Background: Drug resistance is one of the most prevalent causes of death in advanced prostate cancer patients. Combination therapies that target cancer cells via different mechanisms to overcome resistance have gained increased attention in recent years. However, the optimal drug combinations and the underlying mechanisms are yet to be fully explored. Aim and methods: The aim of this study is to investigate drug combinations that inhibit the growth of drug-resistant cells and determine the underlying mechanisms of their actions. In addition, we also established cell lines that are resistant to combination treatments and tested new compounds to overcome the phenomenon of double drug-resistance. Results: Our results show that the combination of enzalutamide (ENZ) and docetaxel (DTX) effectively inhibit the growth of prostate cancer cells that are resistant to either drug alone. The downregulation of transcription factor E2F1 plays a crucial role in cellular inhibition in response to the combined therapy. Notably, we found that the androgen receptor (AR) variant AR3 (a.k.a. AR-V7), but not AR full length (AR-FL), positively regulates E2F1 expression in these cells. E2F1 in turn regulates AR3 and forms a positive regulatory feedforward loop. We also established double drug-resistant cell lines that are resistant to ENZ+DTX combination therapy and found that the expression of both AR3 and E2F1 was restored in these cells. Furthermore, we identified that auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, overcame drug resistance and inhibited the growth of drug-resistant prostate cancer cells both in vitro and in vivo. Conclusion and significance: This proof-of-principle study demonstrates that targeting the E2F1/AR3 feedforward loop via a combination therapy or a multi-targeting drug could circumvent castration resistance in prostate cancer. Prostate cancer is one of the most frequently diagnosed cancers in men. According to the American Cancer Society, 191,930 new cases and 33,330 deaths have been estimated for prostate cancer in United States for 2020. Hormonal therapy is the standard treatment for advanced prostate cancer patients. Unfortunately, the tumors eventually acquire androgen-independence and progress to castration-resistant prostate cancer (CRPC). Docetaxel (DTX) is the first line of treatment for androgen-independent metastatic CRPC. However, a large portion of CRPC patients do not respond to DTX. The new-generation androgen receptor (AR)-targeting agent, enzalutamide (ENZ), significantly increased the overall survival of CRPC patients, either with or without previous chemotherapy [DTX is a taxane compound, and exerts its cytotoxic function via the stabilization of guanosine diphosphate (GDP)-bound tubulin in microtubules. It is widely accepted that the stabilization of microtubules can cause mitotic spindle dis-regulation, thereby leading to the death of rapidly-dividing cancer cells. Microtubules also play important roles in intracellular protein trafficking. The microtubule motor protein dynein is critical for the nuclear translocation of AR, while the taxane-induced microtubule stabilization inhibits the nuclear accumulation and transcriptional activity of AR . The preIn this study, we found that the expression of E2F1 was substantially altered in response to a combined DTX+ENZ treatment in cells that were resistant to either drug alone. It has been reported that E2F expression was downregulated by DTX in HN12 and HN30 head and neck squamous cell lines , and ourTherefore, the aim of the present study was to examine the effect of a combined DTX+ENZ therapy on prostate cancer cells that are resistant to either drug alone. Furthermore, we sought to determine the functional relationship between E2F1 and AR3, and the role of the E2F1/AR3 feedforward loop in drug resistance. This research provides new insights into the mechanisms underlying drug resistance in pre-clinical models.R1/DTX was developed as previously described . R1-ADR The plasmids used in the present study are as follows: the E2F1 expression vector pCMVHA E2F1 was a gift from Kristian Helin  , the E2FThe antibodies used in this study include: Rabbit polyclonal anti-AR3 , Anti-ARThe shRNAs used in this study include:shAR-FL was purchased from Sigma.shAR3:TAGGCTAATGAGGTTT ATTTCTCAAGAG AAAATAAACCTCATTAGCCTTTTTTTTTCshE2F1-1:CCGGCTACTCAGCCTGGAGCAAGAACTCGAGTTCTTGCTCCAGGCTGAGTAGTTTTTGshE2F1-2:CCGGCGCTATGAGACCTCACTGAATCTCGAGATTCAGTGAGGTCTCATAGCGTTTTTGCell growth was measured using cell counting kit-8 (CCK8) assay . Cells were seeded in 96-well plates  at a density of 2000 cells/well. Drugs were added the following day (D1), and then refreshed every two days for 3–6 days as indicated in each experiment. Then, 10 µL of the CCK8 solution was added to each well and the cells were cultured for 1.5 h at 37 °C. Absorbance was measured using a plate reader at 450nm wavelength. Five wells were used for each experimental condition. Spheroid formation was assessed using Ultra-low attachment multiple well plate . Spheroid size was evaluated using the free software ImageJ. Eight thousand cells were suspended in a final volume of 100 μL/well and cultured for four days. Then, half of the old medium was replaced by fresh medium containing the appropriate amounts of agents every two days and cultured for 14 days. Finally, a TUNEL apoptosis assay was performed using the DeadEnd™ Fluorometric TUNEL System  according to the manufacturer’s instructions.Cells were lysed in QIAzol lysis reagent . Total RNA was extracted using a RNeasy mini kit  according to the manufacturer’s instructions. RNA-seq was performed by Arraystar, Inc . Briefly, total RNA from each sample was quantified using a NanoDrop ND-1000. Subsequently, 1–2 μg of total RNA was used to prepare the sequencing library according to the following steps: 1. Total RNA was enriched by oligo (dT) magnetic beads (rRNA removed); and 2. RNA-seq library was prepared using the KAPA Stranded RNA-Seq Library Prep Kit , which incorporates dUTP into the second cDNA strand and renders the RNA-seq library strand-specific. The completed libraries were qualified using an Agilent 2100 Bioanalyzer and quantified using the absolute quantification qPCR method. To sequence the libraries, the barcoded libraries were mixed, denatured to single stranded DNA in NaOH, captured on Illumina flow cell, amplified in situ, and subsequently sequenced for 150 cycles at both ends using the Illumina HiSeq 4000. RNA-seq data was deposited in NCBI’s Sequence Read Archive (BioProject Accession: PRJNA623560). Analyses were performed using BRB-ArrayTools, which was developed by Dr. Richard Simon and the BRB-ArrayTools Development Team. Changes in gene expression were examined by two-tailed two-sample t-test and the Benjamini-Hochberg procedure ,25.Total RNA was extracted using a RNeasy Mini kit  and reverse transcribed using Transcriptor Reverse Transcriptase . Quantitative real-time PCR was performed as previously described .The qRT-PCR primer sequences used in this study include:AR-FL:F: ctactccggaccttacggggacatgcg R:gggctgacattcatagccttcaatgtgtgacAR3:F:CTACTCCGGACCTTACGGGGACATGCG R: TGCCAACCCGGAATTTTTCTCCCE2F1: F: AGCTGGACCACCTGATGAAT R: GTCCTGACACGTCACGTAGGActin: F: GCTCGTCGTCGACAACGGCTC R: CAAACATGATCTGGGTCATCT8 R1-ADR cells were used for each ChIP. The ChIP primer sequences used in this study include:Immunoprecipitation was performed as previously described . 1 × 108ChIP-E2F1-P: F: GTTGGGGGCTACAGGTTGAG R: AAGTCCCGGCCACTTTTACGChIP-FKBP5: F: GCATGGTTTAGGGGTTCTTGC R: AACACCCTGTTCTGAATGTGGCChIP-AR-siteA: F: GACTCGCAAACTGTTGCATT R: TACAGCACTGGAGCGGCTAChIP-AR-siteB: F: CCTAGCAGGGCAGATCTTGT R: TCCCCTTCTCTTGCTCAGAAChIP-AR-siteC: F: GGTAGGAAGTGGCTGAATTCTGGATGA R: CCCTGCCCATGCACCTGCTCLuciferase assays were carried out as previously described . Briefly8 cells were used for each IP. Immunoprecipitation was performed as previously described [For HEK-293T exogenous IP, the cells were transfected with the appropriate plasmids and allowed to grow for 48 h in 100 mm dishes. For R1-ADR endogenous IP, the cells were plated in P150 dishes for three days, 1nM DHT was added, and the cells were incubated for 4 h before being collected as indicated. 