Impaired IFN-γ production and proliferation of NK cells in Multiple Sclerosis

NK cells are multicompetent lymphocytes of the innate immune system with a central role in host defense and immune regulation. Studies in experimental animal models of multiple sclerosis (MS) provided evidence for both pathologic and protective effects of NK cells. Humans harbor two functionally distinct NK-cell subsets exerting either predominantly cytotoxic (CD56dimCD16+) or immunoregulatory (CD56brightCD16−) functions. We analyzed these two subsets and their functions in the peripheral blood of untreated patients with relapsing-remitting MS compared with healthy blood donors. While ex vivo frequencies of CD56brightCD16− and CD56dimCD16+ NK cells were similar in patients and controls, we found that cytokine-driven in vitro accumulation and IFN-γ production of CD56brightCD16− NK cells but not of their CD56dimCD16+ counterparts were substantially diminished in MS. Impaired expansion of CD56brightCD16− NK cells was cell intrinsic because the observed effects could be reproduced with purified NK cells in an independent cohort of patients and controls. In contrast, cytolytic NK-cell activity toward the human erythromyeloblastoid leukemia cell line K562, the allogeneic CD4+ T cell line CEM and allogeneic primary CD4+ T-cell blasts was unchanged. Thus, characteristic functions of CD56brightCD16− NK cells, namely cytokine-induced NK cell expansion and IFN-γ production, are compromised in the NK cell compartment of MS patient

Reduced frequency of cytotoxic CD56dim CD16+ NK cells leads to impaired antibody-dependent degranulation in EBV-positive classical Hodgkin lymphoma

Around 30–50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL

Investigation of two Fermi-LAT gamma-ray blazars coincident with high-energy neutrinos detected by IceCube

After the identification of the gamma-ray blazar TXS 0506+056 as the first compelling IceCube neutrino source candidate, we perform a systematic analysis of all high-energy neutrino events satisfying the IceCube realtime trigger criteria. We find one additional known gamma-ray source, the blazar GB6 J1040+0617, in spatial coincidence with a neutrino in this sample. The chance probability of this coincidence is 30% after trial correction. For the first time, we present a systematic study of the gamma-ray flux, spectral and optical variability, and multi-wavelength behavior of GB6 J1040+0617 and compare it to TXS 0506+056. We find that TXS 0506+056 shows strong flux variability in the Fermi-LAT gamma-ray band, being in an active state around the arrival of IceCube-170922A, but in a low state during the archival IceCube neutrino flare in 2014/15. In both cases the spectral shape is statistically compatible ($\leq 2\sigma$) with the average spectrum showing no indication of a significant relative increase of a high-energy component. While the association of GB6 J1040+0617 with the neutrino is consistent with background expectations, the source appears to be a plausible neutrino source candidate based on its energetics and multi-wavelength features, namely a bright optical flare and modestly increased gamma-ray activity. Finding one or two neutrinos originating from gamma-ray blazars in the given sample of high-energy neutrinos is consistent with previously derived limits of neutrino emission from gamma-ray blazars, indicating the sources of the majority of cosmic high-energy neutrinos remain unknown.Comment: 22 pages, 11 figures, 2 Table

IceCube Search for Neutrinos Coincident with Compact Binary Mergers from LIGO-Virgo's First Gravitational-Wave Transient Catalog

Using the IceCube Neutrino Observatory, we search for high-energy neutrino emission coincident with compact binary mergers observed by the LIGO and Virgo gravitational wave (GW) detectors during their first and second observing runs. We present results from two searches targeting emission coincident with the sky localization of each gravitational wave event within a 1000 second time window centered around the reported merger time. One search uses a model-independent unbinned maximum likelihood analysis, which uses neutrino data from IceCube to search for point-like neutrino sources consistent with the sky localization of GW events. The other uses the Low-Latency Algorithm for Multi-messenger Astrophysics, which incorporates astrophysical priors through a Bayesian framework and includes LIGO-Virgo detector characteristics to determine the association between the GW source and the neutrinos. No significant neutrino coincidence is seen by either search during the first two observing runs of the LIGO-Virgo detectors. We set upper limits on the time-integrated neutrino emission within the 1000 second window for each of the 11 GW events. These limits range from 0.02-0.7 $\mathrm{GeV~cm^{-2}}$. We also set limits on the total isotropic equivalent energy, $E_{\mathrm{iso}}$, emitted in high-energy neutrinos by each GW event. These limits range from 1.7 $\times$ 10$^{51}$ - 1.8 $\times$ 10$^{55}$ erg. We conclude with an outlook for LIGO-Virgo observing run O3, during which both analyses are running in real time

