Structural insights into protection against a SARS-CoV-2 spike variant by T cell receptor diversity.

T cells play a crucial role in combatting SARS-CoV-2 and forming long-term memory responses to this coronavirus. The emergence of SARS-CoV-2 variants that can evade T cell immunity has raised concerns about vaccine efficacy and the risk of reinfection. Some SARS-CoV-2 T cell epitopes elicit clonally restricted CD8+ T cell responses characterized by T cell receptors (TCRs) that lack structural diversity. Mutations in such epitopes can lead to loss of recognition by most T cells specific for that epitope, facilitating viral escape. Here, we studied an HLA-A2-restricted spike protein epitope (RLQ) that elicits CD8+ T cell responses in COVID-19 convalescent patients characterized by highly diverse TCRs. We previously reported the structure of an RLQ-specific TCR (RLQ3) with greatly reduced recognition of the most common natural variant of the RLQ epitope (T1006I). Opposite to RLQ3, TCR RLQ7 recognizes T1006I with even higher functional avidity than the WT epitope. To explain the ability of RLQ7, but not RLQ3, to tolerate the T1006I mutation, we determined structures of RLQ7 bound to RLQ-HLA-A2 and T1006I-HLA-A2. These complexes show that there are multiple structural solutions to recognizing RLQ and thereby generating a clonally diverse T cell response to this epitope that assures protection against viral escape and T cell clonal loss.

T cell receptors employ diverse strategies to target a p53 cancer neoantigen.

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells.

How does TCR-T cell therapy exhibit a superior anti-tumor efficacy.

TCR-engineered T cells have achieved great progress in solid tumor therapy, some of which have been applicated in clinical trials. Deep knowledge about the current progress of TCR-T in tumor therapy would be beneficial to understand the direction. Here, we classify tumor antigens into tumor-associated antigens, tumor-specific antigens, tumor antigens expressed by oncogenic viruses, and tumor antigens caused by abnormal protein modification; Then we detail the TCR-T cell therapy effects targeting those tumor antigens in clinical or preclinical trials, and propose that neoantigen specific TCR-T cell therapy is expected to be a promising approach for solid tumors; Furthermore, we summarize the optimization strategies, such as tumor microenvironment, TCR pairing and affinity, to improve the therapeutic effect of TCR-T. Overall, this review provides inspiration for the antigen selection and therapy strategies of TCR-T in the future.

Characterization of ibrutinib as a non-covalent inhibitor of SRC-family kinases.

Ibrutinib is a BTK-targeted irreversible inhibitor. In this study, we demonstrate that ibrutinib potently inhibits SRC activity in a non-covalent manner via mass spectrometry and crystallography. The S345C mutation renders SRC to bind covalently with ibrutinib, and restores the potency of ibrutinib against the gatekeeper mutant. The co-crystal structure of ibrutinib/SRC shows Ser345 of SRC did not form covalent bond with ibrutinib, leading to a decrease of potency and loss of the ability to overcome the gatekeeper mutation of SRC. The X-ray crystallographic studies also provide structural insight into why ibrutinib behaves differently against gatekeeper mutants of different kinases.

Association of luteinizing hormone/choriogonadotropin receptor gene polymorphisms with polycystic ovary syndrome risk: a meta-analysis.

To investigate the association between Luteinizing hormone/choriogonadotropin receptor (LHCGR) gene polymorphisms and polycystic ovary syndrome (PCOS). A systematic literature search and meta-analysis using STATA software for included studies. Fourteen case-control studies containing rs13405728, rs4539842, and rs2293275 of LHCGR gene were included, which was comprised of 11,738 PCOS cases and 35,329 controls. Results of the meta-analysis showed a significant association between PCOS and rs13405728 (for G vs. A: OR = 0.735, 95% CI = 0.699-0.773, p<.001; For GG vs. AG + AA: OR = 0.578, 95% CI = 0.436-0.767, p<.001; For GG + AG vs. AA: OR = 0.817, 95% CI = 0.741-0.901, p<.001) in Asian populations, and rs4539842 (for ins/ins vs. ins/non + non/non: OR = 0.686, 95% CI = 0.483-0.974, p=.035) and rs2293275 (for AA vs. AG + GG: OR = 4.115, 95% CI = 1.033-16.38, p=.045) in Caucasian populations, respectively. LHCGR gene variations are population specifically associated with PCOS, which indicated these SNPs in LHCGR may contribute to the pathogenesis of PCOS and could be used as potential biomarkers to predict the risk of PCOS.

