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T cell maturation and activation depend upon T cell receptor (TCR) interactions with a wide variety of antigenic peptides displayed in a given major histocompatibility complex (MHC) context. Complementarity-determining region 3 (CDR3) is the most variable part of the TCRα and -β chains, which govern interactions with peptide–MHC complexes. However, it remains unclear how the CDR3 landscape is shaped by individual MHC context during thymic selection of naïve T cells. We established two mouse strains carrying distinct allelic variants of H2-A and analyzed thymic and peripheral production and TCR repertoires of naïve conventional CD4 + T (T conv ) and naïve regulatory CD4 + T (T reg ) cells. Compared with tuberculosis-resistant C57BL/6 (H2-A b ) mice, the tuberculosis-susceptible H2-A j mice had fewer CD4 + T cells of both subsets in the thymus. In the periphery, this deficiency was only apparent for T conv and was compensated for by peripheral reconstitution for T reg . We show that H2-A j favors selection of a narrower and more convergent repertoire with more hydrophobic and strongly interacting amino acid residues in the middle of CDR3α and CDR3β, suggesting more stringent selection against a narrower peptide–MHC-II context. H2-A j and H2-A b mice have prominent reciprocal differences in CDR3α and CDR3β features, probably reflecting distinct modes of TCR fitting to MHC-II variants. These data reveal the mechanics and extent of how MHC-II shapes the naïve CD4 + T cell CDR3 landscape, which essentially defines adaptive response to infections and self-antigens.

T cell recognition of a cognate peptide-MHC complex (pMHC) presented on the surface of infected or malignant cells, is of utmost importance for mediating robust and long-term immune responses. Accurate predictions of cognate pMHC targets for T Cell Receptors (TCR) would greatly facilitate identification of vaccine targets for both pathogenic diseases as well as personalized cancer immunotherapies. Predicting immunogenic peptides therefore has been at the centre of intensive research for the past decades but has proven challenging. Although numerous models have been proposed, performance of these models has not been systematically evaluated and their success rate in predicting epitopes in the context of human pathology, has not been measured and compared. In this study, we evaluated the performance of several publicly available models, in identifying immunogenic CD8+ T cell targets in the context of pathogens and cancers. We found that for predicting immunogenic peptides from an emerging virus such as SARS-CoV-2, none of the models perform substantially better than random or offer considerable improvement beyond HLA ligand prediction. We also observed suboptimal performance for predicting cancer neoantigens. Through investigation of potential factors associated with ill performance of models, we highlight several data- and model-associated issues. In particular, we observed that cross-HLA variation in the distribution of immunogenic and non-immunogenic peptides in training data of the models seem to substantially confound the predictions. We additionally compared key parameters associated with immunogenicity between pathogenic peptides and cancer neoantigens and observed evidence for differences in the thresholds of binding affinity and stability, which suggested the need to modulate different features in identifying immunogenic pathogen vs. cancer peptides. Overall, we demonstrate that accurate and reliable prediction of immunogenic CD8+ T cell targets remains unsolved, thus we hope our work will guide users and model developers regarding potential pitfalls and unsettled questions in existing immunogenicity predictors.

Antigen recognition by T-cells is guided by the T-cell receptor (TCR) heterodimer formed by α and β chains. A huge diversity of TCR sequences should be maintained by the immune system in order to be able to mount an effective response towards foreign pathogens, so, due to cooperative binding of α and β chains to the pathogen, any constraints on chain pairing can have a profound effect on immune repertoire structure, diversity and antigen specificity. By integrating available structural data and paired chain sequencing results we were able to show that there are almost no constraints on pairing in TCRαβ complexes, allowing naive T-cell repertoire to reach the highest possible diversity. Additional analysis reveals that the specific choice of contacting amino acids can still have a profound effect on complex conformation. Moreover, antigen-driven selection can distort the uniform landscape of chain pairing, while small, yet significant, differences in the pairing can be attributed to various specialized T-cell subsets such as MAIT and iNKT T-cells, as well as other TCR sets specific to certain antigens., Author summary In the present paper we study chain pairing preferences in the T-cell receptor (TCR) heterodimer complex. The TCR molecule is formed by α and β chains and binding of both of these chains to an antigen presented by the major histocompatibility complex (MHC) molecule is required in order to trigger an immune response against foreign pathogens and neoantigens. We show that chain pairing in the TCR complex is nearly random ensuring a highly diverse set of TCRs required for recognition of a vast set of antigens. Our results also show that chain pairing preferences can nevertheless influence TCR complex geometry and biases in TCR chain pairing can be used to identify antigen-driven selection or selection towards specialized subsets of T-cells such as mucosal-associated and natural killer invariant T-cells.