3 × 10escribed . Immunofescribed .12 × r2 (r1 < r2). Results of tumor volumes were expressed as means ± SEM. The effect of treatment was analyzed by one-way ANOVA. Post-hoc analysis was performed using Tukey’s test. Relative body weight was analyzed by repeated measures ANOVA with group as between subject factor and time as within subject factor. Immunohistochemical staining of mouse xenograft tumors was carried out as previously described [The animal studies were performed according to protocol ##0917012 (approved on 09/20/2019) reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Maryland School of Medicine, Baltimore, MD, USA. The R1-DDR xenograft was carried out as previously described . Animalsescribed .≈ 1.4 μM, LN-ADR: IC50 ≈ 2.6 μM) in comparison to ENZ treatment alone . We also showed that the combined DTX+ENZ therapy exerted a better inhibitory effect than DTX alone in ENZ-resistant R1-ENZR cells  on AR promoter described by Valdez et al. . In compCross-resistance is common in CRPC patients ,35. We fIn a search for potential therapeutic agents to overcome double drug-resistance, we found that auranofin, a gold-based compound that is an FDA-approved drug for the treatment of rheumatoid arthritis, efficiently inhibited double drug-resistant cell growth A. In recin vivo. As shown in We also examined the effects of auranofin on double drug-resistant cells To elucidate the molecular mechanisms and investigate new strategies for delaying and circumventing drug resistance, we developed cell models that were resistant to either DTX or ENZ. We tried several combinations of a variety of drugs and found that only DTX+ENZ effectively reduced the survival of the drug-resistant cells. This finding is consistent with a previous report which showed antiandrogens inhibited drug efflux activity and reversed docetaxel resistance in advanced prostate cancer . In ordeAR signaling, especially the AR variant AR3/AR-V7-driven molecular events, are critical for therapeutic resistance in aggressive prostate cancer. While the expression and activities of E2F1 and AR are often correlated, the details of this co-regulation are still not fully understood. It has been reported that a low dose of androgen induced E2F activity, while a high dose of androgen inhibited E2F activity . One expSeveral recent reports have shown that ENZ is able to radiosensitize prostate cancer in vitro and in vivo by impairing the DNA damage response ,48. SimiIn this study, we generated DTX and ENZ double drug-resistant prostate cancer cells, which were highly resistant to several compounds that we tested. However, we found that auranofin—an FDA-approved drug for the treatment of rheumatoid arthritis—effectively inhibited double drug-resistant prostate cancer cell growth in vitro and in vivo. Furthermore, this effect is at least partially a result of the downregulation of the E2F1-AR3 axis. The main mechanism of action of auranofin is via the inhibition of redox enzymes. Increasing evidence shows that auranofin has anti-cancer effects . A recenIn summary, this is a proof-of-principle study to demonstrate that simultaneously targeting E2F1 and AR3 via a combined therapy or a multi-targeting drug could circumvent castration resistance in prostate cancer. We also showed that the differential regulation of E2F1 expression by AR3, but not by AR-FL, in R1-ADR cells, suggesting that AR3 may function independently of AR-FL in different genetic contexts via the differential recruitment of co-factors.


Gephyrin A91T and G375D displayed altered clustering patterns in COS-7 cells and nullified the inhibitory synapse-promoting effect of gephyrin in cultured neurons. However, only the G375D mutation reduced gephyrin interaction with GABAA receptors and neuroligin-2 in mouse brain; it also failed to normalize deficits in GABAergic synapse maintenance and neuronal hyperactivity observed in hippocampal dentate gyrus-specific gephyrin-deficient mice. Our results provide insights into biochemical, cell-biological, and network-activity effects of the pathogenic G375D mutation.Gephyrin is critical for the structure, function, and plasticity of inhibitory synapses. Gephyrin mutations have been linked to various neurological disorders; however, systematic analyses of the functional consequences of these mutations are lacking. Here, we performed molecular dynamics simulations of gephyrin to predict how six reported point mutations might change the structural stability and/or function of gephyrin. Additional   •Gephyrin G375D reduces the formation of E- domain-mediated gephyrin oligomers•in vivoGephyrin G375D impairs GABAergic synapse maintenance •in vivoGephyrin G375D increases susceptibility to kainic acid-induced seizures •A receptors and Nlgn2 in vivoGephyrin G375D displays reduced interactions with GABA Molecular Biology; Neuroscience; Structural Biology The gephyrin mutants, A91T and G375D, which displayed altered binding free energies ARs or GlyRs, and further compromises molybdenum cofactor synthesis activity  are known to bind to GABAnd GlyRs . G375 isnd GlyRs C and 1E.nd GlyRs B. HoweveBecause single point mutations in gephyrin conferred no significant conformational changes on monomeric or multimeric structures of gephyrin , we nextAR- and GlyR-binding subdomain of another E-domain -tagged constructs of gephyrin WT and its mutants (To assess whether the mutations in the  mutants A. Immuno mutants B. Recomb mutants . As prev mutants C. Under  mutants C and 2D. mutants C and 2D.in vitro (DIV10), and immunostained transfected neurons for the GABAergic presynaptic marker, VGAT (vesicular GABA transporter), and the postsynaptic GABAA receptor γ2 subunit (GABAARγ2) at DIV14. An assessment of the subcellular localization of gephyrin mutants, visualized by monitoring GFP immunofluorescence, revealed that gephyrin WT was mainly distributed to GABAergic synaptic sites, whereas both A91T and G375D were diffusely distributed along dendrites of the transfected neurons, compared to gephyrin WT and other mutants -induced epileptic mouse model in which mice stereotactically injected with the indicated AAVs were intraperitoneally administered KA (15 mg/kg), and then monitored for seizure-related behaviors by video recording A. The sein vivo.In line with these differential behavioral responses, electroencephalography (EEG) and local field potential (LFP) recordings revealed higher-amplitude and longer-lasting KA-induced ictal activity in DG-specific gephyrin-KO mice compared with control mice F–4J. Thein vivo, including higher-order oligomer formation, interactions with key proteins of GABAergic synapses, and neuronal activity in the hippocampal DG area.In the present study, we applied MD simulations to predict the possible structural alterations produced by disease-associated point mutations that might be responsible for impairing the ability of gephyrin to form proper synaptic scaffolds. Of the six point mutations examined, A91T and G375D displayed reduced binding free energies for G-domain trimerization and E-domain dimerization, respectively, and failed to enhance the density of GABAergic synapses in cultured neurons, and hence were selected for extensive functional analyses. G375D, but not A91T, consistently exhibited loss-of-function phenotypes in all experimental settings. Although a previous report proposed impaired synaptic localization and decreased receptor binding as the mechanistic basis for the pathogenicity of the G375D mutant , the preOur atomic-scale MD simulations indicated that the G375D mutation does not affect the structure of gephyrin in the E-domain dimer, but selectively reduces the binding free energy of the E-domain dimer compared with WT gephyrin . This imin vivo. Notably, using circular dichroism analyses, Dejanovic et al. found that bacterially produced recombinant gephyrin G375D proteins showed no folding defects .We found no synapse-promoting effects of G375D at GABAergic synapses in cultured hippocampal neurons . This re defects . In cont defects I and 1J.asticity . To the A receptor α3 subunit, Cbs, Nlgn2, GABARAP, Pin1, and IQSEC3, to coordinate inhibitory postsynaptic organization  and Nlgn2, but not Cbs or IQSEC3, from gephyrin-containing complexes in hippocampal DG neurons .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ji Won Um (All unique/stable reagents generated in this study are available from the Lead contact upon reasonable request and with a completed Materials Transfer Agreement.This study did not generate data sets.All methods can be found in the accompanying


