SARS-CoV-2-reactive T-cell receptors isolated from convalescent COVID-19 patients confer potent T-cell effector function

Both B cells and T cells are involved in an effective immune response to SARS-CoV-2, the disease-causing virus of COVID-19. While B cells—with the indispensable help of CD4⁺ T cells—are essential to generate neutralizing antibodies, T cells on their own have been recognized as another major player in effective anti-SARS-CoV-2 immunity. In this report, we provide insights into the characteristics of individual HLA-A*02:01- and HLA-A*24:02-restricted SARS-CoV-2-reactive TCRs, isolated from convalescent COVID-19 patients. We observed that SARS-CoV-2-reactive T-cell populations were clearly detectable in convalescent samples and that TCRs isolated from these T cell clones were highly functional upon ectopic re-expression. The SARS-CoV-2-reactive TCRs described in this report mediated potent TCR signaling in reporter assays with low nanomolar EC50 values. We further demonstrate that these SARS-CoV-2-reactive TCRs conferred powerful T-cell effector function to primary CD8⁺ T cells as evident by a robust anti-SARS-CoV-2 IFN-γ response and in vitro cytotoxicity. We also provide an example of a long-lasting anti-SARS-CoV-2 memory response by reisolation of one of the retrieved TCRs 5 months after initial sampling. Taken together, these findings contribute to a better understanding of anti-SARS-CoV-2 T-cell immunity and may contribute to paving the way toward immunotherapeutics approaches targeting SARS-CoV-2.     

