6-C-Linked trehalose glycolipids signal through Mincle and exhibit potent adjuvant activity.

Many studies have investigated the Mincle-mediated agonist activity of α,α'-trehalose-6,6́-glycolipids, however, none have considered how the position, or absence, of the ester moiety influences Mincle-mediated agonist activity. We prepared a variety of 6-C-linked α,α'-trehalose glycolipids containing inverted esters, ketone, carboxy or no carbonyl moieties. Modelling studies indicated that these derivatives bind to the CRD of Mincle in a manner similar to that of the prototypical Mincle agonist, trehalose dibehenate (TDB), with NFAT-GFP reporter cell assays confirming that all compounds, apart from derivatives with short alkyl chains, led to robust Mincle signalling. It was also observed that a carbonyl moiety was needed for good Mincle-mediated signalling. The ability of the compounds to induce mIL-1 ß and mIL-6 production by bone marrow-derived macrophages (BMDMs) further demonstrated the agonist activity of the compounds, with the presence of a carbonyl moiety and longer lipid chains augmenting cytokine production. Notably, a C20 inverted ester led to levels of mIL-1ß that were significantly greater than those induced by TDB. The same C20 inverted ester also led to a significant increase in hIL-1ß and hIL-6 by human monocytes, and exhibited no toxicity, as demonstrated using BMDMs in an in vitro Sytox Green assay. The ability of the inverted ester to enhance antigen-mediated immune responses was then determined. In these studies, the inverted ester was found to augment the OVA-specific Th1/Th7 immune response in vitro, and exhibit adjuvanticity that was better than that of TDB in vivo, as evidenced by a significant increase in IgG antibodies for the inverted ester but not TDB when using OVA as a model antigen.

Memory B cells and memory T cells induced by SARS-CoV-2 booster vaccination or infection show different dynamics and efficacy to the Omicron variant.

Although BNT162b2 vaccination was shown to prevent infection and reduce COVID-19 severity, and the persistence of immunological memory generated by the vaccination has not been well elucidated. We evaluated memory B and T cell responses to the SARS-CoV-2 spike protein before and after the third BNT162b2 booster. Although the antibody titer against the spike receptor-binding domain (RBD) decreased significantly 8 months after the second vaccination, the number of memory B cells continued to increase, while the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the antibody titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, while memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- cTfh1 was positively correlated with RBD-specific antibody-secreting B cells. Furthermore, T cell-dependent antibody production from reactivated memory B cells in vitro was correlated to the Tfh-like cytokine levels. For the response to variant RBDs, although 60%-80% of memory B cells could bind to the Omicron RBD, their binding affinity was low, while memory T cells show an equal response to the Omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate antibody production and T cell responses after Omicron strain infection, which prevents severe illness and death due to COVID-19.

InterClone: Store, Search and Cluster Adaptive Immune Receptor Repertoires

B and T cell receptor repertoire data has the potential to fundamentally change the way we diagnose and treat a wide range of diseases. However, there are few resources for storing or analyzing repertoire data. InterClone provides tools for storing, searching, and clustering repertoire datasets. Efficiency is achieved by encoding the complementarity-determining regions of sequences as mmseqs2 databases. Single chain search or cluster results can be merged into paired (alpha-beta or heavy-light) results for analysis of single-cell sequencing data. We illustrate the use of InterClone with two recently reported examples: 1) searching for SARS-CoV-2 infection-enhancing antibodies in bulk COVID-19 and healthy donor repertoires; 2) identification of SARS-CoV-2 specific TCRs by clustering paired and bulk sequences from COVID-19, BNT162b2 vaccinated and healthy unvaccinated donors. The core functions of InterClone have been implemented as a web server and integrated database (https://sysimm.org/interclone). All source code is available upon request.

Identification, crystallization and epitope determination of public TCR shared and expanded in COVID-19 patients

T cells play pivotal roles in protective immunity against SARS-CoV-2 infection. Follicular helper T (Tfh) cells mediate the production of antigen-specific antibodies; however, T cell receptor (TCR) clonotypes used by SARS-CoV-2-specific Tfh cells have not been well characterized. Here, we first identified and crystallized public TCR of Tfh clonotypes that are shared and expanded in unhospitalized COVID-19-recovered patients. These clonotypes preferentially recognized SARS-CoV-2 spike (S) protein epitopes which are conserved among emerging SARS-CoV-2 variants. These clonotypes did not react with S proteins derived from common cold human coronaviruses, but cross-reacted with symbiotic bacteria, which might confer the publicity. Among SARS-CoV-2 S epitopes, S864-882, presented by frequent HLA-DR alleles, could activate multiple public Tfh clonotypes in COVID-19-recovered patients. Furthermore, S864-882-loaded HLA tetramer preferentially bound to CD4+ T cells expressing CXCR5. In this study, we identified and crystallized public TCR for SARS-CoV-2 that may contribute to the prevention of COVID-19 aggravation.