Proton pump inhibitors stabilize the expression of PD-L1 on cell membrane depending on the phosphorylation of GSK3ß.

BACKGROUND: Preclinical and clinical evidence indicates that proton pump inhibitors (PPIs) may indirectly diminish the microbiome diversity, thereby reducing the effectiveness of immune checkpoint inhibitors (ICIs). Conversely, recent publications have shown that PPIs could potentially enhance the response to ICIs. The precise mechanism through which PPIs modulate the ICIs remains unclear. In this study, we discovered a novel molecular function of PPIs in regulating immune invasion, specifically through inducing PD-L1 translocation in various tumor cells. METHODS: C57BL/6 mice subcutaneous transplantation model is used to verify the potential efficacy of PPIs and PD-L1 antibody. Western blotting analysis and phosphorylated chip are used to verify the alteration of PD-L1-related pathways after being treated with PPIs. The related gene expression is performed by qRT-PCR and luciferase reporter analysis. We also collected 60 clinical patients diagnosed with esophageal cancer or reflux esophagitis and then detected the expression of PD-L1 in the tissue samples by immunohistochemistry. RESULTS: We observed that the IC50 of tumor cells in response to PPIs was significantly higher than that of normal epithelial cells. PPIs significantly increased the expression of PD-L1 on cell membrane at clinically relevant concentrations. Furthermore, pre-treatment with PPIs appeared to synergize the efficiency of anti-PD-L1 antibodies in mouse models. However, PPI administration did not alter the transcription or total protein level of PD-L1 in multiple tumor cells. Using a phosphorylated protein chip, we identified that PPIs enhanced the phosphorylation of GSK3ß, then leading to PD-L1 protein translocation to the cell membranes. The capacity of PPIs to upregulate PD-L1 was negated following GSK3ß knockout. Furthermore, our clinical data showed that the PPIs use resulted in increased PD-L1 expression in esophageal cancer patients. CONCLUSION: We mainly address a significant and novel mechanism that the usage of PPIs could directly induce the expression of PD-L1 by inducing GSK3ß phosphorylation and facilitate primary tumor progression and metastasis.

Evolving roles of CD38 metabolism in solid tumour microenvironment.

Given that plenty of clinical findings and reviews have already explained in detail on the progression of CD38 in multiple myeloma and haematological system tumours, here we no longer give unnecessary discussion on the above progression. Though therapeutic antibodies have been regarded as a greatest breakthrough in multiple myeloma immunotherapies due to the durable anti-tumour responses in the clinic, but the role of CD38 in the immunologic regulation and evasion of non-hematopoietic solid tumours are just initiated and controversial. Therefore, we will focus on the bio-function of CD38 enzymatic substrates or metabolites in the variety of non-hematopoietic malignancies and the potential therapeutic value of targeting the CD38-NAD+ or CD38-cADPR/ADPR signal axis. Though limited, we review some ongoing researches and clinical trials on therapeutic approaches in solid tumour as well.

Multiple SARS-CoV-2 Variants Exhibit Variable Target Cell Infectivity and Ability to Evade Antibody Neutralization.

The continuous emergence of SARS-coronavirus 2 (SARS-CoV-2) variants, especially the variants of concern (VOC), exacerbated the impact of the coronavirus disease 2019 (COVID-19) pandemic. As the key of viral entry into host cells, the spike (S) protein is the major target of therapeutic monoclonal antibodies (mAbs) and polyclonal antibodies elicited by infection or vaccination. However, the mutations of S protein in variants may change the infectivity and antigenicity of SARS-CoV-2, leading to the immune escape from those neutralizing antibodies. To characterize the mutations of S protein in newly emerging variants, the proteolytic property and binding affinity with receptor were assessed, and the vesicular stomatitis virus (VSV)-based pseudovirus system was used to assess the infectivity and immune escape. We found that some SARS-CoV-2 variants have changed significantly in viral infectivity; especially, B.1.617.2 is more likely to infect less susceptible cells than D614G, and the virus infection process can be completed in a shorter time. In addition, neutralizing mAbs and vaccinated sera partially or completely failed to inhibit host cell entry mediated by the S protein of certain SARS-CoV-2 variants. However, SARS-CoV-2 variant S protein-mediated viral infection can still be blocked by protease inhibitors and endocytosis inhibitors. This work provides a deeper understanding of the rise and evolution of SARS-CoV-2 variants and their immune evasion.

