T Cells Directed against the Metastatic Driver Chondromodulin-1 in Ewing Sarcoma: Comparative Engineering with CRISPR/Cas9 vs. Retroviral Gene Transfer for Adoptive Transfer.

Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.

RBD-Based ELISA and Luminex Predict Anti-SARS-CoV-2 Surrogate-Neutralizing Activity in Two Longitudinal Cohorts of German and Spanish Health Care Workers.

The ability of antibodies to neutralize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important correlate of protection. For routine evaluation of protection, however, a simple and cost-efficient anti-SARS-CoV-2 serological assay predictive of serum neutralizing activity is needed. We analyzed clinical epidemiological data and blood samples from two cohorts of health care workers in Barcelona and Munich to compare several immunological readouts for evaluating antibody levels that could be surrogates of neutralizing activity. We measured IgG levels against SARS-CoV-2 spike protein (S), its S2 subunit, the S1 receptor binding domain (RBD), and the full length and C terminus of nucleocapsid (N) protein by Luminex, and against RBD by enzyme-linked immunosorbent assay (ELISA), and assessed those as predictors of plasma surrogate-neutralizing activity measured by a flow cytometry assay. In addition, we determined the clinical and demographic factors affecting plasma surrogate-neutralizing capacity. Both cohorts showed a high positive correlation between IgG levels to S antigen, especially to RBD, and the levels of plasma surrogate-neutralizing activity, suggesting RBD IgG as a good correlate of plasma neutralizing activity. Symptomatic infection, with symptoms such as loss of taste, dyspnea, rigors, fever and fatigue, was positively associated with anti-RBD IgG positivity by ELISA and Luminex, and with plasma surrogate-neutralizing activity. Our serological assays allow for the prediction of serum neutralization activity without the cost, hazards, time, and expertise needed for surrogate or conventional neutralization assays. Once a cutoff is established, these relatively simple high-throughput antibody assays will provide a fast and cost-effective method of assessing levels of protection from SARS-CoV-2 infection. IMPORTANCE Neutralizing antibody titers are the best correlate of protection against SARS-CoV-2. However, current tests to measure plasma or serum neutralizing activity do not allow high-throughput screening at the population level. Serological tests could be an alternative if they are proved to be good predictors of plasma neutralizing activity. In this study, we analyzed the SARS-CoV-2 serological profiles of two cohorts of health care workers by applying Luminex and ELISA in-house serological assays. Correlations of both serological tests were assessed between them and with a flow cytometry assay to determine plasma surrogate-neutralizing activity. Both assays showed a high positive correlation between IgG levels to S antigens, especially RBD, and the levels of plasma surrogate-neutralizing activity. This result suggests IgG to RBD as a good correlate of plasma surrogate-neutralizing activity and indicates that serology of IgG to RBD could be used to assess levels of protection from SARS-CoV-2 infection.

Gene Signatures of T-Cell Activation Can Serve as Predictors of Functionality for SARS-CoV-2-Specific T-Cell Receptors.

The importance of T cells in controlling SARS-CoV-2 infections has been demonstrated widely, but insights into the quality of these responses are still limited due to technical challenges. Indeed, understanding the functionality of the T-cell receptor (TCR) repertoire of a polyclonal antigen-specific population still requires the tedious work of T-cell cloning or TCR re-expression and subsequent characterization. In this work, we show that it is possible to discriminate highly functional and bystander TCRs based on gene signatures of T-cell activation induced by recent peptide stimulation. SARS-CoV-2-specific TCRs previously identified by cytokine release after peptide restimulation and subsequent single-cell RNA sequencing were re-expressed via CRISPR-Cas9-mediated gene editing into a Jurkat-based reporter cell line system suitable for high-throughput screening. We could observe differences in SARS-CoV-2 epitope recognition as well as a wide range of functional avidities. By correlating these in vitro TCR engineered functional data with the transcriptomic profiles of the corresponding TCR-expressing parental T cells, we could validate that gene signatures of recent T-cell activation accurately identify and predict truly SARS-CoV-2-specific TCRs. In summary, this work paves the way for alternative approaches useful for the functional analysis of global antigen-specific TCR repertoires with largely improved throughput.

Two-Year Follow-Up on Chemosensory Dysfunction and Adaptive Immune Response after Infection with SARS-CoV-2 in a Cohort of 44 Healthcare Workers.