From the Cover: Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs.Far-field nanoscopy (1, 2) methods discern features within subdiffraction distances by briefly forcing their molecules to two distinguishable states for the time period of detection. Typically, fluorophores are switched between a signaling “on” and a nonsignaling (i.e., dark) “off” state. Depending on the switching and fluorescence registration strategy used, these superresolution techniques can be categorized into coordinate-stochastic and coordinate-targeted approaches (2). The latter group of methods, comprising the so-called RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] (1, 3–7) approaches, have been realized using patterns of switch-off light with one or more zero-intensity points or lines, to single out target point (zero-dimensional) or line (1D) coordinates in space where the fluorophores are allowed to assume the on state. The RESOLFT idea can also be implemented in the inverse mode, by using switch-on light and confining the off state. In any case, probing the presence of molecules in new sets of points or lines at every scanning step produces images.Owing to the nature of the on and off states involved––first excited electronic and ground state––stimulated emission depletion (STED) (3) and saturated structured illumination microscopy (SSIM) (8), which both qualify as variants of the RESOLFT principle, typically apply light intensities in the range of MW/cm² and above. Especially when imaging sensitive samples where photoinduced changes must be avoided, RESOLFT is preferably realized with fluorophores which lead to the same factor of resolution improvement at much lower intensities of state-switching light. Reversibly switchable fluorescent proteins (RSFPs) are highly suitable for this purpose (4–7, 9), as transitions between their metastable on and off states require 5 orders of magnitude lower threshold intensities than STED/SSIM to guarantee switch-off. Suitable spectral properties, relatively fast millisecond switching kinetics, and high photostability of recently developed yellow-green-emitting RSFPs like rsEGFP (5), rsEGFP2 (7), and rsEGFP(N205S) (10) compared with early RSFPs have indeed enabled RESOLFT nanoscopy in living cells and tissues. To date, RSFP-based RESOLFT has achieved resolution improvements by factors of 4–5 in rsEGFP2-labeled samples (7). To further reduce the imaging time, massive parallelization of scanning has been reported (10). However, the diffraction-limited axial resolution and lack of background suppression restrict applications to thin samples.Imaging applications typically require careful tuning of imaging parameters including speed, contrast, photosensitivity, and spatial resolution, depending on the information that is sought. Light-sheet fluorescence microscopy (LSFM) (11–15) stands out by its ability to balance most of these parameters for 3D imaging of living specimens. Recently reenacted as the selective plane illumination microscope (13), this microscopy mode has sparked increasing interest notably because of its short acquisition times in 3D imaging and low phototoxicity in living specimens. It excites fluorophores only in a thin diffraction-limited slice of the sample, perpendicular to the direction of fluorescence detection. The LS is generated by a cylindrical lens which focuses an expanded laser beam in only one direction onto the specimen or into the back-focal plane of an illumination objective. Alternatively, a single beam is quickly moved as a “virtual” LS (16) across a specimen section.In such conventional LSFM imaging, the lateral resolution is determined by the numerical aperture (N.A.) of the detection objective (17), whereas axial resolution is given by the LS thickness, provided the latter is thinner than the axial extent of the point-spread function describing the imaging process from the focal plane of the detecting lens to the camera. In a previous study, the axial resolution of LSFM was pushed to the diffraction limit by using the full aperture of the illumination objective with Gaussian beams; this was carried out for practically useful combinations of N.A. (e.g., 0.8 for both illumination and detection objectives) permissible in light of the geometrical constraints given by the objective lens dimensions (18). High-N.A. illumination comes with short Rayleigh ranges of Gaussian beams, which inherently limit the field of view (FOV) along the direction of illumination. Scanned Bessel beams for diffraction-limited excitation with a virtual LS (19–21) typically offer larger FOVs (22), but side lobes broaden the scanned LS in the axial direction and contribute to phototoxicity outside of the focal plane of detection (20). A more complex approach has used Bessel-beam excitation in combination with structured illumination to obtain near-isotropic (but still diffraction-limited) resolution as measured on fluorescent beads (20), albeit at the cost of acquisition time and reduced contrast due to fluorescence generated by the side lobes. In different work, axial resolution has also been improved about fourfold by acquiring two complementary orthogonal views of the sample using two alternating LSs, followed by computationally fusing image information with a deconvolution incorporating both views (23). LS approaches have also helped suppress out-of-focus background for single-molecule imaging in biological situations (e.g., in ref. 24), including at superresolution (25–27).Slight axial resolution improvement beyond the diffraction barrier has been demonstrated by overlapping a Gaussian excitation LS with a STED LS featuring a zero-intensity plane (28). Due to scattering and possibly additional aberrations caused by the wavelength difference between excitation and STED light, the maximal achievable resolution in biological specimens was severely limited. This was the case even in fixed samples. A successful application of LS-STED to living cells or organisms has not been reported. The relatively high average STED laser power required for high resolution gains calls for developing a coordinate-targeted superresolution LS approach with low-power operation, meaning a concept that does not solely rely on changing the way the light is directed to––or collected from––the sample, but a concept that harnesses an “on–off” transition for improved feature separation.     

Structural Variations of the Cell Wall Precursor Lipid II and Their Influence on Binding and Activity of the Lipoglycopeptide Antibiotic Oritavancin

Oritavancin is a semisynthetic derivative of the glycopeptide antibiotic chloroeremomycin with activity against Gram-positive pathogens, including vancomycin-resistant staphylococci and enterococci. Compared to vancomycin, oritavancin is characterized by the presence of two additional residues, a hydrophobic 4′-chlorobiphenyl methyl moiety and a 4-epi-vancosamine substituent, which is also present in chloroeremomycin. Here, we show that oritavancin and its des-N-methylleucyl variant (des-oritavancin) effectively inhibit lipid I- and lipid II-consuming peptidoglycan biosynthesis reactions in vitro. In contrast to that for vancomycin, the binding affinity of oritavancin to the cell wall precursor lipid II appears to involve, in addition to the d-Ala-d-Ala terminus, other species-specific binding sites of the lipid II molecule, i.e., the crossbridge and d-isoglutamine in position 2 of the lipid II stem peptide, both characteristic for a number of Gram-positive pathogens, including staphylococci and enterococci. Using purified lipid II and modified lipid II variants, we studied the impact of these modifications on the binding of oritavancin and compared it to those of vancomycin, chloroeremomycin, and des-oritavancin. Analysis of the binding parameters revealed that additional intramolecular interactions of oritavancin with the peptidoglycan precursor appear to compensate for the loss of a crucial hydrogen bond in vancomycin-resistant strains, resulting in enhanced binding affinity. Augmenting previous findings, we show that amidation of the lipid II stem peptide predominantly accounts for the increased binding of oritavancin to the modified intermediates ending in d-Ala-d-Lac. Corroborating our conclusions, we further provide biochemical evidence for the phenomenon of the antagonistic effects of mecA and vanA resistance determinants in Staphylococcus aureus, thus partially explaining the low frequency of methicillin-resistant S. aureus (MRSA) acquiring high-level vancomycin resistance.     