Cancer immunotherapy has recently undergone rapid development into a validated therapy for clinical use. The adoptive transfer of engineered autologous T cells, such as chimeric antigen receptor (CAR) T cells, has been remarkably successful in patients with leukemia and lymphoma with cluster of differentiation (CD)19 expression. Because of the higher number of antigen choices and reduced incidence of cytokine release syndrome (CRS) than CAR-T cells, T cell receptor (TCR)-T cells are also considered a promising immunotherapy. More therapeutic targets for other cancers need to be explored due to the human leukocyte antigen (HLA)-restricted recognition of TCR-T. Major histocompatibility complex (MHC), class I-related (MR1)-restricted T cells can recognize metabolites presented by MR1 in the context of host cells infected with pathogens. MR1 is expressed by all types of human cells. Recent studies have shown that one clone of a MR1-restricted T (MR1-T) cell can recognize many types of cancer cells without HLA-restriction. These studies provide additional information on MR1-T cells for cancer immunotherapy. This review describes the complexity of MR1-T cell TCR in diseases and the future of cancer immunotherapy. Unlike en (HLA) . The MR1en (HLA) . Studiesen (HLA) ,8. CompaTCR recognition is vital to the body as a defense mechanism against infection and tumors. The TCR can recognize peptide antigens, as well as a wide range of other antigens. Unlike the conventional MHC-I and MHC-II molecules that have been extensively studied, some MHC-like molecules  have been attracting increasing attention in terms of antigen presentation . HLA-E, HLA-E is a homologous MHC class Ib molecule that is widely expressed at low levels and can present peptides derived from MHC class Ia molecules  to the natural killer (NK) cells where they bind to cognate receptor molecules like the NK cell lectin-like receptor C1/2 (NKG2A/C)–CD94 complex . The CD1MR1 is characterized by three α domains, a transmembrane domain, and a cytoplasmic tail. Analysis of the crystal structure of MR1 shows that it associates with β-2-microglobulin at the α3 domain . The TCRd-ribitylaminouracil (5-A-RU), the product of an intermediate in the riboflavin biosynthesis pathway, have been detected by the TCR of MAIT cells. 5-A-RU can produce 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU) and 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU) derivative antigens by enzymatic reactions with methylglyoxal or glyoxal [MR1 antigen presentation commonly occurs in various bacterial and yeast infections, but not in viral infections, which were first found to be sensed by MAIT cells . Derivat glyoxal . MR1 [5, glyoxal  can bind glyoxal ,17. Ther glyoxal .+ and CD8+ T cells. Other T cells with the capability of recognizing non-peptide antigen presentation have not been reported to date. CD8+ and CD4+ T cells can recognize short peptide antigens presented by MHC class I (8–11 aa) and MHC class II (15 aa) antigens [+ and CD8+ T cells that possess diverse TCR repertoires for polymorphic HLA structures, MAIT cells have a quite conserved TCR repertoire, which is not restricted by HLA. In humans, MAIT cells constitute 10% of blood T cells and 45% of liver T cells [+ TRAJ33+, with two alternative TRAJ genes (TRAJ12 and TRAJ20) [− MAIT cells such as non-classical MAIT cells  and atypical MR1-restricted T cells [MAIT belongs to a population of unconventional T cells that play important roles in the human immune system, including responses to bacterial and viral infection diseases, autoimmune disease, cancer, allergies, and inflammatory disorders . Currentantigens . MAIT ceantigens ,20. Unli T cells . The fir T cells . By 1999 T cells . Subsequ T cells ,27, whic T cells , and in  T cells . MAIT ce TRAJ20) . It is r T cells .MAIT cells can act directly against a range of bacterial and fungal species via the riboflavin-biosynthesis pathway, even against some cell-invading intracellular bacteria and parasites . BecausePrevious studies have described the role of MAIT cells in tumor immunity. A meta-analysis of expression signatures from ∼18000 human tumors with overall survival outcomes across 39 malignancies found that KLRB1 (encoding CD161) expression is a favorable prognostic gene, which is also a marker of MAIT cells . Colorec–/– that received allogeneic bone marrow transplantation develop more severe graft-versus-host disease (GVHD) than normal mice [MAIT cells also play a role in hematological malignancies. More exhausted MAIT cells are detected in newly diagnosed multiple myeloma (MM) patients and are also restored in refractory or relapsed patients. MM patients have few MAIT cells in blood and bone marrow compared with healthy subjects . MAIT cemal mice . A lowermal mice .+ MAIT cells, but other TRAV1-2−MR1-T cells. The diversity of antigens’ specificities of these TRAV1-2− MR1-T cells indicates a new area for future studies. The discovery of novel MR1-T cell subsets and distinct antigens reflects the structural diversity of MR1 presentation, which could be more diverse than 5-OP-RU and 6-FP . They then screened 47 microbiota-associated bacterial species from different phyla and found that only bacterial species that encoded the riboflavin pathway were stimulatory to MAIT-TCRs. Most species that were high-stimulators belonged to the phyla Bacteroidetes and Proteobacteria, whereas low/non-stimulator species were primarily Actinobacteria or Firmicutes. The activation of MAIT cells by high- vs. low-stimulating bacteria was also correlated with the level of riboflavin they secreted or after bacterial infection of macrophages. There is a highly significant difference among bacterial species in terms of their MAIT-TCR stimulatory capacity. Furthermore, T cells can act as APC for antigen-specific activation of MAIT cells and tuning of their effector functions. Huang et al. [Mycobacterium tuberculosis lysate. TCR sequence analysis allowed the authors to preferentially select expanded conventional CD8+ T cells as well as invariant NK T cells and MAIT cells.However, whether MAIT cells discriminate between many species of the human microbiota remains unclear. Tastan et al.  developeg et al.  developeLepore et al.  found anin vivo and prolong the survival of mice. In addition, MC.7.G5 transferred patient T cells can kill autologous and non-autologous melanoma cells.Crowther et al.  found a + T cells of volunteers with multiple sclerosis. By recognizing different antigens occurring in distinct target cells and exhibiting a variety of effector functions, the MR1-restricted T cells have been shown to drive inflammatory responses, support B-cell function, mediate DC licensing, promote tissue remodeling, and contribute to the maintenance of mucosal homeostasis by enhancing innate defenses at the epithelial barrier. Therefore, future studies are expected to uncover the roles of MR1-restricted T cells in other human diseases.Contentti et al.  identifiNumerous studies have shown that MR1-T cells are more diverse than MAIT ‘invariant’ cells. The diversity of the MR1 repertoire is complex and associated with different antigens, suggesting that MR1-T cells exhibit more functions than expected ,43 Tabl.