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Genes differentially expressed between IgG control and anti-PD-L1 treatment.

Supplementary title page. Figure S1: CD38 on anti-PD-L1 resistant tumor cells is up-regulated, which is associated with tumor progression. Figure S2: anti-PD-L1 resistant tumors demonstrate the distinct mRNA and protein profiling for immune signature, reflecting the upregulation of CD38. Figure S3: PD-1/PD-L1 blockade results in CD38 up-regulation and acquired resistance in KP-derived lung and melanoma transplantation tumors. Figure S4: Tumor-associated PD-L1 promotes tumor growth but PD-L1 knockout cancer cells still form tumors. Figure S5: PD-L1 knockout effect on tumor growth is CD8 T cell-dependent. Figure S6: CD38 up-regulation after anti-PD-L1 treatment is associated with all-trans retinoic acid signaling. Figure S7: IFN-b, which is enriched in anti-PD-L1 treated tumors, up-regulates CD38 expression on multiple cancer cell lines. Figure S8: IRF1, which links ATRA and IFN-b, is upregulated after anti-PD-L1 treatment. Figure S9: IFN-g, TNF-a, IL-2, and IL-1b don''t regulate CD38 expression on lung cancer cells. Figure S10: The anti-PD-L1 resistant tumors demonstrate an immune suppressive microenvironment. Figure S11: CD38 substantially changes in vivo tumor formation of PD-L1KO cancer cells, but does not change in vitro cell growth rate and cell cycle. Figure S12: The elimination of PD-L1KOCD38negative cancer cells is CD8+ T cell dependent. Figure S13: CD38 on tumor cells inhibits CD8+ T cell function and protects tumor cells from CD8+ T cell killing. Figure S14: CD38-mediated CD8+TIL dysfunction is not affected by tumor growth rate/tumor size. Figure S15: CD38 expression in cancer cell lines and patient tissues, which is associated with the differentiated immune features. Figure S16: CD38 or PD-L1 expression is not correlated with overall survival in early-stage lung cancer. Figure S17: Pre-treatment levels of CD38 and PD-L1 expression in NSCLC patients who received anti-PD-1/PD-L1 therapy, as divided by clinical outcome. Figure S18: The effect of cancer immunotherapy by anti-CD38 is CD8 T cell dependent. Figure S19: Combined inhibitors of CD38 and PD-L1 inhibits tumor growth and metastases. Figure S20: The co-inhibition of PD-L1 and CD38 leads to a favorable antitumor immune microenvironment. Figure S21: Sequential treatment of anti-PD-L1 and anti-CD38 results in enhanced immune response in tumor microenvironment. Figure S22: anti-mouse CD38 antibody (NIMR-5) does not directly kill tumor cells through ADCC and CDC, but causes CD38 internalization. Table S2: Tumor immune markers with the greatest differential transcriptional levels after anti-PD-L1 treatment. Table S6: The most changed immune-related genes after anti-PD-L1 treatment. Table S9:The correlation between CD38 and suppressive immune markers in LUAD dataset. Table S10: The correlation between CD38 and suppressive immune markers in LUSC dataset. Table S11: The available NSCLC patients with CD38 and PD-L1 tumor cell IHC staining and responses to anti-PD-1 therapy. Table S12: The co-inhibition effect of PD-L1 and CD38 on tumor growth and metastasis. Supplementary Material and Methods: Reagents, Cells and Mice, CRISPR/Cas9 Editing, Antibody-mediated Cell Depletion, CD8+ T Cell Adoptive Transfer, mRNA Profiling of Murine Tumors, Flow Cytometry, Nanostring Analysis, qRT-PCR and Western Blotting, Liquid Chromatography-Mass Spectrometry (LC-MS) Analysis, Histologic Analysis, ELISA and RPPA, ADCC/CDC and Internalization Assays, Human Samples, Statistics. Supplementary references