Corrigendum: Characterization of SARS-CoV-2 Variants N501Y.V1 and N501Y.V2 Spike on Viral Infectivity.

[This corrects the article DOI: 10.3389/fcimb.2021.720357.].

Characterization of SARS-CoV-2 Variants N501Y.V1 and N501Y.V2 Spike on Viral Infectivity.

SARS-coronavirus 2 (SARS-CoV-2), pathogen of coronavirus disease 2019 (COVID-19), is constantly evolving to adapt to the host and evade antiviral immunity. The newly emerging variants N501Y.V1 (B.1.1.7) and N501Y.V2 (B.1.351), first reported in the United Kingdom and South Africa respectively, raised concerns due to the unusually rapid global spread. The mutations in spike (S) protein may contribute to the rapid spread of these variants. Here, with a vesicular stomatitis virus (VSV)-based pseudotype system, we demonstrated that the pseudovirus bearing N501Y.V2 S protein has higher infection efficiency than pseudovirus with wildtype (WT) and D614G S protein. Moreover, pseudovirus with N501Y.V1 or N501Y.V2 S protein has better thermal stability than WT and D614G, suggesting these mutations of variants may increase the stability of SARS-CoV-2 S protein and virion. However, the pseudovirus bearing N501Y.V1 or N501Y.V2 S protein has similar sensitivity to inhibitors of protease and endocytosis with WT and D614G. These findings could be of value in preventing the spread of virus and developing drugs for emerging SARS-CoV-2 variants.

Combinatorial screening of a panel of FDA-approved drugs identifies several candidates with anti-Ebola activities.

Ebola virus (EBOV), pathogen of Ebola hemorrhagic fever (EHF), is an enveloped filamental RNA virus. Recently, the EHF crisis occurred in the Democratic Republic of the Congo again highlights the urgency for its clinical treatments. However, no Food and Drug Administration (FDA)-approved therapeutics are currently available. Drug repurposing screening is a time- and cost-effective approach for identifying anti-EBOV therapeutics. Here, by combinatorial screening using pseudovirion and minigenome replicon systems we have identified several FDA-approved drugs with significant anti-EBOV activities. These potential candidates include azithromycin, clomiphene, chloroquine, digitoxin, epigallocatechin-gallate, fluvastatin, tetrandrine and tamoxifen. Mechanistic studies revealed that fluvastatin inhibited EBOV pseudovirion entry by blocking the pathway of mevalonate biosynthesis, while the inhibitory effect of azithromycin on EBOV maybe due to its intrinsic cationic amphiphilic structure altering the homeostasis of later endosomal vesicle similar as tamoxifen. Moreover, based on structure and pathway analyses, the anti-EBOV activity has been extended to other family members of statins, such as simvastatin, and multiple other cardiac glycoside drugs, some of which exhibited even stronger activities. More importantly, in searching for drug interaction, we found various synergy between several anti-EBOV drug combinations, showing substantial and powerful synergistic against EBOV infection. In conclusion, our work illustrates a successful and productive approach to identify new mechanisms and targets for treating EBOV infection by combinatorial screening of FDA-approved drugs.

Tissue-specific deconvolution of immune cell composition by integrating bulk and single-cell transcriptomes.

MOTIVATION: Many methods have been developed to estimate immune cell composition from tissue transcriptomes. One common characteristic of these methods is that they are trained using a set of general immune cell transcriptomes that ignores tissue specificities. However, as immune cells are localized in different tissues, they may have distinct expression profiles. Hence, calculations that use general signature matrices may hinder the deconvolution accuracy. RESULTS: This study used single cell RNA-sequencing (scRNA-Seq) data from different mouse tissues instead of general signature expression values to generate tissue-specific signature gene matrices that are used as the input of the deconvolution model. First, the transcriptome of immune cells in each tissue was extracted from scRNA-Seq data and used to construct the entire expression matrix of tissue immune cells. Then, after comparing different gene selection strategies, the expressions of 162 seq-ImmuCC derived signature genes in tissue immune cell scRNA-Seq data were regarded as the tissue specific signature matrices. Finally, a modest improvement in performance was observed in multiple tissues that refer to a traditional general signature matrix in the deconvolution model. With the fast accumulation of scRNA-Seq data, the introduction of these data into an estimation of immune cell compositions for different tissues will open a new window for avoiding tissue bias for immune cell expression. AVAILABILITY AND IMPLEMENTATION: The signature matrices were available at https://github.com/wuaipinglab/ImmuCC/tree/master/tissue_immucc/SignatureMatrix). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Targeting the tumor microenvironment to overcome immune checkpoint blockade therapy resistance.

