Die Rolle von Natürlichen Killerzellen und Natürlichen Killer T-Zellen in der HCV Infektion

Worldwide 71 million people are chronically infected with HCV and each year approximately 400 000 people die from HCV associated liver diseases. The most important risk factor in western industrialised countries to acquire HCV infection is intravenous drug use. For this reason, we utilised a cohort of people who inject drugs (PWID) to comparatively analyse PWID developing chronic infection (HCV RNA positive), spontaneously resolving HCV infection (HCV RNA negative) and PWID remaining anti HCV seronegative (anti HCV negative), to provide novel insights into the contribution of innate and adaptive immune mechanisms to HCV infection outcome. The role of Natural Killer T (NKT) cells, which share characteristics of innate and adaptive immune cells, for the outcome of HCV infection is poorly defined. Invariant NKT (iNKT) cells, a subset of NKT cells, recognise glycolipid antigens such as α galactosylceramide (αGalCer) presented by the non-classical MHC molecule CD1d. Decreased iNKT cell frequencies have been reported in chronically HCV infected patients, however, contradicting reports exist. In this study iNKT cell frequencies did not differ between HCV RNA positive (n=28) and HCV RNA negative (n=33) PWID. Interestingly, phenotypic analysis of iNKT cells of chronically infected PWID showed significantly higher expression of the activation marker CD38, however, this was not associated with apparent differences in PMA/ionomycin stimulated effector functions. In addition, treatment with a combination of IL12, -15 and -18, which is described to activate NK cells, led to a significant increase in CD38+ iNKT cells. Since IFN stimulated genes are induced in patients with chronic infection the influence of IFNs on iNKT cell activation was addressed as well. Although no direct effect of IFNα on iNKT cell activation was observed, CD1d was upregulated on peripheral monocytes, the cell type with the highest expression of CD1d in the periphery. However, CD1d levels on monocytes, did not differ between PWID with chronic and resolved HCV infection. Nevertheless, presence of monocytes promoted iNKT cell expansion and activation, as depletion of monocytes significantly reduced the frequency of CD38+ iNKT cells. Furthermore, CD1d transcript levels were significantly higher in HCV infected liver compared to HBV-infected liver. Collectively the data demonstrate that iNKT cells are activated in chronically HCV infected PWID, possibly via type I interferon-induced upregulation of CD1d on monocytes or other liver resident cells or via direct activation by pro inflammatory cytokines. However, the functional consequences of increased activation of iNKT cells are not fully defined. While iNKT cells are still poorly covered by scientific reports in HCV infection there is growing evidence for the importance of NK cells for HCV infection outcome. Genetic association studies revealed that specific genetically determined combinations of NK cell-receptors and their ligands are associated with differential outcome of HCV infection. NK cell function is regulated by a set of inhibitory and activating receptors including killer-cell immunoglobulin-like receptors (KIRs). A previous study in our lab identified that KIR3DL1/HLA-Bw4 80(T) was associated with spontaneous clearance of HCV infection in our PWID cohort (n=266) which was validated in a second PWID cohort from North America (n=342). For this reason, this study focused on the functional characterisation of NK cells according to the identified KIR/KIR-ligand genotype. Besides PWID, healthy individuals (n=120) were included in the analysis as well. KIR3DL1+ NK cells from HLA-Bw4 80(T) positive PWID showed superior functionality compared to HLA Bw4 80(I) positive PWID. However, this differential impact was not observed in healthy donors. Moreover, the frequency of individuals with multiple HLA-Bw4 alleles was significantly higher in HCV RNA negative as well as anti HCV negative PWID compared to HCV RNA positive PWID and HLA-Bw4 copy number strongly correlated with the functionality of KIR3DL1+ NK cells in healthy individuals. HLA-Bw4 80(T) and multiple HLA-Bw4 copies in combination with KIR3DL1 are associated with protection against chronic HCV in PWID by distinct mechanisms. Better education of KIR3DL1+ NK cells in the presence of multiple HLA Bw4 copies is beneficial prior to seroconversion whereas HLA Bw4 80(T) may be beneficial during acute hepatitis C. To determine possible underlying mechanisms leading to the superior functionality of KIR3DL1+ NK cells, the DNA methylation pattern of the IFNG locus was analysed, since demethylation of the IFNG locus is generally associated with increased IFNG transcription. Unfortunately, the significantly enhanced IFNγ capacity of KIR3DL1+ NK cells from multiple HLA Bw4 motif carriers was not epigenetically regulated by DNA methylation. However, our data could confirm that NK cell memory features, attributed to NKG2C expressing NK cells in CMV positive individuals, are epigenetically imprinted within the IFNG locus. In summary, these studies contribute novel evidence for the importance of NK cells and iNKT cells for HCV infection outcome and describe possible underlying mechanisms. However, further research is needed to elucidate for instance, the role of HCV peptides presented by HLA alleles harbouring a Bw4 motif on the function of KIR3DL1+ NK cells or the exact mechanism leading to the activated state of iNKT cells during chronic HCV infection.Nach Schätzung der WHO sind zurzeit 71 Millionen Menschen weltweit chronisch mit dem Hepatis C Virus (HCV) infiziert und jährlich sterben etwa 400.000 Menschen an den Folgen einer HCV induzierten Leberkrankungen. In westlichen Industrieländern stellt der intravenöse Drogenkonsum hierbei den höchsten Risikofaktor