The two T cell inhibitory receptors PD-1 and TIM-3 are co-expressed during exhausted T cell differentiation, and recent evidence suggests that their crosstalk regulates T cell exhaustion and ...immunotherapy efficacy; however, the molecular mechanism is unclear. Here we show that PD-1 contributes to the persistence of PD-1
TIM-3
T cells by binding to the TIM-3 ligand galectin-9 (Gal-9) and attenuates Gal-9/TIM-3-induced cell death. Anti-Gal-9 therapy selectively expands intratumoral TIM-3
cytotoxic CD8 T cells and immunosuppressive regulatory T cells (T
cells). The combination of anti-Gal-9 and an agonistic antibody to the co-stimulatory receptor GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) that depletes T
cells induces synergistic antitumor activity. Gal-9 expression and secretion are promoted by interferon β and γ, and high Gal-9 expression correlates with poor prognosis in multiple human cancers. Our work uncovers a function for PD-1 in exhausted T cell survival and suggests Gal-9 as a promising target for immunotherapy.
To explore whether a cross-talk exists between PARP inhibition and PD-L1/PD-1 immune checkpoint axis, and determine whether blockade of PD-L1/PD-1 potentiates PARP inhibitor (PARPi) in tumor ...suppression.
Breast cancer cell lines, xenograft tumors, and syngeneic tumors treated with PARPi were assessed for PD-L1 expression by immunoblotting, IHC, and FACS analyses. The phospho-kinase antibody array screen was used to explore the underlying mechanism of PARPi-induced PD-L1 upregulation. The therapeutic efficacy of PARPi alone, PD-L1 blockade alone, or their combination was tested in a syngeneic tumor model. The tumor-infiltrating lymphocytes and tumor cells isolated from syngeneic tumors were analyzed by CyTOF and FACS to evaluate the activity of antitumor immunity in the tumor microenvironment.
PARPi upregulated PD-L1 expression in breast cancer cell lines and animal models. Mechanistically, PARPi inactivated GSK3β, which in turn enhanced PARPi-mediated PD-L1 upregulation. PARPi attenuated anticancer immunity via upregulation of PD-L1, and blockade of PD-L1 resensitized PARPi-treated cancer cells to T-cell killing. The combination of PARPi and anti-PD-L1 therapy compared with each agent alone significantly increased the therapeutic efficacy
Our study demonstrates a cross-talk between PARPi and tumor-associated immunosuppression and provides evidence to support the combination of PARPi and PD-L1 or PD-1 immune checkpoint blockade as a potential therapeutic approach to treat breast cancer.
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Reactivation of T cell immunity by PD-1/PD-L1 immune checkpoint blockade has been shown to be a promising cancer therapeutic strategy. However, PD-L1 immunohistochemical readout is inconsistent with ...patient response, which presents a clinical challenge to stratify patients. Because PD-L1 is heavily glycosylated, we developed a method to resolve this by removing the glycan moieties from cell surface antigens via enzymatic digestion, a process termed sample deglycosylation. Notably, deglycosylation significantly improves anti-PD-L1 antibody binding affinity and signal intensity, resulting in more accurate PD-L1 quantification and prediction of clinical outcome. This proposed method of PD-L1 antigen retrieval may provide a practical and timely approach to reduce false-negative patient stratification for guiding anti-PD-1/PD-L1 therapy.
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•N-linked glycosylation of PD-L1 hinders its recognition by PD-L1 antibodies•Removal of glycosylation enhances anti-PD-L1 signal in a variety of bioassays•Patient sample deglycosylation prevents false-negative detection of PD-L1•Deglycosylated PD-L1 is a more reliable biomarker to guide immunotherapy
Histological detection of PD-L1 may guide therapy with anti-PD-1/PD-L1 antibodies but some PD-L1-negative tumors respond to these treatments. Lee et al. show that enzymatic deglycosylation of tissue sections improves PD-L1 detection and its predictive value, and could potentially impact patient stratification.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. ...In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring β-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.
•N-linked glycosylation is required for physical contact between PD-L1 and PD-1•EGF/EGFR stimulates PD-L1 glycosylation via B3GNT3 glycosyltransferase•Glycosylated-PD-L1 antibody induces PD-L1 internalization•Glycosylated-PD-L1-ADC possesses potent toxicity as well as bystander effects
Li et al. show that glycosylation of PD-L1 is essential for PD-L1/PD-1 interaction and immunosuppression in triple-negative breast cancer (TNBC). They generate a glycosylation-specific antibody that induces PD-L1 internalization and an antibody-drug conjugate with potent anti-tumor activities in TNBC models.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Block-Cell-Printing for live single-cell printing Zhang, Kai; Chou, Chao-Kai; Xia, Xiaofeng ...