The role of mTOR in ovarian Neoplasms, polycystic ovary syndrome, and ovarian aging.

The mammalian target of rapamycin, mTOR, is a serine-threonine protein kinase downstream of the phosphatidylinositol 3-kinase (PI3K)-AKT axis. The pathway can regulate cell growth, proliferation, and survival by activating ribosomal kinases. Recent studies have implicated the mTOR signaling pathway in ovarian neoplasms, polycystic ovary syndrome (PCOS) and premature ovarian failure (POF). Preclinical investigations have demonstrated that the PI3K/AKT/mTOR pathway is frequently activated in the control of various ovarian functions. mTOR allows cancer cells to escape the normal biochemical system and regulates the balance between apoptosis and survival. Some recent studies have suggested that involvement of the mTOR signaling system is an important pathophysiological basis of PCOS. Overexpression of the mTOR pathway can impair the interaction of cumulus cells, lead to insulin resistance, and affect the growth of follicles directly. The roles of mTOR signaling in follicular development have been extensively studied in recent years; abnormalities in this process lead to a series of pathologies such as POF and infertility. To improve understanding of the role of the mTOR signaling pathway in the pathogenesis and development of ovarian diseases, here we review the roles of mTOR signaling in such diseases and discuss the corresponding therapeutic strategies that target this pathway. Clin. Anat. 31:891-898, 2018. © 2018 Wiley Periodicals, Inc.

Structure of the Forkhead Domain of FOXA2 Bound to a Complete DNA Consensus Site.

FOXA2, a member of the forkhead family of transcription factors, plays essential roles in liver development and bile acid homeostasis. In this study, we report a 2.8 Å co-crystal structure of the FOXA2 DNA-binding domain (FOXA2-DBD) bound to a DNA duplex containing a forkhead consensus binding site (GTAAACA). The FOXA2-DBD adopts the canonical winged-helix fold, with helix H3 and wing 1 regions mainly mediating the DNA recognition. Although the wing 2 region was not defined in the structure, isothermal titration calorimetry assays suggested that this region was required for optimal DNA binding. Structure comparison with the FOXA3-DBD bound to DNA revealed more major groove contacts and fewer minor groove contacts in the FOXA2 structure than in the FOXA3 structure. Structure comparison with the FOXO1-DBD bound to DNA showed that different forkhead proteins could induce different DNA conformations upon binding to identical DNA sequences. Our findings provide the structural basis for FOXA2 protein binding to a consensus forkhead site and elucidate how members of the forkhead protein family bind different DNA sites.

DNA-binding properties of FOXP3 transcription factor.

FOXP3, a lineage-specific forkhead (FKH) transcription factor, plays essential roles in the development and function of regulatory T cells. However, the DNA-binding properties of FOXP3 are not well understood. In this study, FOXP3 fragments containing different domains were purified, and their DNA-binding properties were investigated using electrophoretic mobility shift assay and isothermal titration calorimetry (ITC). Both the FKH and leucine-zipper domains were required for optimal DNA binding for FOXP3. FOXP3 protein not only binds with DNA sequences containing one FKH consensus sequence, but also binds with DNA sequences with two direct repeats of consensus sequences separated by three-nucleotides (DRE3). Our results shed lights on the mechanisms by which FOXP3 recognizes cognate DNA elements, and would facilitate further structural and functional studies of FOXP3.

Expression and purification of the kinase domain of PINK1 in Pichia pastoris.