Persistent chemosensory dysfunction (PCD) is a common symptom of long-COVID. Chemosensory dysfunction (CD) as well as SARS-CoV-2-specific antibody levels and CD8+ T-cell immunity were investigated in a cohort of 44 healthcare workers up to a median of 721 days after a positive PCR test. CD was assessed using questionnaires and psychophysical screening tests. After 721 days, 11 of 44 (25%) participants reported PCD, with five describing an impaired quality of life. One participant reported hyperosmia (increased sense of smell). The risk of PCD at 721 days was higher for participants reporting qualitative changes (parosmia (altered smell), dysgeusia (altered taste), or phantosmia (hallucination of smell)) during initial infection than in those with isolated quantitative losses during the first COVID-19 infection (62.5% vs. 7.1%). The main recovery rate occurred within the first 100 days and did not continue until follow-up at 2 years. No correlation was found between antibody levels and CD, but we observed a trend of a higher percentage of T-cell responders in participants with CD. In conclusion, a significant proportion of patients suffer from PCD and impaired quality of life 2 years after initial infection. Qualitative changes in smell or taste during COVID-19 pose a higher risk for PCD.

SARS-CoV-2 CD8+ T cell killing assays using replicating viruses and transgenic antigens.

The quality of an antigen-specific CD8+ T cell repertoire is crucial for the clearance of intracellular pathogens, in particular for viral infections. Here, we describe killing assays to determine the function of CD8+ T cells engineered with SARS-CoV-2-specific T cell receptors in a near-physiological system for antigen presentation. We detail the use of target cells either infected with replicating SARS-CoV-2 virus or engineered with SARS-CoV-2 open reading frames. For complete details on the use and execution of this protocol, please refer to Moosmann et al. (2022) and Wagner et al. (2022).

CMV seropositivity is a potential novel risk factor for severe COVID-19 in non-geriatric patients.

BACKGROUND: COVID-19 has so far affected more than 250 million individuals worldwide, causing more than 5 million deaths. Several risk factors for severe disease have been identified, most of which coincide with advanced age. In younger individuals, severe COVID-19 often occurs in the absence of obvious comorbidities. Guided by the finding of cytomegalovirus (CMV)-specific T cells with some cross-reactivity to SARS-CoV-2 in a COVID-19 intensive care unit (ICU) patient, we decided to investigate whether CMV seropositivity is associated with severe or critical COVID-19. Herpes simplex virus (HSV) serostatus was investigated as control. METHODS: National German COVID-19 bio-sample and data banks were used to retrospectively analyze the CMV and HSV serostatus of patients who experienced mild (n = 101), moderate (n = 130) or severe to critical (n = 80) disease by IgG serology. We then investigated the relationship between disease severity and herpesvirus serostatus via statistical models. RESULTS: Non-geriatric patients (< 60 years) with severe COVID-19 were found to have a very high prevalence of CMV-seropositivity, while CMV status distribution in individuals with mild disease was similar to the prevalence in the German population; interestingly, this was not detectable in older patients. Prediction models support the hypothesis that the CMV serostatus, unlike HSV, might be a strong biomarker in identifying younger individuals with a higher risk of developing severe COVID-19, in particular in absence of other co-morbidities. CONCLUSIONS: We identified 'CMV-seropositivity' as a potential novel risk factor for severe COVID-19 in non-geriatric individuals in the studied cohorts. More mechanistic analyses as well as confirmation of similar findings in cohorts representing the currently most relevant SARS-CoV-2 variants should be performed shortly.

Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection.

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.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition).

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Dexamethasone therapy and rates of secondary pulmonary and bloodstream infections in critically ill COVID-19 patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a pandemic. Bacterial superinfections seem to be associated with higher mortality in COVID-19 patients in intensive care units (ICUs). However, details on the prevalence and species distribution of secondary infections are limited. Moreover, the increasing use of dexamethasone may pose an additional risk of superinfections. METHODS: We performed a single-center retrospective study of the clinical and microbiological characteristics of 154 COVID-19 patients admitted to the ICU between March 2020 and January 2021, focusing on bacterial infections, use of antimicrobial agents and dexamethasone therapy. RESULTS: The median age was 68 years; 67.5% of the patients were men. Critically ill COVID-19 patients were treated with dexamethasone since July 2020 (second wave), which was not common during the first wave of the pandemic. In the dexamethasone group (n=90, 58.4%), respiratory pathogens were detected more frequently, as were multidrugresistant pathogens. The number of patients with polymicrobial detection of respiratory pathogens was significantly increased (p=0.013). The most frequently detected species were Enterobacterales, Staphylococcus aureus, and Aspergillus fumigatus. The rates of bloodstream infections did not differ between the groups. The use of dexamethasone in ICU COVID-19 patients was associated with higher rates of respiratory infectious complications. CONCLUSIONS: Secondary infections are present in a substantial fraction of critically ill COVID-19 patients. Respiratory pathogens were detectable in the majority of COVID-19 ICU patients. The use of dexamethasone poses a potential risk of secondary pulmonary infections. Infectious complications in patients with dexamethasone therapy could be associated with worse outcomes.

Phantosmia, Parosmia, and Dysgeusia Are Prolonged and Late-Onset Symptoms of COVID-19.

