genes exhibiting high similarity to all genes of Salmonella enterica serovar Typhimurium, which are involved in the oxygen-independent route of B₁₂ biosynthesis, were present in the genome of E. blattae DSM 4481. The dha regulon encodes the key enzymes for the anaerobic conversion of glycerol to 1,3-propanediol, including coenzyme B₁₂-dependent glycerol dehydratase. E. blattae DSM 4481 lacked glycerol dehydratase activity and showed no anaerobic growth with glycerol, but the genome of E. blattae DSM 4481 contained a dha regulon. The E. blattaedha regulon is unusual, since it harbors genes for two types of dihydroxyacetone kinases. The major difference to dha regulons of other enteric bacteria is the inactivation of the dehydratase-encoding gene region by insertion of a 33,339-bp prophage (MuEb). Sequence analysis revealed that MuEb belongs to the Mu family of bacteriophages. The E. blattae strains ATCC 33429 and ATCC 33430 did not contain MuEb. Accordingly, both strains harbored an intact dehydratase-encoding gene region and fermented glycerol. The properties of the glycerol dehydratases and the correlating genes (dhaBCE) of both strains were similar to other B₁₂-dependent glycerol and diol dehydratases, but both dehydratases exhibited the highest affinity for glycerol of all B₁₂-dependent dehydratases characterized so far. In addition to the non-functional genes encoding B₁₂-dependent glycerol dehydratase, the genome of E. blattae DSM 4481 contained the genes for only one other B₁₂-dependent enzyme, the methylcobalamin-dependent methionine synthase.

The calcineurin inhibitor Cyclosporine A (CsA) is one of the crucial immunosuppressive drugs given after organ transplantation. The small therapeutic window of CsA generates the dilemma that efficient and toxic drug doses differ only slightly. Moreover, these threshold concentrations differ considerably between individuals; therefore, functional assays are urgently needed. We explored whether the transcription factor NFATc1, a direct as well as indirect target of CsA, can be used as a potential biomarker to determine the individual immunosuppressive activity of CsA. First, in isolated human T cells we showed that flow cytometry is practicable to measure NFATc1, the most abundant NFATc isoform in activated T cells. Second, for whole blood we developed a flow cytometric assay to determine in parallel the inducible transcription factor NFATc1 and the cytokine IL-2 in stimulated T cells. We found that added CsA inhibits both the expression of NFATc1 and IL-2 in T cells of stimulated whole blood samples with IC 50 values of 200 and 150 nM, respectively. The intra-and inter-assay variability was low, and clinical practicability was good. Further experiments have to demonstrate whether the parallel cytometric measurement of NFATc1 and IL-2 in whole blood is a good predictor of individual CsA efficacy and toxicity in CsA-treated patients. '