PTEN-induced putative kinase 1 (PINK1) is a Ser/Thr kinase that specifically localizes on the mitochondrial membrane. It cooperates with Parkin to regulate mitochondrial quality control. Mutations in PINK1 protein which account for 8-15% of Parkinson's disease (PD), are the second most common cause of early-onset Autosomal Recessive Parkinson's disease (AR-PD). The lack of methods for PINK1 heterologous expression and purification has slowed progress in the AR-PD research field. To pave the way for direct structural study of this important protein, in this study, we developed an efficient expression system of recombinant PINK1 kinase domain (rPINK1) using Pichia pastoris (P. pastoris). Our results showed that rPINK1 is best expressed in P. pastoris at 25 °C induction. Additionally, we determined that the optimal induction time was 72 h and the optimal induction methanol concentration was 1% for the expression of rPINK1 in P. pastoris. Subsequent purification by Ni affinity chromatography (Ni-NTA) and cation-exchange chromatography (Mono S) produced the protein with purity higher than 95%. The pure rPINK1 was active to phosphorylate ubiquitin in a substrate phosphorylation assay. Overall, these studies provide the first effective method for heterologous expression and purification of the rPINK1 with a high purity. These findings can help contribute to further researches on the interactions study and biochemical characterization of PINK1.

Saccharomyces boulardii prevention of the hepatic injury induced by Salmonella Enteritidis infection.

Salmonella enterica subsp. enterica serovar Enteritidis (Salmonella Enteritidis) is the predominant cause of serovar-associated food-borne outbreaks in many countries and causes significant clinical symptoms of liver injury, enteritis, and diarrheal diseases. Saccharomyces boulardii is used in clinical application for prophylaxis and the treatment of a variety of diseases caused by bacterial infection. We used a mouse model of Salmonella Enteritidis infection, which included pretreatment with S. boulardii, to reveal the protection mechanisms of S. boulardii against Salmonella Enteritidis infection, including the translocation of Salmonella Enteritidis to the liver 10 days after Salmonella Enteritidis challenge, and the colonisation of Salmonella Enteritidis and the formation of hepatic tissue lesions in mice after Salmonella Enteritidis challenge on the 10th day. Compared with Salmonella Enteritidis infection in mice, S. boulardii decreased Salmonella Enteritidis translocation to the liver by 96%, and 99% of Salmonella Enteritidis colonised the cecum on the 10th day. Saccharomyces boulardii also abated hepatic tissue injury caused by the infiltration of neutrophilic granulocytes, lymphocytes, and plasmocytes by decreasing the translocation of Salmonella to the liver. These findings demonstrated that S. boulardii is an effective agent in the prevention of the hepatic injury induced by Salmonella Enteritidis infection in a mouse model.

Advances, opportunities and challenges in developing therapeutic cancer vaccines.

Therapeutic cancer vaccines have shown promising efficacy in helping immunotherapy for cancer patients, but the systematic characterization of the clinical application and the method for improving efficacy is lacking. Here, we mainly summarize the classification of therapeutic cancer vaccines, including protein vaccines, nucleic acid vaccines, cellular vaccines and anti-idiotypic antibody vaccines, and subdivide the above vaccines according to different types and delivery forms. Additionally, we outline the clinical efficacy and safety of vaccines, as well as the combination strategies of therapeutic cancer vaccines with other therapies. This review will provide a detailed overview and rationale for the future clinical application and development of therapeutic cancer vaccines.

Structural characterization and AlphaFold modeling of human T cell receptor recognition of NRAS cancer neoantigens.

T cell receptors (TCRs) that recognize cancer neoantigens are important for anti-cancer immune responses and immunotherapy. Understanding the structural basis of TCR recognition of neoantigens provides insights into their exquisite specificity and can enable design of optimized TCRs. We determined crystal structures of a human TCR in complex with NRAS Q61K and Q61R neoantigen peptides and HLA-A1 MHC, revealing the molecular underpinnings for dual recognition and specificity versus wild-type NRAS peptide. We then used multiple versions of AlphaFold to model the corresponding complex structures, given the challenge of immune recognition for such methods. Interestingly, one implementation of AlphaFold2 (TCRmodel2) was able to generate accurate models of the complexes, while AlphaFold3 also showed strong performance, although success was lower for other complexes. This study provides insights into TCR recognition of a shared cancer neoantigen, as well as the utility and practical considerations for using AlphaFold to model TCR-peptide-MHC complexes.