Deficiencies in smell and taste are common symptoms of COVID-19. Quantitative losses are well surveyed. This study focuses on qualitative changes such as phantosmia (hallucination of smell), parosmia (alteration of smell), and dysgeusia (alteration of taste) and possible connections with the adaptive immune system. Subjective experience of deficiency in taste and smell was assessed by two different questionnaires after a median of 100 and 244 days after first positive RT-PCR test. SARS-CoV-2-specific antibody levels were measured with the iFlash-SARS-CoV-2 assay. After 100 days a psychophysical screening test for olfactory and gustatory dysfunction was administered. 30 of 44 (68.2%) participants reported a chemosensory dysfunction (14 quantitative, 6 qualitative, 10 quantitative, and qualitative) during COVID-19, eleven (25.0%) participants (1 quantitative, 7 qualitative, 3 quantitative, and quantity) after 100 days, and 14 (31.8%) participants (1 quantitative, 10 qualitative, 3 quantitative and qualitative) after 244 days. Four (9.1%) participants, who were symptom-free after 100 days reported now recently arisen qualitative changes. Serological and T-cell analysis showed no correlation with impairment of taste and smell. In conclusion, qualitative changes can persist for several months and occur as late-onset symptoms months after full recovery from COVID-19-induced quantitative losses in taste and smell.

Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through 'reverse phenotyping'.

The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

COVID-19 in Patients Receiving CD20-depleting Immunochemotherapy for B-cell Lymphoma.

The clinical and immunological impact of B-cell depletion in the context of coronavirus disease 2019 (COVID-19) is unclear. We conducted a prospectively planned analysis of COVID-19 in patients who received B-cell depleting anti-CD20 antibodies and chemotherapy for B-cell lymphomas. The control cohort consisted of age- and sex-matched patients without lymphoma who were hospitalized because of COVID-19. We performed detailed clinical analyses, in-depth cellular and molecular immune profiling, and comprehensive virological studies in 12 patients with available biospecimens. B-cell depleted lymphoma patients had more severe and protracted clinical course (median hospitalization 88 versus 17 d). All patients actively receiving immunochemotherapy (n = 5) required ICU support including long-term mechanical ventilation. Neutrophil recovery following granulocyte colony stimulating factor stimulation coincided with hyperinflammation and clinical deterioration in 4 of the 5 patients. Immune cell profiling and gene expression analysis of peripheral blood mononuclear cells revealed early activation of monocytes/macrophages, neutrophils, and the complement system in B-cell depleted lymphoma patients, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration of COVID-19. Longitudinal immune cell profiling and functional in vitro assays showed SARS-CoV-2-specific CD8+ and CD4+ T-effector cell responses. Finally, we observed long-term detection of SARS-CoV-2 in respiratory specimens (median 84 versus 12 d) and an inability to mount lasting SARS-CoV-2 antibody responses in B-cell depleted lymphoma patients. In summary, we identified clinically relevant particularities of COVID-19 in lymphoma patients receiving B-cell depleting immunochemotherapies.

Mucosal-Associated Invariant T (MAIT) Cells Are Highly Activated and Functionally Impaired in COVID-19 Patients.

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.

Rates of bacterial co-infections and antimicrobial use in COVID-19 patients: a retrospective cohort study in light of antibiotic stewardship.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. Bacterial co-infections are associated with unfavourable outcomes in respiratory viral infections; however, microbiological and antibiotic data related to COVID-19 are sparse. Adequate use of antibiotics in line with antibiotic stewardship (ABS) principles is warranted during the pandemic. We performed a retrospective study of clinical and microbiological characteristics of 140 COVID-19 patients admitted between February and April 2020 to a German University hospital, with a focus on bacterial co-infections and antimicrobial therapy. The final date of follow-up was 6 May 2020. Clinical data of 140 COVID-19 patients were recorded: The median age was 63.5 (range 17-99) years; 64% were males. According to the implemented local ABS guidelines, the most commonly used antibiotic regimen was ampicillin/sulbactam (41.5%) with a median duration of 6 (range 1-13) days. Urinary antigen tests for Legionella pneumophila and Streptococcus peumoniae were negative in all cases. In critically ill patients admitted to intensive care units (n = 50), co-infections with Enterobacterales (34.0%) and Aspergillus fumigatus (18.0%) were detected. Blood cultures collected at admission showed a diagnostic yield of 4.2%. Bacterial and fungal co-infections are rare in COVID-19 patients and are mainly prevalent in critically ill patients. Further studies are needed to assess the impact of antimicrobial therapy on therapeutic outcome in COVID-19 patients to prevent antimicrobial overuse. ABS guidelines could help in optimising the management of COVID-19. Investigation of microbial patterns of infectious complications in critically ill COVID-19 patients is also required.