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

CD8+ T cell exhaustion dampens antitumor immunity. Although several transcription factors have been identified that regulate T cell exhaustion, the molecular mechanisms by which CD8+ T cells are triggered to enter an exhausted state remain unclear. Here, we show that interleukin-2 (IL-2) acts as an environmental cue to induce CD8+ T cell exhaustion within tumor microenvironments. We find that a continuously high level of IL-2 leads to the persistent activation of STAT5 in CD8+ T cells, which in turn induces strong expression of tryptophan hydroxylase 1, thus catalyzing the conversion to tryptophan to 5-hydroxytryptophan (5-HTP). 5-HTP subsequently activates AhR nuclear translocation, causing a coordinated upregulation of inhibitory receptors and downregulation of cytokine and effector-molecule production, thereby rendering T cells dysfunctional in the tumor microenvironment. This molecular pathway is not only present in mouse tumor models but is also observed in people with cancer, identifying IL-2 as a novel inducer of T cell exhaustion. IL-2 is a classic T cell growth factor. Huang and colleagues demonstrate, however, that chronic IL-2 stimulation leads to a new exhaustion pathway that impairs antitumor immune responses.

Exhausted CD8

It has been recently reported that CD38 expressed on tumor cells of multiple murine and human origins could be upregulated in response to PD-L1 antibody therapy, which led to dysfunction of tumor-infiltrating CD8+ T immune cells due to increasing the production of adenosine. However, the role of tumor expressed-CD38 on neoplastic formation and progression remains elusive. In the present study, we aimed to delineate the molecular and biochemical function of the tumor-associated CD38 in lung adenocarcinoma progression. Our clinical data showed that the upregulation of tumor-originated CD38 was correlated with poor survival of lung cancer patients. Using multiple in vitro assays we found that the enzymatic activity of tumor expressed-CD38 facilitated lung cancer cell migration, proliferation, colony formation, and tumor development. Consistently, our in vivo results showed that inhibition of the enzymatic activity or antagonizing the enzymatic product of CD38 resulted in the similar inhibition of tumor proliferation and metastasis as CD38 gene knock-out or mutation. At biochemical level, we further identified that cADPR, the mainly hydrolytic product of CD38, was responsible for inducing the opening of TRPM2 iron channel leading to the influx of intracellular Ca2+ and then led to increasing levels of NRF2 while decreasing expression of KEAP1 in lung cancer cells. These findings suggested that malignant lung cancer cells were capable of using cADPR catalyzed by CD38 to facilitate tumor progression, and blocking the enzymatic activity of CD38 could be represented as an important strategy for preventing tumor progression.

CD8

Epigenetic regulation of immune cell fate decision and function has attracted much attention recently. Epigenetic modifications, including DNA methylation and chromatin remodeling, are involved in multiple aspects of innate and adaptive immune regulation. Next-generation sequencing (NGS), including RNA-Seq, ChIP-Seq, and ATAC-Seq, could now be performed on a genome-wide scale, even at single-cell resolution, which greatly accelerates our understanding of basic immunology. Along the same line, many computational tools have been developed and deconvolution of the cellular constitution from the omic data of highly heterogeneous tissues has opened another window for understanding the complexity of the immune system.

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-β 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-β 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-β correlated with tumor cell dormancy. Identification of this mechanism for controlling TRC dormancy by IFN-β provides deeper insights into cancer-immune interaction and potential new cancer immunotherapeutic modalities.

The F-box proteins were originally identified as the key component of SKP1-Cullin1-F-box E3 ligase complexes that control the stability of their specific downstream substrates essential for cell growth and survival. However, the involvement of these proteins in type I IFN (IFN-I) signaling during innate immunity has not been investigated. In this study we report that the F-box protein FBXO17 negatively regulates IFN-I signaling triggered by double-strand DNA, RNA, or viral infection. We found that FBXO17 specifically interacts with IFN regulatory factor 3 (IRF3) and decreases its dimerization and nuclear translocation. The decrease of IRF3 dimerization and nuclear translocation is due to the recruitment of protein phosphatase 2 (PP2A) mediated by FBXO17, resulting in IRF3 dephosphorylation. Interestingly, PP2A recruitment does not require the F-box domain but instead the F-box associated region of the protein; thus, the recruitment is independent of the canonical function of the SKP1-Cullin1-F-box family of E3 ligase. Together, our studies identify a previously unreported role of FBXO17 in regulating IFN-I signaling and further demonstrate a novel mechanism for IRF3 deactivation by F-box protein-mediated recruitment of PP2A.

SUMMARY 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., Graphical Abstract, In Brief In brief, Tan et al. find that IFN-induced TRIM5γ recruits TRIM31 to degrade HBx, resulting in suppression of hepatitis B virus replication.

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.