Structural basis for T cell recognition of cancer neoantigens and implications for predicting neoepitope immunogenicity.

Adoptive cell therapy (ACT) with tumor-specific T cells has been shown to mediate durable cancer regression. Tumor-specific T cells are also the basis of other therapies, notably cancer vaccines. The main target of tumor-specific T cells are neoantigens resulting from mutations in self-antigens over the course of malignant transformation. The detection of neoantigens presents a major challenge to T cells because of their high structural similarity to self-antigens, and the need to avoid autoimmunity. How different a neoantigen must be from its wild-type parent for it to induce a T cell response is poorly understood. Here we review recent structural and biophysical studies of T cell receptor (TCR) recognition of shared cancer neoantigens derived from oncogenes, including p53R175H, KRASG12D, KRASG12V, HHATp8F, and PIK3CAH1047L. These studies have revealed that, in some cases, the oncogenic mutation improves antigen presentation by strengthening peptide-MHC binding. In other cases, the mutation is detected by direct interactions with TCR, or by energetically driven or other indirect strategies not requiring direct TCR contacts with the mutation. We also review antibodies designed to recognize peptide-MHC on cell surfaces (TCR-mimic antibodies) as an alternative to TCRs for targeting cancer neoantigens. Finally, we review recent computational advances in this area, including efforts to predict neoepitope immunogenicity and how these efforts may be advanced by structural information on peptide-MHC binding and peptide-MHC recognition by TCRs.

SARS-CoV-2 infection establishes a stable and age-independent CD8+ T cell response against a dominant nucleocapsid epitope using restricted T cell receptors.

The resolution of SARS-CoV-2 replication hinges on cell-mediated immunity, wherein CD8+ T cells play a vital role. Nonetheless, the characterization of the specificity and TCR composition of CD8+ T cells targeting non-spike protein of SARS-CoV-2 before and after infection remains incomplete. Here, we analyzed CD8+ T cells recognizing six epitopes from the SARS-CoV-2 nucleocapsid (N) protein and found that SARS-CoV-2 infection slightly increased the frequencies of N-recognizing CD8+ T cells but significantly enhanced activation-induced proliferation compared to that of the uninfected donors. The frequencies of N-specific CD8+ T cells and their proliferative response to stimulation did not decrease over one year. We identified the N222-230 peptide (LLLDRLNQL, referred to as LLL thereafter) as a dominant epitope that elicited the greatest proliferative response from both convalescent and uninfected donors. Single-cell sequencing of T cell receptors (TCR) from LLL-specific CD8+ T cells revealed highly restricted Vα gene usage (TRAV12-2) with limited CDR3α motifs, supported by structural characterization of the TCR-LLL-HLA-A2 complex. Lastly, transcriptome analysis of LLL-specific CD8+ T cells from donors who had expansion (expanders) or no expansion (non-expanders) after in vitro stimulation identified increased chromatin modification and innate immune functions of CD8+ T cells in non-expanders. These results suggests that SARS-CoV-2 infection induces LLL-specific CD8+ T cell responses with a restricted TCR repertoire.

Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors.

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.

FGFR-TKI resistance in cancer: current status and perspectives.

Fibroblast growth factor receptors (FGFRs) play key roles in promoting the proliferation, differentiation, and migration of cancer cell. Inactivation of FGFRs by tyrosine kinase inhibitors (TKI) has achieved great success in tumor-targeted therapy. However, resistance to FGFR-TKI has become a concern. Here, we review the mechanisms of FGFR-TKI resistance in cancer, including gatekeeper mutations, alternative signaling pathway activation, lysosome-mediated TKI sequestration, and gene fusion. In addition, we summarize strategies to overcome resistance, including developing covalent inhibitors, developing dual-target inhibitors, adopting combination therapy, and targeting lysosomes, which will facilitate the transition to precision medicine and individualized treatment.

Systemic Analysis of the DNA Replication Regulator MCM Complex in Ovarian Cancer and Its Prognostic Value.