Characteristics of the diffuse astrophysical electron and tau neutrino flux with six years of IceCube high energy cascade data

We report on the first measurement of the astrophysical neutrino flux using particle showers (cascades) in IceCube data from 2010 -- 2015. Assuming standard oscillations, the astrophysical neutrinos in this dedicated cascade sample are dominated ($\sim 90 \%$) by electron and tau flavors. The flux, observed in the sensitive energy range from $16\,\mathrm{TeV}$ to $2.6\,\mathrm{PeV}$, is consistent with a single power-law model as expected from Fermi-type acceleration of high energy particles at astrophysical sources. We find the flux spectral index to be $\gamma=2.53\pm0.07$ and a flux normalization for each neutrino flavor of $\phi_{astro} = 1.66^{+0.25}_{-0.27}$ at $E_{0} = 100\, \mathrm{TeV}$, in agreement with IceCube's complementary muon neutrino results and with all-neutrino flavor fit results. In the measured energy range we reject spectral indices $\gamma\leq2.28$ at $\ge3\sigma$ significance level. Due to high neutrino energy resolution and low atmospheric neutrino backgrounds, this analysis provides the most detailed characterization of the neutrino flux at energies below $\sim100\,{\rm{TeV}}$ compared to previous IceCube results. Results from fits assuming more complex neutrino flux models suggest a flux softening at high energies and a flux hardening at low energies (p-value $\ge 0.06$). The sizable and smooth flux measured below $\sim 100\,{\rm{TeV}}$ remains a puzzle. In order to not violate the isotropic diffuse gamma-ray background as measured by the Fermi-LAT, it suggests the existence of astrophysical neutrino sources characterized by dense environments which are opaque to gamma-rays.Comment: 4 figures, 4 tables, includes supplementary materia

Identification and characterisation of the Macrophage/Microglia Activation Factor in the damaged hippocampus