Identification of HLA-DR-bound peptides presented by human bronchoalveolar lavage cells in sarcoidosis

Sarcoidosis is an inflammatory disease of unknown etiology, most commonly affecting the lungs. Activated CD4+ T cells accumulate in the lungs of individuals with sarcoidosis and are considered to be of central importance for inflammation. We have previously shown that Scandinavian sarcoidosis patients expressing the HLA-DR allele DRB1*0301 are characterized by large accumulations in the lungs of CD4+ T cells expressing the TCR AV2S3 gene segment. This association afforded us a unique opportunity to identify a sarcoidosis-specific antigen recognized by AV2S3+ T cells. To identify candidates for the postulated sarcoidosis-specific antigen, lung cells from 16 HLA-DRB1*0301pos patients were obtained by bronchoalveolar lavage. HLA-DR molecules were affinity purified and bound peptides acid eluted. Subsequently, peptides were separated by reversed-phase HPLC and analyzed by liquid chromatography-mass spectrometry. We identified 78 amino acid sequences from self proteins presented in the lungs of sarcoidosis patients, some of which were well-known autoantigens such as vimentin and ATP synthase. For the first time, to our knowledge, we have identified HLA-bound peptides presented in vivo during an inflammatory condition. This approach can be extended to characterize HLA-bound peptides in various autoimmune settings.     

Mersacidin eradicates methicillin-resistant Staphylococcus aureus (MRSA) in a mouse rhinitis model

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) often colonize the anterior nares, and nasal carriage remains the main source of bacterial dissemination. The aim of this study was to assess the in vivo activity of the lantibiotic mersacidin against MRSA colonizing nasal epithelia. METHODS: The efficiency of mersacidin in the eradication of MRSA was tested employing mice pre-treated with hydrocortisone and inoculated intranasally either three or six times with a bacterial suspension. RESULTS: In mersacidin-treated animals, pre-colonized with MRSA, bacteria could not be detected in blood, lungs, liver, kidney, spleen or nasal scrapings and there were no lesions manifested after intraperitoneal drug application. Blood samples from infected mice obtained 2 h after mersacidin therapy revealed anti-MRSA activity in a serum bactericidal test. Moreover, elevated interleukin-1beta and tumour necrosis factor-alpha titres were noticed in the pre-infected but not in cured animals. In contrast, mersacidin did not induce differences in the cytokine profiles of treated uninfected control mice. CONCLUSIONS: In the mouse rhinitis model, mersacidin was able to eradicate MRSA colonization. The site of action (epithelium versus blood) of mersacidin needs to be further explored.     