The ability of immune checkpoint inhibitors (ICIs) to reactivate the killing function of the immune system to tumor cells has led to long lasting immune response presenting highly promising clinical advances. Recently, immune checkpoint inhibitors related resistance due to the specialized tumor microenvironment has also drawn a widely attention. To overcome resistance to immune checkpoint blockade therapy, understanding the relationship of this type of therapy and tumor microenvironment is necessary and critical. This review will focus on how the tumor environment influences the effectiveness of the immunotherapeutic check inhibitors. Finally, we provide a briefly succinct glimpse into the most exciting pre-clinical discoveries and ongoing clinical trials to overcome the resistance of ICIs.

Type-I-IFN-Stimulated Gene TRIM5γ Inhibits HBV Replication by Promoting HBx Degradation.

To understand the molecular mechanisms that mediate the anti-hepatitis B virus (HBV) effect of interferon (IFN) therapy, we conduct high-throughput bimolecular fluorescence complementation screening to identify potential physical interactions between the HBx protein and 145 IFN-stimulated genes (ISGs). Seven HBx-interacting ISGs have consistent and significant inhibitory effects on HBV replication, among which TRIM5γ suppresses HBV replication by promoting K48-linked ubiquitination and degradation of the HBx protein on the K95 ubiquitin site. The B-Box domain of TRIM5γ under overexpression conditions is sufficient to trigger HBx degradation and is responsible both for interacting with HBx and recruiting TRIM31, which is an ubiquitin ligase that triggers HBx ubiquitination. High expression levels of TRIM5γ in IFN-α-treated HBV patients might indicate a better therapeutic effect. Thus, our studies identify a crucial role for TRIM5γ and TRIM31 in promoting HBx degradation, which may facilitate the development of therapeutic agents for the treatment of patients with IFN-resistant HBV infection.

Noncanonical Role of FBXO6 in Regulating Antiviral Immunity.

The evolutionarily conserved F-box family of proteins are well known for their role as the key component of SKP1-Cullin1-F-box (SCF) E3 ligase in controlling cell cycle, cell proliferation and cell death, carcinogenesis, and cancer metastasis. However, thus far, there is only limited investigation on their involvement in antiviral immunity. In contrast to the canonical function of FBXO6 associated with SCF E3 ligase complex, we report, in this study, that FBXO6 can also potently regulate the activation of IFN-I signaling during host response to viral infection by targeting the key transcription factor IFN-regulatory factor 3 (IRF3) for accelerated degradation independent of SCF in human embryonic kidney cells (HEK293T) and human lung cancer epithelial cells (A549). Structure and function delineation has further revealed that FBXO6 interacts with IAD domain of IRF3 through its FBA region to induce ubiquitination and degradation of IRF3 without the involvement of SCF. Thus, our studies have identified a general but, to our knowledge, previously unrecognized role and a novel noncanonical mechanism of FBXO6 in modulating IFN-I-mediated antiviral immune responses, which may protect the host from immunopathology of overreactive and harmful IFN-I production.

Inhibition of Influenza A Virus Replication by TRIM14 via Its Multifaceted Protein-Protein Interaction With NP.

Influenza A virus (IAV) is a worldwide ongoing health threat causing diseases in both humans and animals. The interaction between IAV and host is a dynamic and evolving process that influences the pathogenicity and host specificity of the virus. TRIM14, a member of tripartite motif (TRIM) family, has been demonstrated to possess a strong capability of regulating type I interferon and NF-κB induction in host defense against viral infection. In this study, we found that TRIM14 could restrict the replication of IAV in a type I interferon and NF-κB independent manner. Mechanistically, different domains of TRIM14 could selectively interact with the viral nucleoprotein (NP), resulting in disparate influences on the RNP formation and viral replication. In particular, the PRYSPRY domain of TRIM14 exhibited a potent inhibitory activity on NP protein stability and IAV replication. On the contrary, the ΔS2 domain could rather antagonize the function of PRYSPRY domain and promote the IAV RNP formation by stabilizing NP. At the biochemical level, TRIM14-NP interaction could induce the K48-linked ubiquitination and proteasomal degradation of NP. Moreover, due to the rapid degradation of newly synthesized NP, TRIM14 could effectively block the translocation of NP from cytoplasm to nucleus thus further restrain the propagation of IAV in host cells. Taken together, our study has unraveled a previously unknown mechanism of TRIM14 mediated inhibition on RNP formation and influenza virus replication, and provides a new paradigm of complex and multifaceted host-pathogen interaction between ISG and viral protein.

