Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments (CCFs) in senescent cells. The activation of cGAS, in turn triggers the production of SASP factors via Stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence upon irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.

The importance of pre-existing immune responses to seasonal endemic coronaviruses (HCoVs) for the susceptibility to SARS-CoV-2 infection and the course of COVID-19 is subject of an ongoing scientific debate. Recent studies postulate that immune responses to previous HCoV infections can either have a slightly protective or no effect on SARS-CoV-2 pathogenesis and, consequently, be neglected for COVID-19 risk stratification. Challenging this notion, we provide evidence that pre-existing, anti-nucleocapsid antibodies against endemic α-coronaviruses and S2 domain-specific anti-spike antibodies against β-coronavirus HCoV-OC43 are elevated in patients with COVID-19 compared to pre-pandemic donors. This finding is particularly pronounced in males and in critically ill patients. Longitudinal evaluation reveals that antibody cross-reactivity or polyclonal stimulation by SARS-CoV-2 infection are unlikely to be confounders. Thus, specific pre-existing immunity to seasonal coronaviruses may increase susceptibility to SARS-CoV-2 and predispose individuals to an adverse COVID-19 outcome, guiding risk management and supporting the development of universal coronavirus vaccines.

Myeloid-derived suppressor cells (MDSC) are critical in regulating immune responses by suppressing antigen presenting cells (APC) and T cells. We previously observed that incubation of peripheral blood monocytes with interleukin (IL)-10 during their differentiation to monocyte-derived dendritic cells (moDCs) results in the generation of an APC population with a CD14 C HLA-DR low phenotype (IL-10-APC) with reduced stimulatory capacity similar to human MDSC. Co-incubation experiments now revealed that the addition of IL-10-APC to moDC caused a reduction of DC-induced T-cell proliferation, of the expression of maturation markers, and of secreted cytokines and chemokines such as TNF-a, IL-6, MIP-1a and Rantes. Addition of IL-10-APC increased the immunosuppressive molecule osteoactivin and its corresponding receptor syndecan-4 on moDC. Moreover, CD14 C HLA-DR low MDSC isolated from healthy donors expressed high levels of osteoactivin, which was even further upregulated by the auxiliary addition of IL-10. Using transcriptome analysis, we identified a set of molecules and pathways mediating these effects. In addition, we found that IL-10-APC as well as human isolated MDSC expressed higher levels of programmed death (PD)-1, PD-ligand-1 (PD-L1), glucocorticoid-induced-tumor-necrosis-factor-receptor-related-protein (GITR) and GITR-ligand. Inhibition of osteoactivin, syndecan-4, PD-1 or PD-L1 on MDSC by using blocking antibodies restored the stimulatory capacity of DC in co-incubation experiments. Activation of MDSC with Dectin-1 ligand curdlan reduced the expression of osteoactivin and PD-L1. Our results demonstrate that osteoactivin/syndecan-4 and PD-/PD-L1 are key molecules that are profoundly involved in the inhibitory effects of MDSC on DC function and might be promising tools for clinical application.

Purpose To determine risk factors for coronavirus disease 2019 (COVID-19) in healthcare workers (HCWs), characterize symptoms, and evaluate preventive measures against SARS-CoV-2 spread in hospitals. Methods In a cross-sectional study conducted between May 27 and August 12, 2020, after the first wave of the COVID-19 pandemic, we obtained serological, epidemiological, occupational as well as COVID-19-related data at a quaternary care, multicenter hospital in Munich, Germany. Results 7554 HCWs participated, 2.2% of whom tested positive for anti-SARS-CoV-2 antibodies. Multivariate analysis revealed increased COVID-19 risk for nurses (3.1% seropositivity, 95% CI 2.5–3.9%, p = 0.012), staff working on COVID-19 units (4.6% seropositivity, 95% CI 3.2–6.5%, p = 0.032), males (2.4% seropositivity, 95% CI 1.8–3.2%, p = 0.019), and HCWs reporting high-risk exposures to infected patients (5.5% seropositivity, 95% CI 4.0–7.5%, p = 0.0022) or outside of work (12.0% seropositivity, 95% CI 8.0–17.4%, p < 0.0001). Smoking was a protective factor (1.1% seropositivity, 95% CI 0.7–1.8% p = 0.00018) and the symptom taste disorder was strongly associated with COVID-19 (29.8% seropositivity, 95% CI 24.3–35.8%, p < 0.0001). An unbiased decision tree identified subgroups with different risk profiles. Working from home as a preventive measure did not protect against SARS-CoV-2 infection. A PCR-testing strategy focused on symptoms and high-risk exposures detected all larger COVID-19 outbreaks. Conclusion Awareness of the identified COVID-19 risk factors and successful surveillance strategies are key to protecting HCWs against SARS-CoV-2, especially in settings with limited vaccination capacities or reduced vaccine efficacy.

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CD4+ T cells are central mediators of adaptive and innate immune responses and constitute a major reservoir for human immunodeficiency virus (HIV) in vivo. Detailed investigations of resting human CD4+ T cells have been precluded by the absence of efficient approaches for genetic manipulation limiting our understanding of HIV replication and restricting efforts to find a cure. Here we report a method for rapid, efficient, activation-neutral gene editing of resting, polyclonal human CD4+ T cells using optimized cell cultivation and nucleofection conditions of Cas9–guide RNA ribonucleoprotein complexes. Up to six genes, including HIV dependency and restriction factors, were knocked out individually or simultaneously and functionally characterized. Moreover, we demonstrate the knock in of double-stranded DNA donor templates into different endogenous loci, enabling the study of the physiological interplay of cellular and viral components at single-cell resolution. Together, this technique allows improved molecular and functional characterizations of HIV biology and general immune functions in resting CD4+ T cells.

Forward genetic screening associates phenotypes with genotypes by randomly inducing mutations and then identifying those that result in phenotypic changes of interest. Here we present spatially resolved CRISPR screening (SPARCS), a platform for microscopy-based genetic screening for spatial cellular phenotypes. SPARCS uses automated high-speed laser microdissection to physically isolate phenotypic variants in situ from virtually unlimited library sizes. We demonstrate the potential of SPARCS in a genome-wide CRISPR-KO screen on autophagosome formation in 40 million cells. Coupled to deep learning image analysis, SPARCS recovered almost all known macroautophagy genes in a single experiment and discovered a role for the ER-resident protein EI24 in autophagosome biogenesis. Harnessing the full power of advanced imaging technologies, SPARCS enables genome-wide forward genetic screening for diverse spatial phenotypes in situ.

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