T cell immunity is crucial for control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and has been studied widely on a quantitative level. However, the quality of responses, in particular of CD8⁺ T cells, has only been investigated marginally so far. Here, we isolate T cell receptor (TCR) repertoires specific for immunodominant SARS-CoV-2 epitopes restricted to common human Leukocyte antigen (HLA) class I molecules in convalescent individuals. SARS-CoV-2-specific CD8⁺ T cells are detected up to 12 months after infection. TCR repertoires are diverse, with heterogeneous functional avidity and cytotoxicity toward virus-infected cells, as demonstrated for TCR-engineered T cells. High TCR functionality correlates with gene signatures that, remarkably, could be retrieved for each epitope:HLA combination analyzed. Overall, our data demonstrate that polyclonal and highly functional CD8⁺ TCRs—classic features of protective immunity—are recruited upon mild SARS-CoV-2 infection, providing tools to assess the quality of and potentially restore functional CD8⁺ T cell immunity.

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprises mild courses of disease as well as progression to severe disease, characterised by lung and other organ failure. The immune system is considered to play a crucial role for the pathogenesis of COVID-19, although especially the contribution of innate-like T cells remains poorly understood. Here, we analysed the phenotype and function of mucosal-associated invariant T (MAIT) cells, innate-like T cells with potent antimicrobial effector function, in patients with mild and severe COVID-19 by multicolour flow cytometry. Our data indicate that MAIT cells are highly activated in patients with COVID-19, irrespective of the course of disease, and express high levels of proinflammatory cytokines such as IL-17A and TNFα ex vivo. Of note, expression of the activation marker HLA-DR positively correlated with SAPS II score, a measure of disease severity. Upon MAIT cell-specific in vitro stimulation, MAIT cells however failed to upregulate expression of the cytokines IL-17A and TNFα, as well as cytolytic proteins, that is, granzyme B and perforin. Thus, our data point towards an altered cytokine expression profile alongside an impaired antibacterial and antiviral function of MAIT cells in COVID-19 and thereby contribute to the understanding of COVID-19 immunopathogenesis.

Background and purpose: During acute coronavirus disease 2019 (COVID‐19) infection, neurological signs, symptoms and complications occur. We aimed to assess their clinical relevance by evaluating real‐world data from a multinational registry.Methods: We analyzed COVID‐19 patients from 127 centers, diagnosed between January 2020 and February 2021, and registered in the European multinational LEOSS (Lean European Open Survey on SARS‐Infected Patients) registry. The effects of prior neurological diseases and the effect of neurological symptoms on outcome were studied using multivariate logistic regression.Results: A total of 6537 COVID‐19 patients (97.7% PCR‐confirmed) were analyzed, of whom 92.1% were hospitalized and 14.7% died. Commonly, excessive tiredness (28.0%), headache (18.5%), nausea/emesis (16.6%), muscular weakness (17.0%), impaired sense of smell (9.0%) and taste (12.8%), and delirium (6.7%) were reported. In patients with a complicated or critical disease course (53%) the most frequent neurological complications were ischemic stroke (1.0%) and intracerebral bleeding (ICB; 2.2%). ICB peaked in the critical disease phase (5%) and was associated with the administration of anticoagulation and extracorporeal membrane oxygenation (ECMO). Excessive tiredness (odds ratio [OR] 1.42, 95% confidence interval [CI] 1.20–1.68) and prior neurodegenerative diseases (OR 1.32, 95% CI 1.07–1.63) were associated with an increased risk of an unfavorable outcome. Prior cerebrovascular and neuroimmunological diseases were not associated with an unfavorable short‐term outcome of COVID‐19.Conclusion: Our data on mostly hospitalized COVID‐19 patients show that excessive tiredness or prior neurodegenerative disease at first presentation increase the risk of an unfavorable short‐term outcome. ICB in critical COVID‐19 was associated with therapeutic interventions, such as anticoagulation and ECMO, and thus may be an indirect complication of a life‐threatening systemic viral infection.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent–targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry–based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D-driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. Significance: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Background: To assess the prevalence of Herpes simplex and Cytomegalovirus infection in respiratory samples of critically-ill COVID-19 patients, its role in outcome and mortality and the influence of dexamethasone treatment in the early stage of SARS-CoV-2 infection. Methods: All mechanically ventilated COVID-19 patients treated on ICU between March 2020 and January 2021 were included. Respiratory specimens were tested for Herpes simplex virus (HSV) type 1, 2 and Cytomegalovirus (CMV) by quantitative real-time PCR. Clinical parameters were compared in the cohort cohorts with and without HSV-1-infection.Results: 134 patients with a median age of 72.5 years (73.0% male, n=98) were included. HSV-1 reactivation occurred in 61 patients (45.5%), after median 9 (7-13) days of mechanical ventilation. The main factor for reactivation was length of stay on ICU (24 days vs 13 days, p<0.001) and duration of mechanical ventilation (417 vs 214 hours, p<0.001). Treatment with dexamethasone and a history of immunosuppression did not associate with HSV-infection in the univariate analysis (39 vs 41, p=0.462 and 27.9% vs 23.3%, p=0.561, respectively). Both ICU and hospital mortality were not significantly different in the cohorts with and without HSV-infection (57.4% vs 45.2%, p=0.219).Conclusions: Our study shows a high prevalence of HSV-infection in critically-ill COVID-19 patients which was unexpectedly higher than the prevalence of CMV-infections and unrelated to dexamethasone treatment. The main risk factors for HSV and CMV in the studied cohorts were the length of ICU stay and duration of mechanical ventilation. Therefore, we recommend routine monitoring of critically ill COVID-19 patients for these viral co-infections and consider treatment in those patients.

Purpose: To evaluate the diagnostic reliability and practicability of self-collected oropharyngeal swab samples for the detection of SARS-CoV-2 infection as self-sampling could enable broader testing availability and reduce both personal protective equipment and potential exposure. Methods: Hospitalized SARS-CoV-2-infected patients were asked to collect two oropharyngeal swabs (SC-OPS1/2), and an additional oropharyngeal swab was collected by a health care professional (HCP-OPS). SARS-CoV-2 PCR testing for samples from 58 participants was performed, with a 48-h delay in half of the self-collected samples (SC-OPS2). The sensitivity, probability of concordance, and interrater reliability were calculated. Univariate and multivariate analyses were performed to assess predictive factors. Practicability was evaluated through a questionnaire. Results: The test sensitivity for HCP-OPS, SC-OPS1, and SC-OPS2 was 88%, 78%, and 77%, respectively. Combining both SC-OPS results increased the estimated sensitivity to 88%. The concordance probability between HCP-OPS and SC-OPS1 was 77.6% and 82.5% between SC-OPS1 and SC-OPS2, respectively. Of the participants, 69% affirmed performing future self-sampling at home, and 34% preferred self-sampling over HCP-guided testing. Participants with both positive HCP-OPS1 and SC-OPS1 indicating no challenges during self-sampling had more differences in viral load levels between HCP-OPS1 and SC-OPS1 than those who indicated challenges. Increasing disease duration and the presence of anti-SARS-CoV-2-IgG correlated with negative test results in self-collected samples of previously confirmed SARS-CoV-2 positive individuals. Conclusion: Oropharyngeal self-sampling is an applicable testing approach for SARS-CoV-2 diagnostics. Self-sampling tends to be more effective in early versus late infection and symptom onset, and the collection of two distinct samples is recommended to maintain high test sensitivity.