dar, sich mit HCV zu infizieren. Um neue Erkenntnisse über die Rolle des natürlichen und angeboren Immunsystems in der HCV Infektion zu gewinnen, wurde einer Kohorte von intravenös Drogen gebrauchenden Menschen („people who inject drugs“ PWID) analysiert. Hierbei wurden PWID mit chronischer HCV-Infektion (HCV RNA positiv), ausgeheilter HCV-Infektion (HCV RNA negativ) und HCV seronegative PWID (anti HCV negativ) miteinander verglichen. Welche Rolle Natürliche Killer T (NKT) Zellen, die sowohl Eigenschaften von innaten als auch adaptiven Immunzellen aufweisen, bei dem Verlauf einer HCV Infektion spielen ist nicht klar definiert. Invariante NKT (iNKT) Zellen stellen eine Subgruppe der NKT Zellen dar und erkennen Glycolipid Antigene wie z.B. α galactosylceramid (αGalCer), die durch das nicht klassische MHC Molekül CD1d präsentiert werden. Die Untersuchung der iNKT Zellfrequenzen zeigte keine Unterschiede zwischen HCV RNA positiven (n=28) und HCV RNA negativen (n=33) PWID. Interessanterweise ergab die phänotypische Analyse, dass sich iNKT Zellen von chronisch HCV infizierten PWID durch eine signifikant erhöhte Expression des Aktivierungsmarkers CD38 auszeichnen. Allerdings konnten keine funktionellen Unterschiede mit diesem aktivierten Phänotyp assoziiert werden. Zusätzlich wurde die Auswirkung von Interferonen auf iNKT Zellen in dieser Studie adressiert. Während kein direkter Effekt von IFNα oder IFNλ festgestellt werden konnte, führte eine Kombination von NK Zell aktivierenden Zytokinen (IL12, IL15 und IL18) zu einer signifikanten Erhöhung der CD38+ iNKT Zellen. Auch wenn gezeigt werden konnte, dass der Ligand von iNKT Zellen durch IFNα Stimulation hochreguliert wird, konnte kein Unterschied in der CD1d Expression auf peripheren Monozyten, welche in der Peripherie den Zelltyp mit der höchsten CD1d Expression darstellen, zwischen HCV RNA positiven und HCV RNA negativen PWID festgestellt werden. Dennoch nehmen Monozyten eine wichtige Rolle in der Expansion und Aktivierung von iNKT Zellen ein, da eine Depletierung selbiger eine signifikant verminderte Frequenz von CD38+ iNKT Zellen zur Folge hat. Des Weiteren ergaben Leberanalysen, dass HCV infizierte Lebern im Vergleich zu HBV infizierten Lebern eine signifikant erhöhte CD1d Transkription aufweisen. Zusammengefasst weisen unsere Daten darauf hin, dass iNKT Zellen in der chronischen HCV Infektion aktiviert sind. Sowohl eine Typ I Interferon induzierte Hochregulation von CD1d auf Monozyten oder anderen Zellen in der Leber als auch das proinflammatorische Zytokine Milieu sind potentiell für die Modulation des Aktivierungsstatus von iNKT Zellen in der HCV Infektion verantwortlich. Während es kaum wissenschaftliche Berichte gibt, die sich mit iNKT Zellen im Kontext der HCV Infektion auseinandersetzen, steigt die Zahl an Belegen, die NK Zellen eine wichtige Bedeutung für die Prävention und Kontrolle einer HCV Infektion zusprechen. Verschiedene Assoziationsstudien konnten bereits zeigen, dass genetisch determinierten Kombinationen von NK Zell Rezeptoren und ihren Liganden mit der HCV Verlaufsprognose assoziiert sind. Die NK Zell Funktion wird durch aktivierende und inhibierende Rezeptoren einschließlich der Killerzell Immunglobulin ähnlichen Rezeptoren (KIR) reguliert. In einer vorangegangenen Studie wurde mit Hilfe einer multivariaten logistischen Regressionsanalyse KIR3DL1/HLA-Bw4 80(T) mit der spontanen Ausheilung einer HCV Infektion in unserer PWID Kohorte (n=266) assoziiert. Diese Assoziation konnte in einer zweiten anti HCV positiven PWID Kohorte (n=342) aus Nordamerika validiert werden. Auf Grund dessen wurden in der vorliegenden Studie NK Zellen anhand ihres KIR/KIR Liganden Genotyps funktionell charakterisiert. Neben PWID wurden auch gesunde Proben (n=120) in der Analyse inkludiert. Es konnte gezeigt werden, dass KIR3DL1+ NK Zellen von HLA-Bw4 80(T) positiven PWID verglichen mit HLA Bw4 80(I) positiven PWID eine signifikant erhöhte NK Zell Funktionalität aufweisen. Hingegen konnte kein funktioneller Einfluss dieses Genotyps in gesunden Individuen beobachtet werden. Darüber hinaus wurde gezeigt, dass multiple HLA Bw4 Allele, deren Frequenz signifikant in HCV RNA negativen und anti HCV negativen PWID im Vergleich zu HCV RNA positiven PWID erhöht ist, auch mit einer starken NK Zell Funktionalität einhergehen. Zusammenfassend können sowohl HLA Bw4 80(T) als auch multiple Kopien von HLA Bw4 in Kombination mit KIR3DL1 mit der Protektion gegen eine chronische Hepatitis C assoziiert werden. Diese wird über verschiedene Mechanismen vermittelt: während eine bessere NK Zell Lizensierung durch die Anwesenheit von multiplen HLA Bw4 Kopien vor der Serokonversion vorteilhaft ist, könnte HLA Bw4 80(T) während eine akuten HCV Infektion von Vorteil sein. Epigenetische Veränderung könnten eine mögliche Erklärung für die erhöhte IFNγ Produktion von KIR3DL1+ NK Zellen im Kontext von multiplen HLA Bw4 Allelen bieten. Hierfür wurde die DNA Demethylierung des IFNG Lokus, welche generell mit einer erhöhten Transkription assoziiert ist, analysiert. Es konnte jedoch kein Zusammenhang zwischen dem Methylierungsstatus und der Anzahl an HLA Bw4 Kopien hergestellt werden. Hingegen konnten vorausgegangene Studien bestätigt werden, die besagen, dass Memory Eigenschaften von NKG2C exprimierenden NK Zellen in CMV positiven Individuen epigenetisch reguliert sind. Abschließend lässt sich feststellen, dass die hier präsentierten Studien neue Beweise für die Bedeutsamkeit von NK und iNKT Zellen für den HCV Infektionsverlauf liefern und Hinweise auf mögliche zugrundeliegende Mechanismen geben. Dennoch sind weitere Arbeiten notwendig die z.B. klären ob HCV Peptide, die durch Bw4 Motiv tragende HLA Allele präsentiert werden, einen Einfluss auf die Funktion von KIR3DL1+ NK Zellen haben oder welche Mechanismen genau während der chronischen Infektion zu einer Aktivierung von iNKT Zellen führen

