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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages BA.2.12.1,BA.4 and BA.5 exhibit higher transmissibility than the BA.2 lineage1. The receptor binding and immune-evasion capability of these recently emerged variants require immediate investigation. Here,coupled with structural comparisons of the spike proteins,we show that BA.2.12.1,BA.4 and BA.5 (BA.4 and BA.5 are hereafter referred collectively to as BA.4/BA.5) exhibit similar binding affinities to BA.2 for the angiotensin-converting enzyme 2 (ACE2) receptor. Of note,BA.2.12.1 and BA.4/BA.5 display increased evasion of neutralizing antibodies compared with BA.2 against plasma from triple-vaccinated individuals or from individuals who developed a BA.1 infection after vaccination. To delineate the underlying antibody-evasion mechanism,we determined the escape mutation profiles2,epitope distribution3 and Omicron-neutralization efficiency of 1,640 neutralizing antibodies directed against the receptor-binding domain of the viral spike protein,including 614 antibodies isolated from people who had recovered from BA.1 infection. BA.1 infection after vaccination predominantly recalls humoral immune memory directed against ancestral (hereafter referred to as wild-type (WT)) SARS-CoV-2 spike protein. The resulting elicited antibodies could neutralize both WT SARS-CoV-2 and BA.1 and are enriched on epitopes on spike that do not bind ACE2. However,most of these cross-reactive neutralizing antibodies are evaded by spike mutants L452Q,L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1. Nevertheless,these neutralizing antibodies are largely evaded by BA.2 and BA.4/BA.5 owing to D405N and F486V mutations,and react weakly to pre-Omicron variants,exhibiting narrow neutralization breadths. The therapeutic neutralizing antibodies bebtelovimab4 and cilgavimab5 can effectively neutralize BA.2.12.1 and BA.4/BA.5,whereas the S371F,D405N and R408S mutations undermine most broadly sarbecovirus-neutralizing antibodies. Together,our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection,suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants. View Publication

Notoginsenoside R1 (NGR1) is the main monomeric component extracted from the dried roots and rhizomes of Panax notoginseng,and exerts pharmacological action against myocardial infarction (MI). Owing to the differences in compound distribution,absorption,and metabolism in vivo,exploring a more effective drug delivery system with a high therapeutic targeting effect is crucial. In the early stages of MI,CD11b-expressing monocytes and neutrophils accumulate at infarct sites. Thus,we designed a mesoporous silica nanoparticle-conjugated CD11b antibody with loaded NGR1 (MSN-NGR1-CD11b antibody),which allowed NGR1 precise targeted delivery to the heart in a noninvasively manner. By increasing targeting to the injured myocardium,intravenous injection of MSN-NGR1-CD11b antibody nanoparticle in MI mice improved cardiac function and angiogenesis,reduced cell apoptosis,and regulate macrophage phenotype and inflammatory factors and chemokines. In order to further explore the mechanism of NGR1 protecting myocardium,cell oxidative stress model and oxygen-glucose deprivation (OGD) model were established. NGR1 protected H9C2 cells and primary cardiomyocytes against oxidative injury induced by H2O2 and OGD treatment. Further network pharmacology and molecular docking analyses suggested that the AKT,MAPK and Hippo signaling pathways were involved in the regulation of NGR1 in myocardial protection. Indeed,NGR1 could elevate the levels of p-Akt and p-ERK,and promote the nuclear translocation of YAP. Furthermore,LY294002 (AKT inhibitor),U0126 (ERK1/2 inhibitor) and Verteporfin (YAP inhibitor) administration in H9C2 cells indicated the involvement of AKT,MAPK and Hippo signaling pathways in NGR1 effects. Meanwhile,MSN-NGR1-CD11b antibody nanoparticles enhanced the activation of AKT and MAPK signaling pathways and the nuclear translocation of YAP at the infarcted site. Our research demonstrated that MSN-NGR1-CD11b antibody nanoparticle injection after MI enhanced the targeting of NGR1 to the infarcted myocardium and improved cardiac function. More importantly,our pioneering research provides a new strategy for targeting drug delivery systems to the ischemic niche. View Publication