Understanding the hallmarks of the adaptive immune response to SARS-CoV-2 is critical for fighting the COVID-19 pandemic. We assessed the antibody and T-cell reactivity in COVID-19 convalescent patients and healthy donors sampled both prior to and during the pandemic. The numbers of SARS-CoV-2-specific T cells were increased in healthy donors examined during COVID-19. Combined with the absence of symptoms and humoral response across that group, this finding suggests that some individuals might be protected by T-cell cross-reactivity. In convalescent patients we observed public and diverse T-cell response to SARS-CoV-2 epitopes, revealing T-cell receptor motifs with germline-encoded features. Bulk CD4+ and CD8+ T-cell responses to Spike glycoprotein were mediated by groups of homologous T-cell receptors, some of them shared across multiple donors. Overall, our results demonstrate that T-cell response to SARS-CoV-2, including the identified set of specific T-cell receptors, can serve as a useful biomarker for surveying viral exposure and immunity. Funding: Mikhail Shugay was supported by RFBR grant No 19-34-70011. Alexander Ivanov was supported by the Ministry of Science and Higher Education of the Russian Federation [Agreement No. 075-15-2019-1660]. Conflict of Interest: The authors declare no conflicts of interest. Ethical Approval: All donors signed the informed consent approved by the National Research Center for Hematology ethical committee before the enrollment. Additionally, 10 healthy hematopoietic stem cell donor samples and 10 healthy donor serum samples were obtained from the blood bank with the approval of the local ethical committee.

The PBAF chromatin-remodeling complexes are multi-protein machines, regulating expression of genes involved in proliferation and differentiation. PHF10 is a subunit of the PBAF essential for its association with chromatin. Mammalian PHF10 is expressed as four ubiquitous isoforms, which are alternatively incorporated in the complex and differ by their influence on transcription of target genes. PHF10 have different domain structure and two of them (PHF10-S isoforms) lack C-terminal PHD domains, which enables their phosphorylation by CK-1. Here we have found that PBAF subunits have low turnover rate, except for PHF10 which has much lower half-life, and is degraded by β-TrCP. The β-TrCP knockdown stabilizes PBAF core subunits - BRG1 and BAF155 and specific subunits - PHF10, BAF200, BAF180 and BRD7. PHF10 isoforms contain two non-canonical β-TrCP degrons and are degraded by β-TrCP in a phospho-dependent manner. But phosphorylation of PHF10-S degrons by CK-1, contrary to previously described degrons, prevents their degradation. Targeted molecular docking demonstrated that phosphorylated forms of PHF10 bind to β-TrCP with much lower affinity than non-phosphorylated ones, contrary to previously described degrons. This unorthodox mechanism proposes that phosphorylation of β-TrCP degrons by CK-1 could not only degrade a set of proteins, but also stabilize a different set of targets.

Synthesis of β-lactamases is one of the common mechanisms of bacterial resistance to β-lactam antibiotics such as penicillins and cephalosporins. The widespread use of antibiotics resulted in appearance of numerous extended-spectrum β-lactamase variants or inhibitor-resistant β-lactamases. In TEM type β-lactamases mutations of 92 residues have been described. Several mutations are functionally important and they determine the extended substrate specificity. However, roles of the most so-called associated mutations, located far from the active site, remain unknown. We have investigated the role of associated mutations in structure of β-lactamase TEM-72, which contains two key mutations (G238S, E240K) and two associated mutations (Q39K, M182T) by means of molecular dynamics simulation. Appearance of the key mutations (in 238 and 240 positions) caused destabilization of the protein globule, characterized by increased mobility of amino acid residues. Associated mutations (Q39K, M182T) exhibited opposite effect on the protein structure. The mutation M182T stabilized, while the mutation Q39K destabilized the protein. It appears that the latter mutation promoted optimization of the conformational mobility of β-lactamase and may influence the enzyme activity.

β-lactamases are hydrolytic enzymes primarily responsible for occurrence and abundance of bacteria resistant to β-lactam antibiotics. TEM type β-lactamases are formed by the parent enzyme TEM-1 and more than two hundred of its mutants. Positions for the known amino acid substitutions cover ∼30% of TEM type enzyme's sequence. These substitutions are divided into the key mutations that lead to changes in catalytic properties of β-lactamases, and the secondary ones, which role is poorly understood. In this study, Residue Interaction Networks were constructed from molecular dynamic trajectories of β-lactamase TEM-1 and its variants with two key substitutions, G238S and E240K, and their combinations with secondary ones (M182T and Q39K). Particular attention was paid to a detailed analysis of the interactions that affect conformation and mobility of the Ω-loop, representing a part of the β-lactamase active site. It was shown that key mutations weakened the stability of contact inside the Ω-loop thus increasing its mobility. Combination of three amino acid substitutions, including the 182 residue, leads to the release of R65 promoting its new contacts with N175 and D176. As a result, Ω-loop is fixed on the protein globule. The second distal mutation Q39K prevents changes in spatial position of R65, which lead to the weakening of the effect of M182T substitution and the recovery of the Ω-loop mobility. Thus, the distal secondary mutations are directed for recovering the mobility of enzyme disturbed by the key mutations responsible for expansion of substrate specificity. AbbreviationsESBLextended spectrum beta-lactamasesIRinhibitor resistant beta-lactamasesMDmolecular dynamicsRINresidue interaction networksRMSDroot mean square deviationRMSFroot mean square fluctuations. extended spectrum beta-lactamases inhibitor resistant beta-lactamases molecular dynamics residue interaction networks root mean square deviation root mean square fluctuations. Communicated by Ramaswamy H. Sarma