−CD8− double-negative T cell and subsequently an invariant TCR α-chain paired with a constrained TCR β-chain repertoire (TRBV6 family and TRBV20-1) in humans. In 2013, Reantragoon et al. [Porcelli et al.  first den et al.  found thn et al.  found thn et al. . Identifn et al. . Some ofn et al.  also fou− MR1-restricted T cells contain both MAIT-like and non-MAIT-like cells. These include a MAIT-like population that expresses a public canonical TRAV36/TRAJ34+ or TRAJ37+ TRBV28/TRBJ2-5+ TCR. These TCRs utilize a different docking strategy to the TCRs of classical MAIT cells and thus may also permit recognition of distinct antigens beyond 5-OP-RU.Koay et al.  reported+ B cell leukemia and lymphoma [CAR-T has been proven to be therapeutically effective in patients with CD19lymphoma . Based olymphoma . Howeverlymphoma . Second,TCR-T cell TCR chains have an antigen-binding region, an extracellular constant region, and a transmembrane domain. The TCR chain transduces an intrinsic signal, which is dependent on T-cell accessory signaling molecules to induce T-cell activation. TCR-T cell immunotherapy presents some advantages over CAR-T cell immunotherapy. First, TCR-T cell immunotherapy clinical trials indicate that this approach is more efficacious against solid tumors. Second, TCRs can recognize peptide epitopes derived from any subcellular  compartment protein . Third, The CAR-T and TCR-T cell immunotherapies also have some limitations . TCRs caMR1-T cells can target many kinds of cancer cells but not healthy cells, using a single TCR clone . Unlike in vitro [in vitro) and the mouse breast tumor cell line EMT6 (in vivo). Thus, the MR1 can present different antigens and be recognized by different TCR clones. In addition, various T cell clones show differential transcriptomes and are functionally heterogeneous after stimulation [MR1T cells (clone DGB129) can recognize human leukemia (CCRF-SB and THP1) and melanoma A375-MR1) cancer cell lines through MR1 presentation 5-MR1 canin vitro. This clone does not kill normal tissues or cells such as lung fibroblasts, hepatocytes, and alveolar, intestinal, renal, pancreatic, melanocyte, smooth muscle, bronchial, dendritic, and Langerhans cells. Moreover, this clone is safe in vitro when tested with whole peripheral blood mononuclear cells (PBMCs) and resting or activated purified T and B cells of healthy donors, and in normal renal epithelial cells under tert-butyl hydroperoxide (tBHP) and H2O2 stress or γ irradiation. Even healthy lung epithelial cells are safe with Mycobacterium smegmatis infection. This MR1-T cell (clone MC.7.G5) could kill Jurkat cells in NSG mice and prolong survival. Two metastatic melanoma patient-derived T cells transduced with the TCR of MC.7.G5 can recognize autologous and non-autologous melanomas, but not primary healthy melanocytes or pancreatic and lung cells. Thus, these MR1-T cells may be utilized as a universal tool to safely target cancer cells.Another MR1-T cell (clone MC.7.G5)  was idenA well-studied and functional MR1-T cell clone can be utilized in humans without HLA-restriction. MR1 in mammals is highly conserved . MR1 is MR1-T cell immunotherapy can be safe for clinical use. MR1-T cells utilize the TCR structure to find tumors and activate an intrinsic signal similar to that of TCR-T cell immunotherapy. The intrinsic signal organizes antigen receptor signaling to amplify and coordinate changes in T cell function and induces cytokine secretion . Unlike MR1-T cells have no restrictions in target finding, and new TCR clones can easily be established. The CAR targeting structures are based on single-chain variable fragment (scFv), and its ligand primarily recognizes cell surface antigens  in an HLA-independent manner. Only ∼27% of the human proteome is represented on the surface of cells and recognized by CAR. The TCR recognizes epitopes derived from the entire proteome, although it still needs to be presented via the HLA. This restricts the utility of TCR-T cell immunotherapy, as presentation peptides continue to change with HLA types. Due to proteome complexity, cancer heterogeneity, gene mutation, epigenetic modification, and oncogene fusion, presentation peptides are too short (8–11 amino acids) to be considered unique. These antigen presentation and recognition problems of CAR-T and TCR-T cells could be easily resolved by MR1-T cells . BecauseBased on the above advantages, allogeneic MR1-T cells could be easily manufactured and utilized off-the-shelf . off-theThe therapeutic MR1-restricted TCR α and β transfer to T cells could undergo mispairing with the original TCR and generate heterodimers. These heterodimers do not undergo thymic selection and thus could be recognized as normal antigens and result in severe GVHD. This could be resolved by knocking out the original TCR α or β chains and expressing only therapeutic TCR . Crowthein vitro functional assay and in vivo experiments verifying the effectivity and safety of MR1 function are limited. There is no clinical trial of the MR1-T cell for now. More fundamental studies on its effectiveness and safety are warranted before this approach can be used in clinical trials.Although the use of MR1-T cells as a novel immunotherapy strategy for cancer has several advantages, several issues need to be resolved. First, the MR1 antigen presentation mechanism remains largely unknown, including the metabolism products that MR1 presents from the tumor metabolome and what makes it unique compared with other approaches. Second, only a few MR1-restricted TCRs have been associated with tumors to date. Different TCRs have different transcriptomes and functions , which ain vitro and kill AML cells from patients’ bone marrow ex vivo. However, the effectiveness and safety of these TCRs have yet to be assessed. MR1-T cells have the therapeutic potential but require further investigations.We recently identified a novel TCR clone of MR1-T cells that can kill AML cell lines (K562 and THP1)


The finding of oxygen-reconstituted active species of SA-Cu/NG at alkaline media successfully unveils the bottleneck puzzle of why the performance of ORR catalysts at alkaline solution is better than that at acidic media, which provides new physical insight into the development of new ORR catalysts.Identification of an active center of catalysts under realistic working conditions of oxygen reduction reaction (ORR) still remains a great challenge and unclear. Herein, we synthesize the Cu single atom embedded on nitrogen-doped graphene-like matrix electrocatalyst (abbreviated as SA-Cu/NG). The results show that SA-Cu/NG possesses a higher ORR capability than 20% Pt/C at alkaline solution while the inferior activity to 20% Pt/C at acidic medium. Based on the experiment and simulation calculation, we identify the atomic structure of Cu-N When energy consumption is rapidly rising, the resulting environmental issues have become serious . Therefo2 in enzymes and the protein lactase, increasing investigations indicate that the single-atom Cu catalysts (Cu-SACs) can also serve as excellent ORR catalysts. For example, Cu-N-C moieties can become highly efficient active sites by adjusting the valance state of Cu atom toward Cu(II) or Cu(I) by engineering coordination environment of Cu-N-C moieties , 382+2p32O, and CuO as counterparts. The valence of Cu in SA-Cu/NG is found between Cu(I) and Cu(II), because the XANES