Concomitant type I IFN and M-CSF signaling reprograms monocyte differentiation and drives pro-tumoral arginase production.

BACKGROUND: Type I IFN-based therapies against solid malignancies have yielded only limited success. How IFN affects tumor-associated macrophage (TAM) compartment to impact the therapeutic outcomes are not well understood. METHODS: The effect of an IFN-inducer poly(I:C) on tumor-infiltrating monocytes and TAMs were analyzed using a transplantable mouse tumor model (LLC). In vitro culture systems were utilized to study the direct actions by poly(I:C)-IFN on differentiating monocytes. RESULTS: We found that poly(I:C)-induced IFN targets Ly6C+ monocytes and impedes their transition into TAMs. Such an effect involves miR-155-mediated suppression of M-CSF receptor expression, contributing to restricting tumor growth. Remarkably, further analyses of gene expression profile of IFN-treated differentiating monocytes reveal a strong induction of Arg1 (encoding arginase-1) in addition to other classical IFN targets. Mechanistically, the unexpected Arg1 arm of IFN action is mediated by a prolonged STAT3 signaling in monocytes, in conjunction with elevated macrophage colony-stimulating factor (M-CSF) signaling. Functionally, induction of ARG1 limited the therapeutic effect of IFN, as inhibition of arginase activity could strongly synergize with poly(I:C) to enhance CD8+ T cell responses to thwart tumor growth in mice. CONCLUSIONS: Taken together, we have uncovered two functionally opposing actions by IFN on the TAM compartment. Our work provides significant new insights on IFN-mediated immunoregulation that may have implications in cancer therapies.

Identification of TRIM14 as a Type I IFN-Stimulated Gene Controlling Hepatitis B Virus Replication by Targeting HBx.

Hepatitis B virus (HBV) remains a major cause of hepatic disease that threatens human health worldwide. Type I IFN (IFN-I) therapy is an important therapeutic option for HBV patients. The antiviral effect of IFN is mainly mediated via upregulation of the expressions of the downstream IFN-stimulated genes. However, the mechanisms by which IFN induces ISG production and inhibits HBV replication are yet to be clarified. TRIM14 was recently reported as a key molecule in the IFN-signaling pathway that regulates IFN production in response to viral infection. In this study, we sought to understand the mechanisms by which IFN restricts HBV replication. We confirmed that TRIM14 is an ISG in the hepatic cells, and that the pattern-recognition receptor ligands polyI:C and polydAdT induce TRIM14 dependent on IFN-I production. In addition, IFN-I-activated STAT1 (but not STAT3) directly bound to the TRIM14 promoter and mediated the induction of TRIM14. Interestingly, TRIM14 played an important role in IFN-I-mediated inhibition of HBV, and the TRIM14 SPRY domain interacted with the C-terminal of HBx, which might block the role of HBx in facilitating HBV replication by inhibiting the formation of the Smc-HBx-DDB1 complex. Thus, our study clearly demonstrates that TRIM14 is a STAT1-dependent ISG, and that the IFN-I-TRIM14-HBx axis shows an alternative way to understand the mechanism by which IFN-I inhibits virus replication.

CD38-Mediated Immunosuppression as a Mechanism of Tumor Cell Escape from PD-1/PD-L1 Blockade.

Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.

seq-ImmuCC: Cell-Centric View of Tissue Transcriptome Measuring Cellular Compositions of Immune Microenvironment From Mouse RNA-Seq Data.

The RNA sequencing approach has been broadly used to provide gene-, pathway-, and network-centric analyses for various cell and tissue samples. However, thus far, rich cellular information carried in tissue samples has not been thoroughly characterized from RNA-Seq data. Therefore, it would expand our horizons to better understand the biological processes of the body by incorporating a cell-centric view of tissue transcriptome. Here, a computational model named seq-ImmuCC was developed to infer the relative proportions of 10 major immune cells in mouse tissues from RNA-Seq data. The performance of seq-ImmuCC was evaluated among multiple computational algorithms, transcriptional platforms, and simulated and experimental datasets. The test results showed its stable performance and superb consistency with experimental observations under different conditions. With seq-ImmuCC, we generated the comprehensive landscape of immune cell compositions in 27 normal mouse tissues and extracted the distinct signatures of immune cell proportion among various tissue types. Furthermore, we quantitatively characterized and compared 18 different types of mouse tumor tissues of distinct cell origins with their immune cell compositions, which provided a comprehensive and informative measurement for the immune microenvironment inside tumor tissues. The online server of seq-ImmuCC are freely available at http://wap-lab.org:3200/immune/.