1R, 1′S-isotetrandrine, a naturally occurring plant alkaloid found in Mahonia of Berberidaceae, possesses anti-inflammatory, antibacterial, and antiviral properties, but the antioxidative activity and mechanism action remain unclear. In this study, we demonstrated the antioxidative effect and mechanism of 1R, 1'S-isotetrandrine against tert-butyl hydroperoxide-induced oxidative damage in HepG2 cells. We found that 1R, 1′S-isotetrandrine suppressed cytotoxicity, reactive oxygen species generation, and glutathione depletion. Additionally, our study confirmed that 1R, 1′S-isotetrandrine significantly increased the antioxidant enzyme heme oxygenase-1 expression and nuclear translocation of factor-erythroid 2 p45-related factor 2 (Nrf2). Specifically, the nuclear translocation of Nrf2 induced by 1R, 1′S-isotetrandrine was associated with Nrf2 negative regulatory protein Keap1 inactivation and phosphorylation of both extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase. Preincubation with thiol-reducing agents reduced 1R, 1′S-isotetrandrine-induced heme oxygenase-1 expression, and treatment with either extracellular signal-regulated protein kinase or c-Jun NH2-terminal kinase inhibitors attenuated the levels of 1R, 1′S-isotetrandrine-induced Nrf2 activation and heme oxygenase-1 expression. Furthermore, the cytoprotective effect of 1R, 1′S-isotetrandrine was abolished by heme oxygenase-1, extracellular signal-regulated protein kinase, and c-Jun NH2-terminal kinase inhibitors. These results indicated that the 1R, 1′S-isotetrandrine ameliorated tert-butyl hydroperoxide-induced oxidative damage through upregulation of heme oxygenase-1 expression by the dissociation of Nrf2 from Nrf2-Keap1 complex via extracellular signal-regulated protein kinase and c-Jun NH2-terminal kinase activation and Keap1 inactivation.

Influenza virus mRNA synthesis by the RNA-dependent RNA polymerase involves binding and cleavage of capped cellular mRNA by the PB2 and PA subunits, respectively, and extension of viral mRNA by PB1. However, the mechanism for such a dynamic process is unclear. Using high-throughput mutagenesis and sequencing analysis, we have not only generated a comprehensive functional map for the microdomains of individual subunits but also have revealed the PA linker to be critical for polymerase activity. This PA linker binds to PB1 and also forms ionic interactions with the PA C-terminal channel. Nearly all mutants with five-amino-acid insertions in the linker were nonviable. Our model further suggests that the PA linker plays an important role in the conformational changes that occur between stages that favor capped mRNA binding and cleavage and those associated with viral mRNA synthesis. IMPORTANCE The RNA-dependent RNA polymerase of influenza virus consists of the PB1, PB2, and PA subunits. By combining genome-wide mutagenesis analysis with the recently discovered crystal structure of the influenza polymerase heterotrimer, we generated a comprehensive functional map of the entire influenza polymerase complex. We identified the microdomains of individual subunits, including the catalytic domains, the interaction interfaces between subunits, and nine linkers interconnecting different domains. Interestingly, we found that mutants with five-amino-acid insertions in individual linkers were nonviable, suggesting the critical roles these linkers play in coordinating spatial relationships between the subunits. We further identified an extended PA linker that binds to PB1 and also forms ionic interactions with the PA C-terminal channel.

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.

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.

Farrerol, (S)-2,3-dihydro-5,7-dihydroxy-2-(4-hydroxyphenyl)-6,8-dimethyl-4-benzopyrone, isolated from rhododendron, has been shown to have antioxidative potential, but the molecular mechanism underlying this activity remains unclear. The inducible expression of heme oxygenase-1 (HO-1), a potent antioxidative and cytoprotective enzyme, is known to play an important role in cytoprotection in a variety of pathological models. In this study, we evaluated the antioxidative potential of farrerol against oxidative damage and investigated its antioxidative mechanism in RAW 264.7 cells. The molecular mechanism underlying the cytoprotective function of farrerol was determined by analyzing intracellular signaling pathways, transcriptional activation and the inhibitory effect of HO-1 on ROS production. Farrerol induced antioxidant enzymes mRNA expression, HO-1 protein expression and nuclear translocation of NF-E2-related factor 2 in RAW 264.7 macrophage cells. Farrerol down-regulated the expression of the Keap1 protein and the thiol reducing agents attenuated farrerol-induced HO-1 expression. Further investigation utilizing Western blotting and specific inhibitors of Akt, p38, JNK and ERK demonstrated that Akt, p38, and ERK axis of signaling pathway mediates HO-1 expression. Moreover, tert-butyl hydroperoxide (t-BHP)-induced oxidative damage was ameliorated by farrerol treatment in a dose-dependent manner, which was abolished by Akt, p38, ERK and HO-1 inhibitors (Snpp). It is hence likely that farrerol inactivated KEAP-1 or activated the Akt, p38 and ERK to facilitate the release of Nrf2 from Keap1 and subsequent reduced the intracellular production of reactive oxygen species via the induction of HO-1 expression. These results support the central role of HO-1 in the cytoprotective effect of farrerol.

