MHC Class II Presentation in Autoimmunity.

Antigen presentation by major histocompatibility complex class II (MHC-II) molecules is crucial for eliciting an efficient immune response by CD4+ T cells and maintaining self-antigen tolerance. Some MHC-II alleles are known to be positively or negatively associated with the risk of the development of different autoimmune diseases (ADs), including those characterized by the emergence of autoreactive T cells. Apparently, the MHC-II presentation of self-antigens contributes to the autoimmune T cell response, initiated through a breakdown of central tolerance to self-antigens in the thymus. The appearance of autoreactive T cell might be the result of (i) the unusual interaction between T cell receptors (TCRs) and self-antigens presented on MHC-II; (ii) the posttranslational modifications (PTMs) of self-antigens; (iii) direct loading of the self-antigen to classical MHC-II without additional nonclassical MHC assistance; (iv) the proinflammatory environment effect on MHC-II expression and antigen presentation; and (v) molecular mimicry between foreign and self-antigens. The peculiarities of the processes involved in the MHC-II-mediated presentation may have crucial importance in the elucidation of the mechanisms of triggering and developing ADs as well as for clarification on the protective effect of MHC-II alleles that are negatively associated with ADs.

Genetically engineered CD80-pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement.

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide-MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols.

Alterations in SARS-CoV-2 Omicron and Delta peptides presentation by HLA molecules.

The T-cell immune response is a major determinant of effective SARS-CoV-2 clearance. Here, using the recently developed T-CoV bioinformatics pipeline (https://t-cov.hse.ru) we analyzed the peculiarities of the viral peptide presentation for the Omicron, Delta and Wuhan variants of SARS-CoV-2. First, we showed the absence of significant differences in the presentation of SARS-CoV-2-derived peptides by the most frequent HLA class I/II alleles and the corresponding HLA haplotypes. Then, the analysis was limited to the set of peptides originating from the Spike proteins of the considered SARS-CoV-2 variants. The major finding was the destructive effect of the Omicron mutations on PINLVRDLPQGFSAL peptide, which was the only tight binder from the Spike protein for HLA-DRB1*03:01 allele and some associated haplotypes. Specifically, we predicted a dramatical decline in binding affinity of HLA-DRB1*03:01 and this peptide both because of the Omicron BA.1 mutations (N211 deletion, L212I substitution and EPE 212-214 insertion) and the Omicron BA.2 mutations (V213G substitution). The computational prediction was experimentally validated by ELISA with the use of corresponding thioredoxin-fused peptides and recombinant HLA-DR molecules. Another finding was the significant reduction in the number of tightly binding Spike peptides for HLA-B*07:02 HLA class I allele (both for Omicron and Delta variants). Overall, the majority of HLA alleles and haplotypes was not significantly affected by the mutations, suggesting the maintenance of effective T-cell immunity against the Omicron and Delta variants. Finally, we introduced the Omicron variant to T-CoV portal and added the functionality of haplotype-level analysis to it.

Pre-Steady-State Kinetics of the SARS-CoV-2 Main Protease as a Powerful Tool for Antiviral Drug Discovery.

The design of effective target-specific drugs for COVID-19 treatment has become an intriguing challenge for modern science. The SARS-CoV-2 main protease, Mpro, responsible for the processing of SARS-CoV-2 polyproteins and production of individual components of viral replication machinery, is an attractive candidate target for drug discovery. Specific Mpro inhibitors have turned out to be promising anticoronaviral agents. Thus, an effective platform for quantitative screening of Mpro-targeting molecules is urgently needed. Here, we propose a pre-steady-state kinetic analysis of the interaction of Mpro with inhibitors as a basis for such a platform. We examined the kinetic mechanism of peptide substrate binding and cleavage by wild-type Mpro and by its catalytically inactive mutant C145A. The enzyme induces conformational changes of the peptide during the reaction. The inhibition of Mpro by boceprevir, telaprevir, GC-376, PF-00835231, or thimerosal was investigated. Detailed pre-steady-state kinetics of the interaction of the wild-type enzyme with the most potent inhibitor, PF-00835231, revealed a two-step binding mechanism, followed by covalent complex formation. The C145A Mpro mutant interacts with PF-00835231 approximately 100-fold less effectively. Nevertheless, the binding constant of PF-00835231 toward C145A Mpro is still good enough to inhibit the enzyme. Therefore, our results suggest that even noncovalent inhibitor binding due to a fine conformational fit into the active site is sufficient for efficient inhibition. A structure-based virtual screening and a subsequent detailed assessment of inhibition efficacy allowed us to select two compounds as promising noncovalent inhibitor leads of SARS-CoV-2 Mpro.