To date,driver genes for pancreatic cancer treatment are difficult to pursue therapeutically. Targeting mutated KRAS,the most renowned driver gene in pancreatic cancer,is an active area of study. We discovered a gene named SEMA3C was highly expressed in pancreatic cancer cell lines and patients with a G12D mutation in KRAS. High expression of SEMA3C in patients was significantly associated with the decreased survival of pancreatic cancer patients based on the TCGA database. In pancreatic cancer cells,SEMA3C knockdown or inhibition exhibited growth/colony inhibition and cell cycle arrest. In addition,SEMA3C inhibition sensitized KRAS or MEK1/2 inhibition in pancreatic cancer cells. Overexpression of SEMA3C resulted in the induction of autophagy,whereas depletion of SEMA3C compromised induction of autophagy. SEMA3C modified the PD-L1 expression in tumor and immune cells and is correlated with the M2-like macrophage marker ARG1/CD163 expression,which could reshape the tumor microenvironment. Inhibition of SEMA3C decreased tumor formation in the xenograft model in vivo. Taken together,our data suggest that SEMA3C plays a substantial role in promoting cancer cell survival by regulating the autophagy process and impacting the tumor environment immune response. SEMA3C can be used as a novel target or marker with therapeutic or diagnostic potential in pancreatic cancer especially in tumors harboring the specific KRAS G12D mutation. View Publication

BACKGROUND Osteoclasts play a crucial role in the maintenance,repair,and remodeling of bones of the adult vertebral skeleton due to their bone resorption capability. Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are associated with increased activity of osteoclasts. OBJECTIVES Our study aimed to investigate the dynamic proteomic changes during osteoclast differentiation in healthy donors,in RA,and PsA. METHODS Blood samples of healthy donors,RA,and PsA patients were collected,and monocytes were isolated and differentiated into osteoclasts in vitro using macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor $\kappa$B ligand (RANK-L). Mass spectrometry-based proteomics was used to analyze proteins from cell lysates. The expression changes were analyzed with Gene Set Enrichment Analysis (GSEA). RESULTS The analysis of the proteomic changes revealed that during the differentiation of the human osteoclasts,expression of the proteins involved in metabolic activity,secretory function,and cell polarity is increased; by contrast,signaling pathways involved in the immune functions are downregulated. Interestingly,the differences between cells of healthy donors and RA/PsA patients are most pronounced after the final steps of differentiation to osteoclasts. In addition,both in RA and PsA the differentiation is characterized by decreased metabolic activity,associated with various immune pathway activities; furthermore by accelerated cytokine production in RA. CONCLUSIONS Our results shed light on the characteristic proteomic changes during human osteoclast differentiation and expression differences in RA and PsA,which reveal important pathophysiological insights in both diseases. View Publication

Protein kinase CK2 is a serine/threonine kinase composed of two catalytic subunits (CK2$\alpha$ and/or CK2$\alpha$') and two regulatory subunits (CK2$\beta$). CK2 promotes cancer progression by activating the NF-$\kappa$B,PI3K/AKT/mTOR,and JAK/STAT pathways,and also is critical for immune cell development and function. The potential involvement of CK2 in CD8+ T cell function has not been explored. We demonstrate that CK2 protein levels and kinase activity are enhanced upon mouse CD8+ T cell activation. CK2$\alpha$ deficiency results in impaired CD8+ T cell activation and proliferation upon TCR stimulation. Furthermore,CK2$\alpha$ is involved in CD8+ T cell metabolic reprogramming through regulating the AKT/mTOR pathway. Lastly,using a mouse Listeria monocytogenes infection model,we demonstrate that CK2$\alpha$ is required for CD8+ T cell expansion,maintenance,and effector function in both primary and memory immune responses. Collectively,our study implicates CK2$\alpha$ as an important regulator of mouse CD8+ T cell activation,metabolic reprogramming,and differentiation both in vitro and in vivo. View Publication

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset,which express na{\{i}}ve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of na{\"{i}}ve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy." View Publication