Broad and diverse mechanisms used by deubiquitinase family members in regulating the type I interferon signaling pathway during antiviral responses.

The innate immune response conferred by type I interferons is essential for host defense against viral infection but needs to be tightly controlled to avoid immunopathology. We performed a systematic functional screening by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) knockout and overexpression to investigate the roles of the deubiquitinating enzyme (DUB) family in regulating antiviral immunity. We demonstrated that the expression of a large fraction of DUBs underwent complex temporal alteration, suggesting a dynamic program of feedback regulation. Moreover, we identified previously unrecognized roles of a subset of DUBs, including USP5, USP14, USP22, USP48, USP52, COPS5, and BRCC3, in inhibiting antiviral immunity at various levels. We explored an unexpected mechanism where multiple DUBs, such as USP5 and USP22, form diverse signalosomes with E3 ligases or DUBs to alter the substrates' ubiquitination state instead of directly cleaving the ubiquitin chains on substrates via their protease activity. Altogether, our study has revealed a panoramic view of the broad and dynamic involvement of DUB family proteins in regulating antiviral responses.

STAT3/p53 pathway activation disrupts IFN-ß-induced dormancy in tumor-repopulating cells.

Dynamic interaction with the immune system profoundly regulates tumor cell dormancy. However, it is unclear how immunological cues trigger cancer cell-intrinsic signaling pathways for entering into dormancy. Here, we show that IFN-ß treatment induced tumor-repopulating cells (TRC) to enter dormancy through an indolamine 2,3-dioxygenase/kynurenine/aryl hydrocarbon receptor/p27-dependent (IDO/Kyn/AhR/p27-dependent) pathway. Strategies to block this metabolic circuitry did not relieve dormancy, but led to apoptosis of dormant TRCs in murine and human melanoma models. Specifically, blocking AhR redirected IFN-ß signaling to STAT3 phosphorylation through both tyrosine and serine sites, which subsequently facilitated STAT3 nuclear translocation and subsequent binding to the p53 promoter in the nucleus. Upregulation of p53 in turn disrupted the pentose phosphate pathway, leading to excessive ROS production and dormant TRC death. Additionally, in melanoma patients, high expression of IFN-ß correlated with tumor cell dormancy. Identification of this mechanism for controlling TRC dormancy by IFN-ß provides deeper insights into cancer-immune interaction and potential new cancer immunotherapeutic modalities.

Type I IFN augments IL-27-dependent TRIM25 expression to inhibit HBV replication.

Hepatitis B virus (HBV) can cause chronic hepatitis B, which may lead to cirrhosis and liver cancer. Type I interferon (IFN) is an approved drug for the treatment of chronic hepatitis B. However, the fundamental mechanisms of antiviral action by type I IFN and the downstream signaling pathway are unclear. TRIM25 is an IFN-stimulated gene (ISG) that has an important role in RIG-I ubiquitination and activation. Whether TRIM25 is induced in liver cells by type I IFN to mediate anti-HBV function remains unclear. Here we report that interleukin-27 (IL-27) has a critical role in IFN-induced TRIM25 upregulation. TRIM25 induction requires both STAT1 and STAT3. In TRIM25 knockout HepG2 cells, type I IFN production was consistently attenuated and HBV replication was increased, whereas overexpression of TRIM25 in HepG2 cells resulted in elevated IFN production and reduced HBV replication. More interestingly, we found that TRIM25 expression was downregulated in HBV patients and the addition of serum samples from HBV patients could inhibit TRIM25 expression in HepG2 cells, suggesting that HBV might have involved a mechanism to inhibit antiviral ISG expression and induce IFN resistance. Collectively, our results demonstrate that type I IFN -induced TRIM25 is an important factor in inhibiting HBV replication, and the IFN-IL-27-TRIM25 axis may represent a new target for treating HBV infection.