curve for SA-Cu/NG shifts to the higher energy than the Cu foil and Cu2O, while slightly lower energy than standard CuO  bonding, and no Cu-Cu bond is discovered. Impressively, compared with the CuPc standard sample, SA-Cu/NG shows a slightly shift position in maximum intensity, indicating that atomic Cu in the SA-Cu/NG sample coordinates not only with nitrogen but also with the adjacent carbon. However, this phenomenon cannot be analyzed from R space of Cu EXAFS experimentally.The Cu K-edge XANES and the EXAFS of SA-Cu/NG are also used to explore the local structure of SA-Cu/NG with the standard Cu foil, Cudard CuO . To calcdard CuO , where t and CuO  exhibits2 g−1) and excellent pore property (1.97 cm3 g−1)  of 0.8 V (versus RHE), indicating that the N-doped carbon can catalyze ORR but not highly efficient. Impressively, when the small quantity of Cu is introduced into the NG sample, a leap increase of ORR activity is observed for the SA-Cu/NG sample, which indicates that the SA-Cu/NG with mixed valence is highly efficient for ORR. E1/2 of 0.856 V (versus RHE) is obtained for SA-Cu/NG, more excellent than 20% Pt . The excellent activity for SA-Cu/NG is comparable with the majority previously published for nonnoble metal electrocatalysts [Jk) of 6.84 mA/cm2 at 0.85 V (versus RHE), which is 11.4 times of NG (0.6 mA/cm2) and 1.4-folds of 20% Pt/C (5 mA/cm2), respectively  further verifies the more outstanding activity of SA-Cu/NG to 20% Pt/C (92.6 mV decade−1) (n) is obtained. The SA-Cu/NG is 3.95 at 0.7 V (versus RHE), better than NG  and comparable to commercial Pt/C catalyst. This phenomenon indicates the outstanding 4e− selectivity of SA-Cu/NG in 0.1 M KOH. Moreover, 2− yields for SA-Cu/NG are not more than 5.3% within 0.2-0.8 V in 0.1 M KOH, which is extremely lower than the NG sample (<19%). Similarly, n for SA-Cu/NG from RRDE measurement is 3.89-3.96 in the scope of the test  . The selt Figure , in agreApart from the ORR performance, the long-term durability and methanol resistance ability of the catalysts are another pivotal concern for the actual applications for corresponding devices. As expected, the retention of current density of SA-Cu/NG electrodes reaches nearly 92% after the continuous 40000 s testing, apparently more stable than 20% Pt/C of 81.8% Figure . Moreove2 and 100 mA/cm2 compared to that of the 20% Pt/C counterpart  of Cu atom is around four, possibly including Cu-N/Cu-C/Cu-O bonds. To precisely identify the active species, we constructed 32 types of all possible structures of Cu‐NxCyOz, as shown in Figure 2O) with known oxidation states  (marked as Cu-N2C2 in the following content) possesses the highest ORR activity, and the onset potential (UonsetRHE) of all the tested active sites of SA-Cu/NG is apparently inferior to the onset potential 0.80 V of Pt(111). The inferior theoretical activity of all possible active sites of SA-Cu/NG to Pt(111) is entirely contradictory with the experimental results that SA-Cu/NG possesses superior ORR activity to Pt/C  were detected by in situ techniques, CHE model, and microkinetic analysis [in situ generated oxygen adsorbate species may lead to reconstitutions of active sites of SA-Cu/NG and therefore enhance the ORR activity under working potential. Therefore, we systematically calculate preadsorption energies of O∗, OH∗, or OOH∗ intermediates on the catalytic center of SA-Cu/NG under different electrode potentials and generate the surface Pourbaix diagrams . There is another reaction mechanism for ORR, namely, dissociative pathway, where O2 is adsorbed and then dissociated into two O∗ species on the Cu atom. However, the dissociation energy barrier of O2 on SA-Cu/NG is larger than 1 eV  (0.48 eV) [E1/2 of oxygen-reconstituted Cu-N2C2-O structure reaches ∼0.89 V versus RHE and outperforms Pt(111) by ∼50 mV (Jk is larger than that of Pt(111). All theoretical outcomes show qualitatively reasonable consistency with experimental measurements, i.e., the oxygen-reconstituted structure (Cu-N2C2-O) rather than the virgin Cu-N2C2 configuration is the real active center of alkaline ORR under working potential.Previous research has demonstrated that, under the various electrocatalytic working conditions, the adsorbate might be adsorbed at the active center and the modified surface structures would significantly boost the activity of catalysts. For example, some fresh catalytic centers of SACs  . So, we y ∼50 mV . Moreove2C2-O active center. And O2 reacts with water on the other side of graphene through a series of proton-coupled electron transfer, finally resulting in the release of OH−. Actually, the adsorption or reaction process on both sides of SA-Cu/NG is rational, as confirmed in other SACs by previously reported in situ EXAFS [2C2 to Cu-N2C2-O derives from the increased affinity of Cu atom for intermediates. Compared with Cu-N2C2, Cu atom of Cu-N2C2-O breaks up one Cu-C bond to form a three-coordination configuration when binding either OOH∗ or OH∗. And the adsorption strength becomes stronger making the process of forming OOH∗ and OH∗ much more exothermic, which yields a higher onset potential. ∗ and OH∗ on Cu-N2C2-O relative to Cu-N2C2, it is valuable to probe the d-orbital Cu site contributing to ORR. With the upward shifting of d-band of Cu atom from Cu-N2C2 to Cu-N2C2-O  that is about 320 mV inferior to Pt/C in 0.5 M H2SO4 solution. The rather sluggishness performance of SA-Cu/NG for ORR under acidic condition is in line with the inferior theoretical half-wave potential of Cu-N2C2 to Pt(111) in 2C2) rather than the oxygen-reconstituted active species (Cu-N2C2-O) of SA-Cu/NG are responsible for the inferior activity of SA-Cu/NG at acidic medium. In short, the finding of oxygen-reconstituted active species of SA-Cu/NG at alkaline media can successfully unveil the bottleneck puzzle of why the performance of ORR catalysts under alkaline condition is better than that under acidic condition.ORR performance at acidic condition for SA-Cu/NG is further evaluated. As seen in Figure 2C2 configuration, and SA-Cu/NG shows the superior ORR ability to Pt/C under alkaline medium while inferior activity to 20% Pt/C at acidic condition. Together with experimental data and DFT theoretical analysis, we for the first time found that the oxygen-reconstituted Cu-N2C2-O structure under working condition is the realistic ORR active species at alkaline conditions, while the virgin Cu-N2C2 structure is the ORR active moiety at acidic media. The theoretical half-wave potentials of both oxygen-reconstituted Cu-N2C2-O structure and virgin Cu-N2C2 structure are also in perfect agreement with experimental results at alkaline and acidic media, respectively. Adsorption of atomic oxygen intermediate O∗ on the Cu-C site yielded the reconstituted structure, and such O∗ adsorption optimizes the electronic structure of Cu atom by breaking one Cu-C bond during binding of OH∗ and OOH∗ reaction intermediates and therefore boosts ORR activity. The finding of reconstitution of surface active sites unveils the bottleneck puzzle of why the performance of ORR catalysts under alkaline electrolyte is better than that under acidic condition, which also offers a fresh perspective to advisably fabricate high-performance ORR electrocatalysts.In summary, we have synthesized a novel single-atom Cu catalyst (SA-Cu/NG) with Cu-NExperimental details including synthesis of samples, structure, and electrochemical characterizations of SA-Cu/NG and other samples and performance of SA-Cu/NG-based Zn-air batteries are presented in Supplementary Materials. All computational details, including the DFT calculation process, screening of all possible structures of Cu single-atom catalysts, free energy diagram of Cu-SAC, and surface-reconstituted Cu-SACs and surface Pourbaix diagrams of Cu-SACs, are also described in Supplementary Materials.