BACKGROUND Glioblastoma (GBM) is the most common and aggressive form of primary brain tumor. Although numerous postoperative therapeutic strategies have already been developed,including radiotherapy,tumors inevitably recur after several years of treatment. The coinhibitory molecule B7-H4 negatively regulates T cell immune responses and promotes immune escape. Exosomes mediate intercellular communication and initiate immune evasion in the tumor microenvironment (TME). OBJECTIVE This study aimed to determine whether B7-H4 is upregulated by radiation and loaded into exosomes,thus contributing to immunosuppression and enhancing tumor growth. METHODS Iodixanol density-gradient centrifugation and flow cytometry were used to verify exosomal B7-H4. Na{\{i}}ve T cells were differentiated into Th1 cells with or without exosomes. T cell-secreted cytokines and markers of T cell subsets were measured. Mechanistically the roles of B7-H4 and ALIX in GBM were analyzed using databases and tissue samples. Co-immunoprecipitation and pull-down assays were used to tested the direct interactions between ATM and ALIX or STAT3. In vitro ATM kinase assays western blotting and site-directed mutation were used to assess ATM-mediated STAT3 phosphorylation. Finally the contribution of exosomal B7-H4 to immunosuppression and tumor growth was investigated in vivo. RESULTS Exosomes from irradiated GBM cells decreased the anti-tumor immune response of T cell in vitro and in vivo via delivered B7-H4. Mechanistically irradiation promoted exosome biogenesis by increasing the ATM-ALIX interaction. Furthermore the ATM-phosphorylated STAT3 was found to directly binds to the B7-H4 promoter to increase its expression. Finally the radiation-induced increase in exosomal B7-H4 induced FoxP3 expression during Th1 cell differentiation via the activated STAT1 pathway. In vivo exosomal B7-H4 decreased the radiation sensitivity of GBM cells and reduced the survival of GBM mice model. CONCLUSION This study showed that radiation-enhanced exosomal B7-H4 promoted immunosuppression and tumor growth hence defining a direct link between irradiation and anti-tumor immune responses. Our results suggest that co-administration of radiotherapy with anti-B7-H4 therapy could improve local tumor control and identify exosomal B7-H4 as a potential tumor biomarker." View Publication

The biological macromolecule Nocardia rubra cell-wall skeleton (Nr-CWS) has well-established immune-stimulating and anti-tumor activities. However,the role of Nr-CWS on natural killer (NK) cells remains unclear. Here,we explore the function and related mechanisms of Nr-CWS on NK cells. Using a tumor-bearing model,we show that Nr-CWS has slightly effect on solid tumor. In addition,using a tumor metastasis model,we show that Nr-CWS suppresses the lung metastasis induced by B16F10 melanoma cells in mice,which indicates that Nr-CWS may up-regulate the function of NK cells. Further investigation demonstrated that Nr-CWS can increase the expression of TRAIL and FasL on spleen NK cells from Nr-CWS treated B16F10 tumor metastasis mice. The spleen index and serum levels of TNF-$\alpha$,IFN-$\gamma$,and IL-2 in B16F10 tumor metastasis mice treated with Nr-CWS were significantly increased. In vitro,the studies using purified or sorted NK cells revealed that Nr-CWS increases the expression of CD69,TRAIL,and FasL,decreases the expression of CD27,and enhances NK cell cytotoxicity. The intracellular expression of IFN-$\gamma$,TNF-$\alpha$,perforin (prf),granzyme-B (GrzB),and secreted TNF-$\alpha$,IFN-$\gamma$,IL-6 of the cultured NK cells were significantly increased after treatment with Nr-CWS. Overall,the findings indicate that Nr-CWS could suppress the lung metastasis induced by B16F10 melanoma cells,which may be exerted through its effect on NK cells by promoting NK cell terminal differentiation (CD27lowCD11bhigh),and up-regulating the production of cytokines and cytotoxic molecules. View Publication

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example,ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However,additional antitumor benefits from targeting the DDR pathways,which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal,inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here,we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However,STING deficiency in tumor cells does not prevent DC activation,suggesting that transactivation of the STING pathway occurs within DCs. Furthermore,depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells,indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary,in this study,we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation,which is enhanced in ATM-deficient cells. View Publication