Titelblatt und Inhaltsverzeichnis Einleitung Ziel und Fragestellung der vorliegenden Arbeit Material und Methoden Ergebnisse Diskussion LiteraturverzeichnisMakrophagen und Mikrogliazellen spielen eine zentrale Rolle im Abwehrsystem des zentralen Nervensystems (ZNS). Sie werden nach einer ZNS Läsion unmittelbar aktiviert, migrieren zum Ort des Schadens und sind hier für den sekundären neuronalen Zellschaden von herausragender Bedeutung. Das Verständnis der genauen molekularen Mechanismen hierfür ist bisher nur lückenhaft vorhanden. Ziel dieser Dissertation war die Charakterisierung der Regulation und des Expressionsmusters des Makrophagen Aktivierungsfaktor (MAF) nach fokaler ZNS Läsion. MAF wurde erstmals als differenzierungsassoziierter Faktor des Monozyten/Makrophagen-Systems beschrieben. Vorarbeiten zu dieser Arbeit zeigten eine konsistente läsionsassoziierte Hochregulation von MAF auf mRNA Ebene nach entorhinaler Kortexläsion (ECL). Es konnte in dieser Dissertation gezeigt werden, dass (i) MAF differenzierungsabhängig nur in ausdifferenzierten Makrophagen exprimiert wird, (ii), diese Hochregulation von MAF CD11b abhängig induziert wird, (iii) mittels eigenständig generierter polyklonaler, spezifischer Antikörper, dass MAF auf Proteinebene in Makrophagen/Mikroglia nach ECL läsionsassoziiert und auf den deafferenzierenden Hippocampus beschränkt exprimiert wird, (iv) dass MAF in murinen Mikrogliazellen in vitro und in vivo ein intrazellulär vesikuläres Expressionsmuster aufweist, (v) dass MAF hämolytische Eigenschaften besitzt und (vi) strukturell einer neuartigen hochkonservierten Proteinfamilie zuzuordnen ist, die sich durch ein gemeinsames Motiv von sieben transmembranären Spannen auszeichnet. Die CD11b-gesteuerte und läsionsassoziierte Expression von MAF im deafferenzierenden Hippocampus weist auf eine mögliche zentrale Rolle im Rahmen der Differenzierung von Mikrogliazellen zum phagozytierenden Phänotyp hin. MAF könnte ein geeignetes Zielmolekül für das therapeutische Vermeiden oder Begrenzen des durch Makrophagen/Mikroglia verursachten sekundären Zellschadens nach ZNS Läsion sein.After traumatic brain lesion, microglial cells are rapidly activated, migrate towards the sites of injury and cause secondary damage that accounts for most of the loss of brain function. This thesis aimed at characterizing the regulation of the macrophage/microglia activation factor (MAF) which expression was previously demonstrated to be differentiation-associated in myeloid hematopoetic cells and was shown to be consistently upregulated following entorhinal cortex lesion (ECL) at the mRNA level. Using the monocytic cell line U937, we could demonstrate that MAF is upregulated after TPA-induced differentiation into macrophages. The maturation-associated MAF upregulation could be blocked by CD11b antisense transfection. Furthermore, we have generated a specific antibody against MAF. In BV-2 microglial cells, MAF is co-localized with IB-4, a classical microglial marker. In addition, we have analyzed the in vivo expression patterns of MAF after ECL. We could show a substantial upregulation of MAF on most macrophages/microglial cells in the deafferentiated hippocampus, while there was no MAF expression detectable on the contralateral side. In the perilesional region, in most but not all cells MAF is co-localized with CD11b and IB4, two classical markers for microglial cells. Confocal microscopy revealed a lysosome-like expression pattern in BV-2 cells as well as in ECL-associated macrophages/microglial cells in vivo. My data indicate that MAF is upregulated after monocyte maturation in a CD11b- dependent manner. Furthermore, we could demonstrate that MAF is expressed only in selected macrophages/microglial cells around the lesion and in the degenerating hippocampus after ECL. Consistent with these lesion-associated expression patterns, MAF expression in monocytic cells seems to play a functional role in the differentiation to a phagocytosing phenotype and may be, at least partially, required for phagocytotic activity, specifically in lesioned tissue after brain trauma

Innate Immune Recognition of EBV

The ability of Epstein-Barr virus (EBV) to establish latency despite specific immune responses and to successfully persist lifelong in the human host shows that EBV has developed powerful strategies and mechanisms to exploit, evade, abolish, or downsize otherwise effective immune responses to ensure its own survival. This chapter focuses on current knowledge on innate immune responses against EBV and its evasion strategies for own benefit and summarizes the questions that remain to be tackled. Innate immune reactions against EBV originate both from the main target cells of EBV and from nontarget cells, which are elements of the innate immune system. Thus, we structured our review accordingly but with a particular focus on the innate recognition of EBV in its two stages in its life cycle, latent state and lytic replication. Specifically, we discuss (I) innate sensing and resulting innate immune responses against EBV by its main target cells, focusing on (i) EBV transmission between epithelial cells and B cells and their life cycle stages; and (ii) elements of innate immunity in EBV's target cells. Further, we debate (II) the innate recognition and resulting innate immune responses against EBV by cells other than the main target cells, focusing on (iii) myeloid cells: dendritic cells, monocytes, macrophages, and neutrophil granulocytes; and (iv) natural killer cells. Finally, we address (III) how EBV counteracts or exploits innate immunity in its latent and lytic life cycle stages, concentrating on (v) TLRs; (vi) EBERs; and (vii) microRNAs

A distinct subpopulation of human NK cells restricts B cell transformation by EBV