Managing chronic pain in elderly patients requires a CHANGE of approach

In many countries, the number of elderly people has increased rapidly in recent years and this is expected to continue; it has been predicted that almost a quarter of the population in the European Union will be over 65 years of age in 2035. Many elderly people suffer from chronic pain but it is regularly under-treated, partly because managing these patients is often complex. This paper outlines the extent of untreated pain in this population and the consequent reduction in quality of life, before articulating the reasons why it is poorly or inaccurately diagnosed. These include the patient’s unwillingness to complain, atypical pain presentations, multiple morbidities and cognitive decline. Successful pain management depends upon accurate diagnosis, which is based upon a complete history and thorough physical examination, as well as an assessment of psychosocial functioning. Poor physician/patient communication can be improved by using standardized instruments to establish individual treatment targets and measure progress towards them. User-friendly observational instruments may be valuable for patients with dementia. In line with the widely accepted biopsychosocial model of pain, a multidisciplinary approach to pain management is recommended, with pharmacotherapy, psychological support, physical rehabilitation and interventional procedures available if required. Declining organ function and other physiological changes require lower initial doses of analgesics and less frequent dosing intervals, and the physician must be aware of all medications that the patient is taking, in order to avoid drug/drug interactions. Non-adherence to treatment is common, and various strategies can be employed to improve it; involving the elderly patient’s caregivers and family, using medication systems such as pill-boxes, or even sending text messages. In the long term, the teaching of pain medicine needs to be improved – particularly in the use of opioids – both at undergraduate level and after qualification.     

German guidelines for the sequential medical treatment of rheumatoid arthritis with traditional and biologic disease-modifying antirheumatic drugs

The German Society of Rheumatology approved new German guidelines for the sequential medical treatment of rheumatoid arthritis (RA) based on the European League Against Rheumatism (EULAR) recommendations for the management of RA published in 2010. An update of the EULAR systematic literature research was performed in Medline, Embase, and Cochrane databases. Meta-analyses, controlled trials, cohort studies, and registry data addressing traditional and biologic disease-modifying antirheumatic drugs, glucocorticoids, and treatment strategies published between January 2009 and August 2011 were included. Two reviewers independently evaluated and compared the additional data that had been published after the time limit set by the EULAR recommendations. A national guideline working group developed an adapted set of recommendations. The new German guidelines were accepted by vote using an informal Delphi approach. Twelve recommendations and the resulting updated treatment algorithm were developed and approved as a practical orientation for rheumatologists. These recommendations are based on a successive treatment with traditional and biologic disease-modifying drugs depending on the individual progress of the disease and distinct patient characteristics. The German guidelines have been developed on the basis of the internationally well-recognized EULAR recommendations. In addition, more recent evidence from a systematic literature research was considered. They have been developed and approved by a group of national experts aiming at guidance for rheumatologists to reach best medical practice.     

Gait abnormalities differentially indicate pain or structural joint damage in monoarticular antigen-induced arthritis

Gait abnormalities have been suggested to provide an objective measure for joint pain in animal models. Here, we aimed to assess whether parameters of gait analysis correlate with measures of pain-related behavior in experimental monoarthritis. For this purpose, antigen-induced arthritis was induced in the left knee joints of 68 female Lewis rats, of which 30 were treated with tumor necrosis factor-alpha(TNF)-neutralizing compounds. During the course of arthritis, paw print analysis parameters and measures for mechanical and thermal hyperalgesia were obtained. Knee joints harvested on either day 3 or day 21 were scored histologically for signs of inflammation and cartilage and bone destructions. Data were compared to those obtained from 33 immunized control rats and correlated for days 3 and 21. Arthritic rats showed distinct asymmetric gait abnormalities. In the acute stage of antigen-induced arthritis, but not in the chronic phase, there was a significant correlation between the gait parameter ‘left–right distance’ and measures of primary and secondary hyperalgesia. Both in the acute and chronic phases, however, the gait parameter ‘angle between paws’ indicating outward rotation of paws mainly correlated with joint destruction as assessed using histology. Etanercept treatment exhibited pronounced anti-nociceptive and pro-locomotional effects, but the described correlations remained. In conclusion, some parameters of gait analysis may represent a good measure for arthritis pain, mainly in acute inflammation, while others are increasingly influenced by mechanical joint deformation as indicated by cartilage and bone destructions. Thus, gait abnormalities may not unequivocally be suitable for objective pain assessment in all stages of experimental arthritis.