Microliposome maintenance (MCM) 2, MCM3, MCM4, MCM5, MCM6, and MCM7 are DNA replication regulators and are involved in the progression of multiple cancer types, but their role in ovarian cancer is still unclear. The purpose of this study is to clarify the biological function and prognostic value of the MCM complex in ovarian cancer (OS) progression. We analyzed DNA alterations, mRNA and protein levels, protein structure, PPI network, functional enrichment, and prognostic value in OC based on the Oncomine, cBioPortal, TCGA, CPTAC, PDB, GeneMANIA, DAVID, KEGG, and GSCALite databases. The results indicated that the protein levels of these DNA replication regulators were increased significantly. Moreover, survival analysis showed a prognostic signature based on the MCM complex, which performed moderately well in terms of OS prognostic prediction. Additionally, protein structure, functional enrichment, and PPI network analyses indicated that the MCM complex synergistically promoted OC progression by accelerating DNA replication and the cell cycle. In conclusion, our study suggested that the MCM complex might be a potential target and prognostic marker for OC patients.

IL-37 As a Potential Biotherapeutics of Inflammatory Diseases.

Interleukin-37 (IL-37) was discovered as a new member of pro-inflammatory IL-1 superfamily. However, further studies suggested that IL-37 plays a critical anti-inflammatory role in innate and adaptive immunity. IL-37 may suppress the inflammatory process via intracellular SMAD family member 3 (SMAD3) and extracellular IL-18 Receptor alpha (IL-18Rα) signaling pathway, respectively. Meanwhile, the abnormal expression of IL-37 was observed in immune-mediated inflammatory diseases, such as inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, systemic lupus erythematosus, asthma, and multiple sclerosis, which suggest IL-37 is a potential therapeutic target for these diseases. In this review, we summarize the anti-inflammatory mechanism of IL-37 and discuss the critical roles of IL-37 in the pathogenesis of these diseases. Further studies are required to confirm the effectiveness of IL-37 as a novel target for these inflammatory diseases.

BDNF and nicotine dependence: associations and potential mechanisms.

Smoking is the leading preventable cause of death worldwide and tobacco addiction has become a serious public health problem. Nicotine is the main addictive component of tobacco, and the majority of people that smoke regularly develop nicotine dependence. Nicotine addiction is deemed to be a chronic mental disorder. Although it is well known that nicotine binds to the nicotinic acetylcholine receptors (nAChRs) and activates the mesolimbic dopaminergic system (MDS) to generate the pleasant and rewarding effects, the molecular mechanisms of nicotine addiction are not fully understood. Brain-derived neurotrophic factor (BDNF) is the most prevalent growth factor in the brain, which regulates neuron survival, differentiation, and synaptic plasticity, mainly through binding to the high affinity receptor tyrosine kinase receptor B (TrkB). BDNF gene polymorphisms are associated with nicotine dependence and blood BDNF levels are altered in smokers. In this review, we discussed the effects of nicotine on BDNF expression in the brain and summarized the underlying signaling pathways, which further indicated BDNF as a key regulator in nicotine dependence. Further studies that aim to understand the neurobiological mechanism of BDNF in nicotine addcition would provide a valuable reference for quitting smoking and developing the treatment of other addictive substances.

Structural basis for oligoclonal T cell recognition of a shared p53 cancer neoantigen.

Adoptive cell therapy (ACT) with tumor-specific T cells can mediate cancer regression. The main target of tumor-specific T cells are neoantigens arising from mutations in self-proteins. Although the majority of cancer neoantigens are unique to each patient, and therefore not broadly useful for ACT, some are shared. We studied oligoclonal T-cell receptors (TCRs) that recognize a shared neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by HLA-A2. Here we report structures of wild-type and mutant p53-HLA-A2 ligands, as well as structures of three tumor-specific TCRs bound to p53R175H-HLA-A2. These structures reveal how a driver mutation in p53 rendered a self-peptide visible to T cells. The TCRs employ structurally distinct strategies that are highly focused on the mutation to discriminate between mutant and wild-type p53. The TCR-p53R175H-HLA-A2 complexes provide a framework for designing TCRs to improve potency for ACT without sacrificing specificity.