Salmonella Pullorum strains. In this study, the biofilm-forming ability of two Salmonella Pullorum strains S6702 and S11923-3 were compared. The biofilm forming ability of S11923-3 was much stronger than that of S6702. After knocking out the rpoS gene, S11923-3ΔrpoS had significantly reduced biofilm while S6702ΔrpoS demonstrated similar biofilm compared with each parent strain. The analysis of RpoS sequences indicated two amino acid substitutions (L193P and R293C) between S6702 and S11923-3 RpoS. A complementation study confirmed that the expression of S11923-3 RpoS rather than S6702 RpoS could restore the biofilm-forming ability of ΔrpoS strains and the L193P mutation contributed to the restoration of the biofilm-forming ability. Further study indicated that RpoS with the L193P mutant had significantly improved expression level and binding activity to RNAP and csgD gene promoter, which increased the efficacy of the csgD gene promoter and biofilm-forming ability. Therefore, the L193P mutation of RpoS is critical for stronger biofilm formation of Salmonella Pullorum.The role of alternative sigma factor RpoS in regulating biofilm formation may differ in various  S/38 (RpoS), as an alternative sigma factor, is a central regulator enabling many Gram-negative bacteria to adapt to stress conditions and specialized environments  subunits of RNA polymerase (RNAP) that consists of five principal subunits  to directly initiate transcription of different classes of promoters is a major strategy employed by bacteria to modify the expression of genes , 2. The ronments . RpoS caronments .Salmonella biofilm-forming pathway, CsgD controlled by RpoS regulates the expression of the curli fimbriae csgBAC operon and putative transmembrane protein AdrA. AdrA is involved in regulating the production of cellulose by activating basABZC-bcsEFG operons  in distilled water was used for the biofilm assays.The bacterial strains and plasmids used in this study are listed in um S6702  and S119rpoS genes of S. Pullorum strains S6702 and S11923-3 were deleted using lambda red-mediated mutagenesis procedures  based on the pGEX-6P-1-7S and pGEX-6P-1-9S plasmids. The pGEX-6P-1-p7 and pGEX-6P-1-p9 plasmids containing RpoS promotor without Tac promotor were constructed using a ClonExpress II One Step Cloning Kit (Vazyme) with primers listed in S. Pullorum S6702ΔrpoS and S11923-3ΔrpoS by electroporation to produce complementary strains S6702ΔrpoSR7, S6702 ΔrpoSR7B, S6702ΔrpoSR9, S6702ΔrpoSR9B, S6702ΔrpoSRp7, S6702ΔrpoSRp9, S11923-3ΔrpoSR7, S11923-3ΔrpoSR7B, S11923-3ΔrpoSR9, S11923-3ΔrpoSR9B, S11923-3ΔrpoSRp7, and S11923-3ΔrpoSRp9.To construct the complementary strains, the rpoS substitution strains, the rpoS genes with residues 193/293 substitution were amplified using primer pair rpoS-F1/R1 , and named as S6702-9rpoS, S6702-9rpoSB, S11923-3-7rpoS and S11923-3-7rpoSB  was measured in Microplate Reader (Bio-Rad). The assays were conducted three times using duplicate wells in each independent assay.Biofilm formation ability was measured as previously described , 17. OveCurli fimbriae and cellulose production was evaluated by colony morphology in Congo red plates with 40 μg/mL of Congo red (Sangon Biotech) and 20 μg/mL of Coomassie brilliant blue (Sangon Biotech) and LB agar plates with 200 μg/mL of calcofluor (Sigma-Aldrich) after the inoculation plates were incubated at 28°C for 4 days . The colThe biofilm morphology was also determined using scanning electron microscopy as previously described . PolystygyrB, csgD, csgA, and bcsA, respectively. The target genes' mRNA levels were normalized to the gyrB mRNA levels (2−ΔΔCt) (Bacteria were grown in 1/10 TSB medium at 28°C for 24 h in 60 mm dishes (Corning). The supernatant was discarded and the bacteria accumulated in the biofilms under the dishes were scraped. The total RNA was extracted using a Bacterial RNA Kit (Omega). The cDNA was synthesized using a PrimeScript RT reagent Kit with gDNA Eraser (Takara) and quantified via TB Green Premix Ex Taq (Takara). The gene transcript levels were tested in triplicate for real-time PCR in a Linegene 9600 Plus machine (Bioer). Primer pairs of Q-gyrB-F/R, Q-csgD-F/R, Q-csgA-F/R, and Q-bcsA-F/R  were use(2−ΔΔCt) –21.600 = 1.0, then suspended in SDS sample buffer (Beyotime Biotechnology). The samples were resolved in 12% SDS polyacrylamide gels, transferred to PVDF membranes (Millipore), and analyzed by immunoblotting using anti-RpoS (NeoClone) and anti-RpoA (NeoClone) antibodies. Bands were developed using anti-mouse-HRP (Abcam) and an ECL detection system (Tanon).Bacteria were grown in 1/10 TSB in 60 mm dishes at 28°C for 8 and 24 h to test the protein expression. After the dishes were washed with PBS, the bacterial cells were collected and quantified to ODrpoS genes were amplified using rpoS-yF/yR as primers and chromosomal DNA from the S6702 and S11923-3 as templates. The PCR products were inserted between the BamHI and XhoI sites of the pET32a, which were called pET32a-7S and pET32a-9S, respectively. The plasmids were transformed to Escherichia coli BL21 (DE3) for the overexpression and purification of recombinant RpoS. Bacterial cells were grown at 37°C until OD600 of 0.4–0.6 and 1 mM of IPTG was added. The cells were then grown at 16°C for 12 h and harvested by centrifugation at 5,000 g for 10 min. His-tagged proteins were purified by incubation with Ni-NTA resin (Genscript). The resin was washed with at least 8 column volumes (CV) of wash buffer . The protein was eluted with elution buffer  in 1 CV fractions. The fractions were analyzed by SDS-PAGE and the purified RpoS was prepared for binding assays and electrophoretic mobility shift assays (EMSAs).The RNAP-clarified lysates were prepared via the following steps: 50 mL of each bacterial culture incubated in TSB at 37°C overnight was pelleted at 5,000 g for 10 min. The pellets were resuspended in 8 mL pull-down buffer  and then sonicated on ice for up to 15 min at 1 min intervals. Cell debris was cleared by centrifugation at 14,000 rpm for 30 min at 4°C, and the supernatant was passed through a 0.45 μm filter. The clarified lysate protein concentration was measured by Bradford assays (Thermo Fisher Scientific).His-tag Dynabeads  were incubated with 700 μL of purified RpoS protein (2.0 mg/mL) and rotated at 25 rpm for at least 20 min at room temperature. The beads were washed according to the manufacturer's protocols with 300 μL wash buffer . Then the clarified lysates were incubated with the beads for 15 min and washed four times. The samples were then eluted from the beads by incubation in 100 μL of elution buffer . The samples were resolved in SDS-PAGE, transferred onto PVDF membranes (Millipore), and probed with primary anti-RpoS, anti-RpoA, and anti-RpoB antibodies , 23 and csgD gene promoter including 237 