Deletion of the non-adjacent genes UL148 and UL148D impairs human cytomegalovirus-mediated TNF receptor 2 surface upregulation.

Human cytomegalovirus (HCMV) is a prototypical ß-herpesvirus which frequently causes morbidity and mortality in individuals with immature, suppressed, or senescent immunity. HCMV is sensed by various pattern recognition receptors, leading to the secretion of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα). TNFα binds to two distinct trimeric receptors: TNF receptor (TNFR) 1 and TNFR2, which differ in regard to their expression profiles, affinities for soluble and membrane-bound TNFα, and down-stream signaling pathways. While both TNF receptors engage NFκB signaling, only the nearly ubiquitously expressed TNFR1 exhibits a death domain that mediates TRADD/FADD-dependent caspase activation. Under steady-state conditions, TNFR2 expression is mainly restricted to immune cells where it predominantly submits pro-survival, proliferation-stimulating, and immune-regulatory signals. Based on the observation that HCMV-infected cells show enhanced binding of TNFα, we explored the interplay between HCMV and TNFR2. As expected, uninfected fibroblasts did not show detectable levels of TNFR2 on the surface. Intriguingly, however, HCMV infection increased TNFR2 surface levels of fibroblasts. Using HCMV variants and BACmid-derived clones either harboring or lacking the ULb' region, an association between TNFR2 upregulation and the presence of the ULb' genome region became evident. Applying a comprehensive set of ULb' gene block and single gene deletion mutants, we observed that HCMV mutants in which the non-adjacent genes UL148 or UL148D had been deleted show an impaired ability to upregulate TNFR2, coinciding with an inverse regulation of TACE/ADAM17.

Cytomegalovirus kinetics after hematopoietic cell transplantation reveal peak titers with differential impact on mortality, relapse and immune reconstitution.

Even in the era of PCR-based monitoring, prophylaxis, and preemptive therapy, Cytomegalovirus (CMV) viremia remains a relevant cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). However, studies using binary analysis (presence/absence of CMV) reported contradicting data for NRM, overall survival and leukemia relapse. Here, we analyzed CMV replication kinetics in 11 508 whole blood PCR samples of 705 patients with HCT between 2012 and 2017. Using two independent models based on CMV peak titers and on the time point of first CMV reactivation, we stratified patients into risk cohorts. Each cohort had distinct cellular immune reconstitution profiles and differentiated for relevant clinical outcomes. Patients with high CMV peak titers had significantly reduced overall survival (HR 2.13, 95% CI 1.53-2.96; p < .0001), due to high NRM. Early impaired T cell reconstitution was a risk factor for high CMV peak titers, however relevant CMV viremia also related to boosted T cell reconstitution. Importantly, intermediate CMV peak titers associated with a significantly reduced relapse probability (HR 0.53, 95% CI 0.31-0.91; p = .022). In short, CMV kinetics models distinguished relevant clinical outcome cohorts beyond the R+ serostatus with distinct immune reconstitution patterns and resolve in part contradicting results of previous studies exclusively focused on the presence or absence of CMV.

Multiple Knockout of Classical HLA Class II ß-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA.

Comprehensive knockout of HLA class II (HLA-II) ß-chain genes is complicated by their high polymorphism. In this study, we developed CRISPR/Cas9 genome editing to simultaneously target HLA-DRB, -DQB1, and -DPB1 through a single guide RNA recognizing a conserved region in exon 2. Abrogation of HLA-II surface expression was achieved in five different HLA-typed, human EBV-transformed B lymphoblastoid cell lines (BLCLs). Next-generation sequencing-based detection confirmed specific genomic insertion/deletion mutations with 99.5% penetrance in sorted cells for all three loci. No alterations were observed in HLA-I genes, the HLA-II peptide editor HLA-DMB, or its antagonist HLA-DOB, showing high on-target specificity. Transfection of full-length HLA-DPB1 mRNA into knockout BLCLs fully restored HLA-DP surface expression and recognition by alloreactive human CD4 T cells. The possibility to generate single HLA-II-expressing BLCLs by one-shot genome editing opens unprecedented opportunities for mechanistically dissecting the interaction of individual HLA variants with the immune system.

STAT2-Dependent Immune Responses Ensure Host Survival despite the Presence of a Potent Viral Antagonist.

A pathogen encounter induces interferons, which signal via Janus kinases and STAT transcription factors to establish an antiviral state. However, the host and pathogens are situated in a continuous arms race which shapes host evolution toward optimized immune responses and the pathogens toward enhanced immune-evasive properties. Mouse cytomegalovirus (MCMV) counteracts interferon responses by pM27-mediated degradation of STAT2, which directly affects the signaling of type I as well as type III interferons. Using MCMV mutants lacking M27 and mice lacking STAT2, we studied the opposing relationship between antiviral activities and viral antagonism in a natural host-pathogen pair in vitro and in vivo In contrast to wild-type (wt) MCMV, ΔM27 mutant MCMV was efficiently cleared from all organs within a few days in BALB/c, C57BL/6, and 129 mice, highlighting the general importance of STAT2 antagonism for MCMV replication. Despite this effective and relevant STAT2 antagonism, wt and STAT2-deficient mice exhibited fundamentally different susceptibilities to MCMV infections. MCMV replication was increased in all assessed organs (e.g., liver, spleen, lungs, and salivary glands) of STAT2-deficient mice, resulting in mortality during the first week after infection. Taken together, the results of our study reveal the importance of cytomegaloviral interferon antagonism for viral replication as well as a pivotal role of the remaining STAT2 activity for host survival. This mutual influence establishes a stable evolutionary standoff situation with fatal consequences when the equilibrium is disturbed.IMPORTANCE The host limits viral replication by the use of interferons (IFNs), which signal via STAT proteins. Several viruses evolved antagonists targeting STATs to antagonize IFNs (e.g., cytomegaloviruses, Zika virus, dengue virus, and several paramyxoviruses). We analyzed infections caused by MCMV expressing or lacking the STAT2 antagonist pM27 in STAT2-deficient and control mice to evaluate its importance for the host and the virus in vitro and in vivo The inability to counteract STAT2 directly translates into exaggerated IFN susceptibility in vitro and pronounced attenuation in vivo Thus, the antiviral activity mediated by IFNs via STAT2-dependent signaling drove the development of a potent MCMV-encoded STAT2 antagonist which became indispensable for efficient virus replication and spread to organs required for dissemination. Despite this clear impact of viral STAT2 antagonism, the host critically required the remaining STAT2 activity to prevent overt disease and mortality upon MCMV infection. Our findings highlight a remarkably delicate balance between host and virus.