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

<p>Abstract</p> <p>Background</p> <p>We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.</p> <p>Results</p> <p>Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.</p> <p>Conclusion</p> <p>From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.</p

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

HLA-Bw4 80(T) and multiple HLA-Bw4 copies combined with KIR3DL1 associate with spontaneous clearance of HCV infection in people who inject drugs

BACKGROUND AND AIMS: NK cell function is regulated by inhibitory and activating receptors including killer-cell immunoglobulin-like receptors (KIRs). Here, we analyzed the impact of different KIR/KIR-ligand genotypes on the outcome of HCV infection in people who inject drugs (PWID). KIR/KIR-ligand genotypes associated with spontaneous clearance of HCV infection were identified in a cohort of PWID from Germany (n=266) and further validated in a second anti-HCV positive cohort of PWID recruited in North America (n=342). Moreover, NK cells of PWID and healthy donors were functionally characterized according to their KIR/KIR-ligand genotype by flow cytometry.RESULTS: Multivariate logistic regression analysis revealed that KIR3DL1/HLA-Bw4 80(T) was associated with spontaneous clearance of HCV infection in PWID, which was confirmed in the PWID cohort from North America. Moreover, compared with PWID with detectable HCV-RNA the frequency of individuals with multiple HLA-Bw4 alleles was significantly higher in anti-HCV positive PWID with resolved HCV infection (29.7% vs. 15.2%; p=0.0229) and in anti-HCV seronegative PWID (39.2%; p=0.0006). KIR3DL1(+) NK cells from HLA-Bw4 80(T)-positive PWID showed superior functionality compared to HLA-Bw4 80(I)-positive PWID. This differential impact was not observed in healthy donors; however the HLA-Bw4 copy number strongly correlated with the functionality of KIR3DL1(+) NK cells.CONCLUSIONS: HLA-Bw4-80(T) and multiple HLA-Bw4 copies in combination with KIR3DL1 are associated with protection against chronic hepatitis C in PWID by distinct mechanisms. Better licensing of KIR3DL1(+) NK cells in the presence of multiple HLA-Bw4 copies is beneficial prior to seroconversion whereas HLA-Bw4 80(T) may be beneficial during acute hepatitis C. Lay summary NK cells are part of the innate immune system and are regulated by a complex network of activating and inhibiting receptors. The regulating receptor-ligand pairs of an individual are genetically determined. Here, we identified a particular set of ligand and receptor genes that associated with better functionality of NK cells and better outcome upon exposure to HCV in a high risk group.</p