nucleotides in the open reading frame was amplified from S11923-3 and purified with an AxyPrep DNA Gel Extraction Kit (Axygen), and the purified products were PCR labeled using FAM-modified primer. The binding reaction was conducted with non-specific competitor DNA  in buffer (pH 7.4) containing 750 mM of NaCl, 0.5 mM of DTT, 0.5 mM of EDTA, and 50 mM of Tris at 25°C for 30 min. A total of 20 μL of each reaction system contained 4 μL 5 × binding buffer (Beyotime Biotechnology) and 200 ng of poly dI-dC (Sigma-Aldrich). The final mixtures including DNA fragments and RpoS protein were run on a 6% SDS-PAGE. Then the images were scanned and observed using a fluorescence imaging system .EMSAs were conducted as previously described . BrieflycsgD genes from strains S6702 and S11923-3 were amplified using primer pair csgD-F/R and the PCR products were inserted into pUC18 with KpnI and BamHI digestion to form pcsgD. The lacZ gene from plasmid pSV-β-gal was amplified using primer pair lacZ-F/R and the PCR products were inserted into pcsgD with BamHI and PstI digestion to form pcsgD-lacZ, in which the lacZ gene was downstream from the csgD gene promoter. The pcsgD-lacZ was transformed into S6702, S11923-3, and their rpoS deletion mutants. The bacteria harboring the plasmids were cultured in dishes at 28°C for 24 and 48 h without shaking. After collecting and measuring the absorbance at 600 nm, the samples were mixed with reaction buffer  and stop buffer (1 M of Na2CO3). The absorbance was measured at 420 nm. The β-galactosidase activity (Miller units) was calculated as previously described . All of the statistical analyses were assessed using the two-tailed S. Pullorum, we constructed rpoS gene deletion mutants S6702ΔrpoS and S11923-3ΔrpoS. The biofilm-forming ability of strains S6702 and S11923-3 and their rpoS deletion mutants were determined. Crystal violet staining of bacterial strains on polystyrene plates showed that wild-type (WT) strain S6702 and its mutant strain S6702ΔrpoS had similar circle staining in the plate well walls  after incubation at 28°C for 4 days, while the S11923-3Δh colony . Howeverrescence .rpoS and S11923-3ΔrpoS had a small number of bacterial cells and tiny fiber structures surrounding the bacterial surface  or Cys (5/14) in RpoS residue 293.To investigate the differences in RpoS in the two strains, we compared the amino acid sequences of RpoS in strains S6702 and S11923-3. Only two amino acid substitutions of L193P and R293C were found in the S6702 and S11923-3 RpoS . To deteanalyzed . All of S. Pullorum strains were subjected to polystyrene plate culture and crystal violet staining  were significantly increased when compared with that of S6702, while OD550 values of S6702-9rpoSB  were significantly decreased when compared with that of S6702-9rpoS  were significantly decreased when compared with that of S11923-3, while OD550 values of S11923-3-7rpoSB  were significantly increased when compared with that of S11923-3-7rpoS. These data indicated that RpoS residue 193 rather than residue 293 facilitated the biofilm-forming ability in these two strains.Biofilm formation of the substitution strains were also determined by crystal violet staining test, the OD02-9rpoS . In contS. Pullorum strains, six strains were selected for determination of RpoS expression. Samples were collected after 8 h and 24 h of incubation and subjected to immunoblotting analysis. As shown in S. Pullorum.To determine the RpoS expression among different rpoS gene expression, the pGEX-6p-1-p7 and pGEX-6p-1-p9 plasmids with the substitution of Tac promoter by RpoS promoter of S6702 and S11923-3 were constructed and then transformed into S6702ΔrpoS and S11923-3ΔrpoS. The immunoblotting results showed that complement of S11923-3 RpoS had higher expression level than that of S6702 RpoS either in the mutant strains S6702ΔrpoS or S11923-3ΔrpoS  and clarified lysates from mutant strains lacking RpoS was conducted. The RNAP pulled down by His-RpoS from the lysate was confirmed by immunoblotting analyses with anti-RpoS, anti-RpoA, and anti-RpoB antibodies . When thSalmonella and its expression is controlled by RpoS  to form holoenzyme Eσ. Eσ enables specific binding to gene promoters and is required for transcription initiation . S. Typhtor CsgD . Howevertor CsgD . Furthereritidis . In a prtor RpoS . In thisS. Pullorum. It has been shown that accumulation of RpoS coincided with the expression of curli fimbriae and other morphological and physiological changes  or two amino acid substitutions (L193P and R293C) were found. Then complementary strains and substitution strains with combinational substitution of residues 193 and 293 were constructed and their biofilm formation abilities were determined by crystal violet staining . The res changes . In this changes . AccordiE. coli is well-studied  can open the secondary structure, promoting translation (rpoS gene promoter sequences between strains S6702 and S11923-3 (data not shown) and the transcript level of S11923-3 was higher than that of S6702  isolates. Substitution of Ile128 with Pro128 in E. coli abolishes RpoS activity including response to oxidative stresses and ability to bind RNAP , which removed the impact of the csgD promoter. Overall, the results of RpoS activities indicated that S11923-3 RpoS (193P) had stronger effects on biofilm formation in S. Pullorum by enhancing the affinity to RNAP and activating csgD promotor.RpoS combines with RNA polymerase (RNAP) to regulate many genes by binding to gene promotors and initiating transcription that enables bacteria to survive adverse conditions , 38. Resind RNAP . Our stuind RNAP . Amino aind RNAP . Thus, w702 RpoS . In the 702 RpoS . In addicsgBA promoter expression in Salmonella  in RpoS that can enhance the RpoS expression level, binding activity to RNAP and expression activity of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ZF and DP conceived this study. ZF and MH conducted the research. ZF, TQ, and DP analyzed the data. ZF, YD, SC, and DP wrote the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ongoing pandemic of SARS-CoV-2 has brought tremendous crisis on global health care systems and industrial operations that dramatically affect the economic and social life of numerous individuals worldwide. Understanding anti-SARS-CoV-2 immune responses in population with different genetic backgrounds and tracking the viral evolution are crucial for successful vaccine design. In this study, we reported the generation of CD8 T cell epitopes by a total of 80 alleles of three major class I HLAs using NetMHC 4.0 algorithm for the SARS-CoV-2 spike protein, which can be targeted by both B cells and T cells. We found diverse capacities of S protein specific epitope presentation by different HLA alleles with very limited number of predicted epitopes