Although studies have identified the presence of gut-associated cells in the enthesis of joints affected by spondylarthritis (SpA),a direct link through cellular transit between the gut and joint has yet to be formally demonstrated. Using KikGR transgenic mice to label in situ and track cellular trafficking from the distal colon to the joint under inflammatory conditions of both the gut and joint,we demonstrate bona-fide gut-joint trafficking of T cells from the colon epithelium,also called intraepithelial lymphocytes (IELs),to distal sites including joint enthesis,the pathogenic site of SpA. Similar to patients with SpA,colon IELs from the TNF$\Delta$ARE/+ mouse model of inflammatory bowel disease and SpA display heightened TNF production upon stimulation. Using ex vivo stimulation of photo-labeled gut-joint trafficked T cells from the popliteal lymph nodes of KikGR and KikGR TNF$\Delta$ARE/+ we saw that the CD4+ photo-labeled population was highly enriched for IL-17 competence in healthy as well as arthritic mice,however in the TNF$\Delta$ARE/+ mice these cells were additionally enriched for TNF. Using transfer of magnetically isolated IELs from TNF+/+ and TNF$\Delta$ARE/+ donors into Rag1 -/- hosts,we confirmed that IELs can exacerbate inflammatory processes in the joint. Finally,we blocked IEL recruitment to the colon epithelium using broad spectrum antibiotics in TNF$\Delta$ARE/+ mice. Antibiotic-treated mice had reduced gut-joint IEL migration,contained fewer Il-17A and TNF competent CD4+ T cells,and lessened joint pathology compared to untreated littermate controls. Together these results demonstrate that pro-inflammatory colon-derived IELs can exacerbate inflammatory responses in the joint through systemic trafficking,and that interference with this process through gut-targeted approaches has therapeutic potential in SpA. View Publication

While inhibitory Siglec receptors are known to regulate myeloid cells,less is known about their expression and function in lymphocytes subsets. Here we identified Siglec-7 as a glyco-immune checkpoint expressed on non-exhausted effector memory CD8+ T cells that exhibit high functional and metabolic capacities. Seahorse analysis revealed higher basal respiration and glycolysis levels of Siglec-7+ CD8+ T cells in steady state,and particularly upon activation. Siglec-7 polarization into the T cell immune synapse was dependent on sialoglycan interactions in trans and prevented actin polarization and effective T cell responses. Siglec-7 ligands were found to be expressed on both leukemic stem cells and acute myeloid leukemia (AML) cells suggesting the occurrence of glyco-immune checkpoints for Siglec-7+ CD8+ T cells,which were found in patients' peripheral blood and bone marrow. Our findings project Siglec-7 as a glyco-immune checkpoint and therapeutic target for T cell-driven disorders and cancer. View Publication

BACKGROUND Ageing is a complex phenomenon that leads to decreased proliferative activity,loss of function of the cells,and cellular senescence. Senescence of the immune system exacerbates individual's immune response,both humoral and cellular but increases the frequency of infections. We hypothesized that physiological ageing of adaptive immune system occurs in recipients of allogeneic hematopoietic cells transplant (allo-HCT) at faster rate when compared to their respective donors since the small number of donor cells undergo immense proliferative stress restoring recipients hematopoiesis. We compared molecular characterizations of ageing between recipients and donors of allo-HCT: telomeric length and immunophenotypic changes in main lymphocyte subsets - CD4+,CD8+,CD19+,CD56+. RESULTS Median telomeric length (TL) of CD8+ lymphocytes was significantly longer in donors compared to recipients (on average 2,1 kb and 1,7 kb respectively,p??=??0,02). Similar trends were observed for CD4+ and CD19+ although the results did not reach statistical significance. We have also found trends in the immunophenotype between recipients and donors in the subpopulations of CD4+ (na{\{i}}ve and effector memory) CD8+ Eomes+ and B-lymphocytes (B1 and B2). Lower infection risk recipients had also a significantly greater percentage of NK cells (22 3%) than high-risk patients (9 3%) p??=??0 04. CONCLUSION Our data do not support the initial hypothesis of accelerated aging in the long term all-HCT recipients with the exception of the recipients lymphocytes (mainly CD8+) which present some molecular features characteristic for physiological ageing (telomeric shortening immunophenotype) when compared to their respective donors. However a history of lower infection numbers in HCT recipients seems to be associated with increased percentage of NK cells. The history of GVHD seems not to affect the rate of ageing. Therefore it is safe to conclude that the observed subtle differences between recipients' and donors' cells result mainly from the proliferative stress in the early period after allo-HCT and the difference between hosts' and recipients' microenvironments." View Publication