Understanding the hallmarks of the immune response to SARS-CoV-2 is critical for fighting the COVID-19 pandemic. We assessed antibody and T cell reactivity in convalescent COVID-19 patients and healthy donors sampled both prior to and during the pandemic. Healthy donors examined during the pandemic exhibited increased numbers of SARS-CoV-2-specific T cells, but no humoral response. Their probable exposure to the virus resulted in either asymptomatic infection without antibody secretion, or activation of pre-existing immunity. In convalescent patients, we observed a public and diverse T cell response to SARS-CoV-2 epitopes, revealing T cell receptor (TCR) motifs with germline-encoded features. Bulk CD4+ and CD8+ T cell responses to the spike glycoprotein were mediated by groups of homologous TCRs, some of them shared across multiple donors. Overall, our results demonstrate that the T cell response to SARS-CoV-2, including the identified set of TCRs, can serve as a useful biomarker for surveying antiviral immunity., Graphical Abstract, Highlights Healthy donors sampled in 2020 had an increased T cell response to SARS-CoV-2 SARS-CoV-2 glycoprotein S-specific TCR repertoire features public CDR3 motifs Two epitopes are recognized by the majority of the HLA-A2+ COVID-19 convalescents, Shomuradova et al. assessed the immune response to SARS-CoV-2 in convalescent patients and healthy donors. Antigen-specific T cells were increased in convalescents and in donors sampled during the pandemic. The work identified two public epitopes from S-glycoprotein. T cell receptor repertoire profiling of S-glycoprotein specific lymphocytes revealed public CDR3 motifs.

Synthesis of b-lactamases is one of the common mechanisms of bacterial resistance to b-lactam antibiotics including penicillins and cephalosporins. The widespread use of antibiotics results in appearance of numerous extended-spectrum b-lactamase variants or resistance to inhibitors. Mutations of 92 residues of TEM type were found. Several mutations are the key mutations that determine the extension of spectrum of substrates. However, roles of the most associated mutations, located far from active site, remain unknown. We have investigated the role of associated mutations in structure of b-lactamase TEM-72, which contain two key mutation (G238S, E240K) and two associated mutations (Q39K, M182T) by means of simulation of molecular dynamics. The key mutation lead to destabilization of the protein globule, characterized by increased mobility of amino acid residues at high temperature of modelling. Mutation M182T lead to stabilization protein, whereas mutation Q39K is destabilizing mutation. It seems that the last mutation serves for optimization of conformational mobility of b-lactamase and may influence on enzyme activity.Sintez b-laktamaz iavliaetsia odnim iz naibolee obshchikh mekhanizmov rezistentnosti bakteriĭ k b-laktamnym antibiotikam, takim kak penitsilliny i tsefalosporiny. Shirokoe ispol'zovanie antibiotikov privelo k vozniknoveniiu mnozhestva variantov b-laktamaz s rasshirennym spektrom deĭstviia ili ustoĭchivym k ingibitoram. U b-laktamaz TEM tipa uzhe opisany mutatsii 92 aminokislotnykh ostatkov. Riad mutatsiĭ iavliaiutsia funktsional'nymi i otvechaiut za rasshirenie spektra substratnoĭ spetsifichnosti. Odnako rol' bol'shinstva tak nazyvaemykh soputstvuiushchikh mutatsiĭ, raspolozhennykh vdali ot aktivnogo tsentra, ostaetsia neizuchennoĭ. V rabote metodom molekuliarnoĭ dinamiki issledovali rol' soputstvuiushchikh mutatsiĭ v strukture b-laktamaz TEM-72, kotoraia soderzhit dve kliuchevye mutatsii (G238S, E240K) i dve soputstvuiushchie (Q39K, M182T). Pokazano, chto poiavlenie kliuchevykh mutatsiĭ ostatkov 238 i 240 privodilo k destabilizatsii belkovoĭ globuly, chto vyrazhalos' v uvelichenii podvizhnosti aminokislotnykh ostatkov. Soputstvuiushchie mutatsii ostatkov 182 i 39 vliiali na strukturu belka protivopolozhnym obrazom. Mutatsiia M182T uvelichivala ustoĭchivost' belkovoĭ globuly, a mutatsiia Q39K okazyvala destabiliziruiushchiĭ éffekt. Éta mutatsiia, po-vidimomu, sposobstvuet optimizatsii konformatsionnoĭ podvizhnosti b-laktamazy i mozhet vliiat' na rabotu fermenta.