Proceedings of the National Academy of Sciences - PNAS,
02/2014, Volume:
111, Issue:
8
Journal Article
Peer reviewed
Open access
A unique live-cell printing technique, termed “Block-Cell-Printing” (BloC-Printing), allows for convenient, precise, multiplexed, and high-throughput printing of functional single-cell arrays. ...Adapted from woodblock printing techniques, the approach employs microfluidic arrays of hook-shaped traps to hold cells at designated positions and directly transfer the anchored cells onto various substrates. BloC-Printing has a minimum turnaround time of 0.5 h, a maximum resolution of 5 µm, close to 100% cell viability, the ability to handle multiple cell types, and efficiently construct protrusion-connected single-cell arrays. The approach enables the large-scale formation of heterotypic cell pairs with controlled morphology and allows for material transport through gap junction intercellular communication. When six types of breast cancer cells are allowed to extend membrane protrusions in the BloC-Printing device for 3 h, multiple biophysical characteristics of cells—including the protrusion percentage, extension rate, and cell length—are easily quantified and found to correlate well with their migration levels. In light of this discovery, BloC-Printing may serve as a rapid and high-throughput cell protrusion characterization tool to measure the invasion and migration capability of cancer cells. Furthermore, primary neurons are also compatible with BloC-Printing.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Psoriasis affects more than 125 million people worldwide, and the diagnosis and treatment efficacy evaluation of the disease mainly rely on clinical assessments that could be subjective. Our previous ...study showed that the skin erythema level could be quantified using diffuse reflectance spectroscopy (DRS), and the hemoglobin concentration of most psoriatic lesion was higher than that of its adjacent uninvolved skin. While the compromised epidermal barrier function has been taken as the major cause of clinical manifestation of skin dryness and inflammation of psoriasis, very few methods can be used to effectively evaluate this function. In this study, we investigate the near infrared spectroscopic features of psoriatic (n = 21) and normal (n = 21) skin that could link to the epidermal barrier function. From the DRS measurements, it was found that the water bonding status and light scattering properties of psoriasis are significantly different from those of uninvolved or normal skin. The connection between these parameters to the epidermal barrier function and morphology will be discussed. Our results suggest that objective evaluation of epidermal barrier function of psoriasis could be achieved using a simple DRS system.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Nuclear receptors recruit multiple coactivators sequentially to activate transcription. This “ordered” recruitment allows different coactivator activities to engage the nuclear receptor complex at ...different steps of transcription. Estrogen receptor (ER) recruits steroid receptor coactivator-3 (SRC-3) primary coactivator and secondary coactivators, p300/CBP and CARM1. CARM1 recruitment lags behind the binding of SRC-3 and p300 to ER. Combining cryo-electron microscopy (cryo-EM) structure analysis and biochemical approaches, we demonstrate that there is a close crosstalk between early- and late-recruited coactivators. The sequential recruitment of CARM1 not only adds a protein arginine methyltransferase activity to the ER-coactivator complex, it also alters the structural organization of the pre-existing ERE/ERα/SRC-3/p300 complex. It induces a p300 conformational change and significantly increases p300 HAT activity on histone H3K18 residues, which, in turn, promotes CARM1 methylation activity on H3R17 residues to enhance transcriptional activity. This study reveals a structural role for a coactivator sequential recruitment and biochemical process in ER-mediated transcription.
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•ER sequentially recruits SRC-3 and CARM1 coactivators to ER binding sites•CARM1 recruitment replaces one of two SRC-3 proteins from the complex•CARM1 recruitment induces p300 conformational change and promotes H3K18ac•Increased histone H3K18 acetylation in turn enhanced CARM1-mediated H3R17 methylation
Nuclear receptors sequentially recruit different coactivators to regulate transcription. Yi et al. combined cryo-EM structure analysis and biochemical approaches to demonstrate that late-recruited coactivators impact the structure and specific functions of early-recruited nuclear receptor/coactivator complexes to enhance transcription.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to ...high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without spike (S), membrane (M), and envelope (E) genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs.
SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. Additionally, our stable replicon cells represent a new model system to study SARS-CoV-2 replication.
Derailed transmembrane receptor trafficking could be a hallmark of tumorigenesis and increased tumor invasiveness, but receptor dynamics have not been used to differentiate metastatic cancer cells ...from less invasive ones. Using single-particle tracking techniques, we developed a phenotyping asssay named Transmembrane Receptor Dynamics (TReD), studied the dynamics of epidermal growth factor receptor (EGFR) in seven breast epithelial cell lines and developed a phenotyping assay named Transmembrane Receptor Dynamics (TReD). Here we show a clear evidence that increased EGFR diffusivity and enlarged EGFR confinement size in the plasma membrane (PM) are correlated with the enhanced metastatic potential in these cell lines. By comparing the TReD results with the gene expression profiles, we found a clear negative correlation between the EGFR diffusivities and the breast cancer luminal differentiation scores (r = -0.75). Upon the induction of epithelial-mesenchymal transition (EMT), EGFR diffusivity significantly increased for the non-tumorigenic MCF10A (99%) and the non-invasive MCF7 (56%) cells, but not for the highly metastatic MDA-MB-231 cell. We believe that the reorganization of actin filaments during EMT modified the PM structures, causing the receptor dynamics to change. TReD can thus serve as a new biophysical marker to probe the metastatic potential of cancer cells and even to monitor the transition of metastasis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing ...the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1
) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1
elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design.
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