Central tolerance shapes the neutralizing B cell repertoire against a persisting virus in its natural host.

Viral mimicry of host cell structures has been postulated to curtail the B cell receptor (BCR) repertoire against persisting viruses through tolerance mechanisms. This concept awaits, however, experimental testing in a setting of natural virus-host relationship. We engineered mouse models expressing a monoclonal BCR specific for the envelope glycoprotein of lymphocytic choriomeningitis virus (LCMV), a naturally persisting mouse pathogen. When the heavy chain of the LCMV-neutralizing antibody KL25 was paired with its unmutated ancestor light chain, most B cells underwent receptor editing, a behavior reminiscent of autoreactive clones. In contrast, monoclonal B cells expressing the same heavy chain in conjunction with the hypermutated KL25 light chain did not undergo receptor editing but exhibited low levels of surface IgM, suggesting that light chain hypermutation had lessened KL25 autoreactivity. Upon viral challenge, these IgMlow cells were not anergic but up-regulated IgM, participated in germinal center reactions, produced antiviral antibodies, and underwent immunoglobulin class switch as well as further affinity maturation. These studies on a persisting virus in its natural host species suggest that central tolerance mechanisms prune the protective antiviral B cell repertoire.

The PNPLA3 variant I148M reveals protective effects toward hepatocellular carcinoma in mice via restoration of omega-3 polyunsaturated fats.

Alcohol consumption and high caloric diet are leading causes of progressive fatty liver disease. Genetic variant rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3 rs738409 C>G) has been repeatedly described as one of the major risk loci for alcoholic liver cirrhosis (ALC) and hepatocellular carcinoma (HCC) in humans, however, the mechanism behind this association is incompletely understood. We generated mice carrying the rs738409 variant (PNPLA3 I148M) in order to detect genotype-phenotype relationships in mice upon chow and alcohol-high fat/high sugar diet (EtOH/WD). We could clearly demonstrate that the presence of rs738409 per se is sufficient to induce spontaneous development of steatosis after 1 year in mice on a chow diet, whereas in the setting of unhealthy diet feeding, PNPLA3 I148M did not affect hepatic inflammation or fibrosis, but induced a striking lipid remodeling, microvesicular steatosis and protected from HCC formation. Using shot gun lipidomics, we detected a striking restoration of reduced long chain-polyunsaturated fatty acids (LC-PUFA)-containing TGs, docosapentaenoic acid (C22:5 n3) and omega-3-derived eicosanoids (5-HEPE, 20-HEPE, 19,20-EDP, 21-HDHA) in PNPLA3 I148M mice upon EtOH/WD. At the molecular level, PNPLA3 I148M modulated enzymes for fatty acid and TG transport and metabolism. These findings suggest (dietary) lipids as an important and independent driver of hepatic tumorigenesis. Genetic variant in PNPLA3 exerted protective effects in mice, conflicting with findings in humans. Species-related differences in physiology and metabolism should be taken into account when modeling unhealthy human lifestyle, as genetic mouse models may not always allow for translation of insight gained in humans.

Enabling comprehensive optogenetic studies of mouse hearts by simultaneous opto-electrical panoramic mapping and stimulation.

During the last decade, cardiac optogenetics has turned into an essential tool for investigating cardiac function in general and for assessing functional interactions between different myocardial cell types in particular. To advance exploitation of the unique research opportunities offered by this method, we develop a panoramic opto-electrical measurement and stimulation (POEMS) system for mouse hearts. The core of the experimental platform is composed of 294 optical fibers and 64 electrodes that form a cup which embraces the entire ventricular surface of mouse hearts and enables straightforward 'drop&go' experimentation. The flexible assignment of fibers and electrodes to recording or stimulation tasks permits a precise tailoring of experiments to the specific requirements of individual optogenetic constructs thereby avoiding spectral congestion. Validation experiments with hearts from transgenic animals expressing the optogenetic voltage reporters ASAP1 and ArcLight-Q239 demonstrate concordance of simultaneously recorded panoramic optical and electrical activation maps. The feasibility of single fiber optical stimulation is proven with hearts expressing the optogenetic voltage actuator ReaChR. Adaptation of the POEMS system to larger hearts and incorporation of additional sensors can be achieved by redesigning the system-core accordingly.

T cell-specific inactivation of mouse CD2 by CRISPR/Cas9.

The CRISPR/Cas9 system can be used to mutate target sequences by introduction of double-strand breaks followed by imprecise repair. To test its use for conditional gene editing we generated mice transgenic for CD4 promoter-driven Cas9 combined with guide RNA targeting CD2. We found that within CD4(+) and CD8(+) lymphocytes from lymph nodes and spleen 1% and 0.6% were not expressing CD2, respectively. T cells lacking CD2 carryied mutations, which confirmed that Cas9 driven by cell-type specific promoters can edit genes in the mouse and may thus allow targeted studies of gene function in vivo.