NK cells constitute the first line of defense against pathogens and transformed cells. They mature in secondary lymphoid organs, including tonsils, where common pathogens, such as EBV, enter the host and potentially imprint differentiating cells, which then patrol the body via the blood stream. Therefore, we set out to characterize a distinct human NK cell population in tonsils that produces high amounts of the immunomodulatory and antiviral cytokine IFN-γ. We found that the tonsilar IFN-γ(high) NK cell subset is CD56(bright)NKG2A(+)CD94(+)CD54(+)CD62L(-), is present in tonsils ex vivo and is more mature than other CD56(bright) NK cells in tonsils and less mature than other NK cells in blood, shows very low plasticity even after prolonged cytokine stimulation, accumulates in tonsils of EBV carriers, and is able to potently restrict EBV-induced transformation of B cells. Thus, we characterized a distinct and stable IFN-γ(high) NK cell subpopulation that can specifically restrict malignant transformation of EBV-infected B cells. This subset should be exploited for future development of cell-based therapeutic approaches in EBV-associated malignancies

TNF-α Induces Macroautophagy and Regulates MHC Class II Expression in Human Skeletal Muscle Cells*

Macroautophagy, a homeostatic process that shuttles cytoplasmic constituents into endosomal and lysosomal compartments, has recently been shown to deliver antigens for presentation on major histocompatibility complex (MHC) class II molecules. Skeletal muscle fibers show a high level of constitutive macroautophagy and express MHC class II molecules upon immune activation. We found that tumor necrosis factor-α (TNF-α), a monokine overexpressed in inflammatory myopathies, led to a marked up-regulation of macroautophagy in skeletal myocytes. Furthermore, TNF-α augmented surface expression of MHC class II molecules in interferon-γ (IFN-γ)-treated myoblasts. The synergistic effect of TNF-α and IFN-γ on the induction of MHC class II surface expression was not reflected by higher intracellular human leukocyte antigen (HLA)-DR levels and was reversed by macroautophagy inhibition, suggesting that TNF-α facilitates antigen processing via macroautophagy for more efficient MHC class II loading. Muscle biopsies from patients with sporadic inclusion body myositis, a well defined myopathy with chronic inflammation, showed that over 20% of fibers that contained autophagosomes costained for MHC class II molecules and that more than 40% of double-positive muscle fibers had contact with CD4+ and CD8+ immune cells. These findings establish a mechanism through which TNF-α regulates both macroautophagy and MHC class II expression and suggest that macroautophagy-mediated antigen presentation contributes to the immunological environment of the inflamed human skeletal muscle

IRAK4 is essential for TLR9-induced suppression of Epstein-Barr virus <i>BZLF1</i> transcription in Akata Burkitt’s lymphoma cells

<div><p>Burkitt’s lymphoma (BL) is the most common childhood cancer in equatorial Africa, and is endemic to areas where people are chronically co-infected with Epstein-Barr virus (EBV) and the malaria pathogen <i>Plasmodium falciparum</i>. The contribution of these pathogens in the oncogenic process remains poorly understood. We showed earlier that the activation of Toll-like receptor (TLR) 9 by hemozoin, a disposal product formed from the digestion of blood by <i>P</i>. <i>falciparum</i>, suppresses the lytic reactivation of EBV in BL cells. EBV lytic reactivation is regulated by the expression of transcription factor Zta (ZEBRA), encoded by the EBV gene <i>BZLF1</i>. Here, we explore in the BL cell line Akata, the mechanism involved in repression by TLR9 of expression of <i>BZLF1</i>. We show that <i>BZLF1</i> repression is mediated upon TLR9 engagement by a mechanism that is largely independent of <i>de novo</i> protein synthesis. By CRISPR/Cas9-induced inactivation of TLR9, MyD88, IRAK4 and IRAK1 we confirm that <i>BZLF1</i> repression is dependent on functional TLR9 and MyD88 signaling, and identify IRAK4 as an essential element for TLR9-induced repression of <i>BZLF1</i> expression upon BCR cross-linking. Our results unprecedentedly show that TLR9-mediated inhibition of lytic EBV is largely independent of new protein synthesis and demonstrate the central roles of MyD88 and IRAK4 in this process contributing to EBV’s persistence in the host’s B-cell pool.</p></div