The <i>RHOA</i> Mutation G17V Does Not Lead to Increased Migration of Human Malignant T Cells but Is Associated with Matrix Remodelling

Nodal T-follicular helper cell lymphoma, angioimmunoblastic-type (AITL), is characterized by constitutional symptoms, advanced-stage disease, and generalized lymphadenopathy. A genetic hallmark of this lymphoma is the frequent occurrence of the RHOA mutation G17V in neoplastic cells, which is observed in around 60% of patients. Because RHOA is involved in both T-cell receptor downstream signalling and cell migration, we hypothesized that the characteristic presentation of AITL could be the result of enhanced tumor cell migration. Therefore, this study aimed to elucidate the impact of the RHOA variant G17V on the migration of neoplastic T cells. We transfected the T-cell lymphoma cell lines HH and HuT78 to stably express the RHOA-G17V variant. RHOA-G17V-expressing T cells did not exhibit enhanced motility compared to empty-vector-transfected cells in microchannels, a 3D collagen gel, or primary human lymphatic tissue. Cells of the HH cell line expressing RHOA-G17V had an increased number of cells with cleaved collagen compared with the empty-vector-transfected cells. Therefore, we hypothesized that the early spread of AITL tumor cells may be related to remodelling of the extracellular matrix. Accordingly, we observed a significant negative correlation between the relative area of collagen in histological sections from 18 primary AITL and the allele frequency of the RHOA-G17V mutation. In conclusion, our results suggest that the characteristic presentation of AITL with early, widespread dissemination of lymphoma cells is not the result of an enhanced migration capacity due to the RHOA-G17V mutation; instead, this feature may rather be related to extracellular matrix remodelling

Sensitivity of anti-SARS-CoV-2 serological assays in a high-prevalence setting

Evaluation and power of seroprevalence studies depend on the performed serological assays. The aim of this study was to assess four commercial serological tests from EUROIMMUN, DiaSorin, Abbott, and Roche as well as an in-house immunofluorescence and neutralization test for their capability to identify SARS-CoV-2 seropositive individuals in a high-prevalence setting. Therefore, 42 social and working contacts of a German super-spreader were tested. Consistent with a high-prevalence setting, 26 of 42 were SARS-CoV-2 seropositive by neutralization test (NT), and immunofluorescence test (IFT) confirmed 23 of these 26 positive test results (NT 61.9% and IFT 54.8% seroprevalence). Four commercial assays detected anti-SARS-CoV-2 antibodies in 33.3-40.5% individuals. Besides an overall discrepancy between the NT and the commercial assays regarding their sensitivity, this study revealed that commercial SARS-CoV-2 spike-based assays are better to predict the neutralization titer than nucleoprotein-based assays are

Analysis of the long-term impact on cellular immunity in COVID-19-recovered individuals reveals a profound nkt cell impairment

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affected over 120 million people and killed over 2.7 million individuals by March 2021. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remain to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19-convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2-unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of annexin V and 7-aminoactinomycin D (7-AAD) double-positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies and TIM-3 expression on CD4 and CD8 T cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by granzyme B (GzmB) expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully able to proliferate and produce effector cytokines upon T cell receptor (TCR) stimulation. Collectively, we provide a comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease. IMPORTANCE Wuhan was the very first city hit by SARS-CoV-2. Accordingly, the patients who experienced the longest phase of convalescence following COVID-19 reside here. This enabled us to investigate the “immunological scar” left by SARS-CoV-2 on cellular immunity after recovery from the disease. In this study, we characterized the long-term impact of SARS-CoV-2 infection on the immune system and provide a comprehensive picture of cellular immunity of a convalescent COVID-19 patient cohort with the longest recovery time. We revealed that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease; in particular, a profound NKT cell impairment was found in the convalescent phase of COVID-19