for HLA-B*2705, HLA-B*4402 and HLA-B*4403 and as high as 132 epitopes for HLA-A*6601. Our analysis of 1000 S protein sequences from field isolates collected globally over the past few months identified three recurrent point mutations including L5F, D614G and G1124V. Differential effects of these mutations on CD8 T cell epitope generation by corresponding HLA alleles were observed. Finally, our multiple alignment analysis indicated the absence of seasonal CoV induced cross-reactive CD8 T cells to drive these mutations. Our findings suggested that individuals with certain HLA alleles, such as B*44 are more prone to SARS-CoV-2 infection. Studying anti-S protein specific CD8 T cell immunity in diverse genetic background is critical for better control and prevention of the SARS-CoV-2 pandemic. The coronavirus (CoV) is an enveloped, positive-stranded RNA virus that can cause respiratory and enteric diseases in wide range of hosts including human, numerous animals, birds and fish . Four geth, 2020. Like SARS-CoV and MERS-CoV, SARS-CoV-2 was identified as betacoronavirus through genome sequencing and bioinformatic analysis [In December 2019, a novel coronavirus disease (COVID-19), caused by 2019 novel coronavirus (2019-nCoV) and later renamed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified in Wuhan City, Hubei Province, China from patients with severe pneumonia –7. Subseanalysis , 7 and hThe S protein of SARS-CoV contains neutralization epitopes, which are critical for eliciting anti-viral B cell immune responses. Therefore, S protein appears to be a promising antigen target for vaccine development , 10. SimIn this study, we used the NetMHC 4.0 prediction algorithm to analyze a panel of classical human MHC I  restricted CD8 epitopes on the S protein of SARS-CoV-2. Our data showed a significant variation of CD8 T cell epitope repertoires for different HLA-A, HLA-B and HLA-C alleles. In addition, our analysis using a large number of S protein sequences derived from SARS-CoV-2 genomes revealed important mutations including L5F, D614G and G1124V that are although less likely driven by seasonal CoV derived cross-reactive CD8 T cells, may differentially impact CD8 T cell epitope generation by different HLA alleles. Our results indicate genetic variability of three major class I HLAs may regulate S protein specific CD8 T cell responses, leading to differential susceptibility to and severity of the infection of SARS-CoV-2 that keeps mutating.Full-length S protein amino acid sequence (accession number QHD43416.1) of the reference SARS-CoV-2 Wuhan-Hu-1 isolate (accession number MN908947) was downloaded in FASTA format from the NCBI GenBank.CD8 T cell epitope prediction was performed on NetMHC4.0 Web Server that utilizes artificial neural networks (ANNs) , 24. A tTo identify mutations on SARS-CoV-2 S protein, we selected a total of 1000 complete or near-complete S protein sequences of viruses isolated from countries in North America, Europe, Asia, Oceania, Africa and South America. These sequences were deposited in the NCBI database with collection dates ranging from January to June 2020 and showed at least 95% of query coverage for the reference S protein (accession number QHD43416.1). The list of the accession numbers for these sequences was provided in The S protein sequences of four common seasonal CoVs: HCoV-229E (AAG48592.1), HCoV-OC43 (CAA83661.1), HCoV-NL63 (APF29063.1), and HCoV-HKU1 (BBA20983.1) were used for comparison with the reference S protein of SARS-CoV 2 by multiple alignment in MEGA-X using MUSCLE under default conditions. Phylogenetic tree analysis of the S protein of SARS-CoV-2 and four seasonal human CoVs was performed using the maximum likelihood method and JTT model with a bootstrap of 1000 replicates within the Mega-X software.One-way analysis of variance (ANOVA) and Tukey’s multiple comparison test were used for assessment of the means of predicted CD8 T cell epitopes presented by three HLA allele groups. Statistical analysis was performed using GraphPad Prism version 5.0 . A p-value <0.05 was considered statistically significant.The NetMHC 4.0 algorithm has been widely utilized for predicting CD8 T cell epitopes of human and animal pathogens. By using this tool, our analysis for the S protein of reference Wuhan-Hu-1 virus generated a total of 5044 CD8 T cell epitope pool for three major class I HLAs with a total of 80 different alleles . Among tSARS-CoV-2 has evolved since its initial outbreak by changing its viral genome, which may cause mutations in viral proteins including S protein, resulting in the establishment of better fitness , 26. We The amino acid L on position 5 is part of the S protein signal peptide . Signal The amino acid G on position 1124 is within the connector domain of the S protein  that is The mutation at position 614 with a sole D to G switch at a 63% frequency is especially alarming. The result revealed that SARS-Cov-2 isolates with G614 mutation has been adapted and spread efficiently within human population. To our knowledge, the D614 amino acid is not within the essential receptor binding domain or a residue of any validated neutralization epitopes for SARS-CoV-2. In addition, whether it is involved in CD8 T cell response is not known. Through screening the epitopes listed on the Similarly, we reanalyzed the 9-mer CD8 T cell epitope for the reference S protein with a single D614G mutation. The resulting epitopes of each allele were compared to the corresponding epitopes derived from the original reference S protein . The comAlthough we identified major mutations on CD8 T cell epitopes including L5F, D614G and G1124V for the SARS-CoV-2 S protein, the detailed mechanisms driving these mutations are not known. One possibility is that existing cross-reactive CD8 T cell immunity elicited by human seasonal CoVs could promote the virus to mutate and escape immune recognition in general population that are frequently targeted by seasonal CoVs. To test this, we went ahead to evaluate if there are shared CD8 T cell epitopes containing L5, D614 and G1124 from four representative seasonal CoVs [CD8 T cell response specific for S protein of SARS-CoV has been well characterized, yet most of the data has primarily focused on HLA-A*0201 , 36–39. In this study, we intended to provide a broad representation of S protein specific CD8 T cell epitopes of 80 different human HLAs for better understanding anti-SARS-CoV2 T cell immunity. Our results indicated a potential differential anti-S protein CD8 T cell response during COVID-19 infection in individuals with unique HLA alleles as the capacity of these alleles to present potential epitopes varies dramatically . Among tIn addition to HLA-B*2705, B44 alleles also had very low number of epitopes Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.