Deubiquitinase family proteins are broadly involved in modulating antiviral response through dynamic and diverse mechanisms., 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.

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 IFNβ 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. 1066. This article is highlighted in the In This Issue feature, p. 1047

Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating T cells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8

Zika virus (ZIKV) has become a public health threat due to its global transmission and link to severe congenital disorders. The host immune responses to ZIKV infection have not been fully elucidated, and effective therapeutics are not currently available. Herein, we demonstrated that cholesterol-25-hydroxylase (CH25H) was induced in response to ZIKV infection and that its enzymatic product, 25-hydroxycholesterol (25HC), was a critical mediator of host protection against ZIKV. Synthetic 25HC addition inhibited ZIKV infection invitro by blocking viral entry, and treatment with 25HC reduced viremia and conferred protection against ZIKV in mice and rhesus macaques. 25HC suppressed ZIKV infection and reduced tissue damage in human cortical organoids and the embryonic brain of the ZIKV-induced mouse microcephaly model. Our findings highlight the protective role ofCH25H during ZIKV infection and the potential use of 25HC as a natural antiviral agent to combat ZIKV infection and prevent ZIKV-associated outcomes, such as microcephaly.

Huge efforts have been devoted to develop therapeutic monoclonal antibodies targeting human Programmed death-ligand 1 (hPD-L1) for treating various types of human cancers. However, thus far there is no suitable animal model for evaluating the anti-tumor efficacy of such antibodies against hPD-L1. Here we report the generation of a robust and effective system utilizing hPD-L1-expressing mouse tumor cells to study the therapeutic activity and mode of action of anti-human PD-L1 in mice. The model has been validated by using a clinically proven hPD-L1 blocking antibody. The anti-hPD-L1 antibody treatment resulted in potent dose-dependent rejection of the human PD-L1-expressing tumors in mice. Consistent with what have observed in autochthonous mouse tumor models and cancer patients, the hPD-L1 tumor bearing mice treated by anti-hPD-L1 antibody showed rapid activation, proliferation and reinvigoration of the cytolytic effector function of CD8+T cells inside tumor tissues. Moreover, anti-hPD-L1 treatment also led to profound inhibition of Treg expansion and shifting of myeloid cell profiles, showing bona fide induction of multilateral anti-tumor responses by anti-hPD-L1 blockade. Thus, this hPD-L1 mouse model system would facilitate the pre-clinical investigation of therapeutic efficacy and immune modulatory function of various forms of anti-hPD-L1 antibodies.

Mice are some of the widely used experimental animal models for studying human diseases. Defining the compositions of immune cell populations in various tissues from experimental mouse models is critical to understanding the involvement of immune responses in various physiological and patho-physiological conditions. However, non-lymphoid tissues are normally composed of vast and diverse cellular components, which make it difficult to quantify the relative proportions of immune cell types. Here we report the development of a computational algorithm, ImmuCC, to infer the relative compositions of 25 immune cell types in mouse tissues using microarray-based mRNA expression data. The ImmuCC algorithm showed good performance and robustness in many simulated datasets. Remarkable concordances were observed when ImmuCC was used on three public datasets, one including enriched immune cells, one with normal single positive T cells, and one with leukemia cell samples. To validate the performance of ImmuCC objectively, thorough cross-comparison of ImmuCC predicted compositions and flow cytometry results was done with in-house generated datasets collected from four distinct mouse lymphoid tissues and three different types of tumor tissues. The good correlation and biologically meaningful results demonstrate the broad utility of ImmuCC for assessing immune cell composition in diverse mouse tissues under various conditions.

Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

New influenza vaccines that provide effective and broad protection are desperately needed. Live attenuated viruses are attractive vaccine candidates because they can elicit both humoral and cellular immune responses. However, recent formulations of live attenuated influenza vaccines (LAIVs) have not been protective. We combined high-coverage transposon mutagenesis of influenza virus with a rapid high-throughput screening for attenuation to generate W7-791, a live attenuated mutant virus strain. W7-791 produced only a transient asymptomatic infection in adult and neonatal mice even at doses 100-fold higher than the LD50 of the parent strain. A single administration of W7-791 conferred full protection to mice against lethal challenge with H1N1, H3N2, and H5N1 strains, and improved viral clearance in ferrets. Adoptive transfer of Tcells from W7-791-immunized mice conferred heterologous protection, indicating a role for Tcell-mediated immunity. These studies present an LAIV development strategy to rapidly generate and screen entire libraries of viral clones.

Tripartite motif 14 (TRIM14) was reported to function as a mitochondrial signaling adaptor in mediating innate immune responses. However, the involvement of TRIM14 in host defense against viral infection and molecular mechanisms remain unclear. Here, we demonstrated that enforced expression of TRIM14 could potently inhibit the infection and replication of HCV in hepatocytes, whereas TRIM14 knockout cells became more susceptible to HCV infection. Interestingly, further experiments revealed that such anti-HCV activity was independent of activating the NF-κB or interferon pathways but required the C-terminal SPRY domain of no signaling capacity. In searching for mechanisms how TRIM14 exerts its antiviral function we found that TRIM14 interacted with HCV encoded non-structural protein NS5A and could strongly induce its degradation dependent on the NS5A1 subdomain. Interestingly extensive domain mapping analyses revealed that NS5A degradation was mediated by the highly conserved SPRY domain of TRIM14, which might involve the K48 ubiquitination pathway. Collectively, our work uncovered a new mechanism responsible for host defense against HCV infection, and could potentially aid the development of novel anti-HCV therapeutics.

Cancer cells modulate the recruitment and function of inflammatory cells to create an immunosuppressive microenvironment that favors tumor growth and metastasis. However, the tumor-derived regulatory programs that promote intratumoral immunosuppression remain poorly defined. Here, we show in a KrasLA1/+p53R172HΔg/+-based mouse model that bone morphogenetic protein-4 (BMP4) augments the expression of the T cell co-inhibitory receptor ligand PD-L1 in the mesenchymal subset of lung cancer cells, leading to profound CD8+ T cell-mediated immunosuppression, producing tumor growth and metastasis. We previously reported in this model that BMP4 functions as a pro-tumorigenic factor regulated by miR-200 via GATA4/6. Thus, BMP4-mediated immunosuppression is part of a larger miR-200-directed gene expression program in tumors that promotes tumor progression, which could have important implications for cancer treatment.

Recognition of self-peptide–MHC complexes by high-affinity TCRs and CD28 signaling are critical for the development of forkhead-winged helix box transcription factor 3+ regulatory T cells (Tregs) in thymus. However, the type of APCs that are responsible for selecting Tregs has remained unclear. To dissect the role of hematopoietic-derived APCs (HCs) and thymic epithelial cells (TECs) in Treg selection, we constructed bone marrow chimeras with disrupted CD28/B7 signaling in the HC or TEC compartment and analyzed the generation of Tregs in the thymus. We found that both HCs and TECs were independently able to fully reconstitute the Treg population in the thymus of bone marrow chimeras. In addition, Treg selection requires the TCR signal and CD28 costimulation presented in cis on the same APC type in vivo. This study demonstrates a new role, to our knowledge, for HCs in the development of Tregs in thymus.

Summary Despite the clinical successes fostered by immune checkpoint inhibitors, mechanisms underlying PD-1 upregulation in tumor-infiltrating T cells remain an enigma. Here, we show that tumor-repopulating cells (TRCs) drive PD-1 upregulation in CD8 + T cells through a transcellular kynurenine (Kyn)-aryl hydrocarbon receptor (AhR) pathway. Interferon-γ produced by CD8 + T cells stimulates release of high levels of Kyn produced by TRCs, which is transferred into adjacent CD8 + T cells via the transporters SLC7A8 and PAT4. Kyn induces and activates AhR and thereby upregulates PD-1 expression. This Kyn-AhR pathway is confirmed in both tumor-bearing mice and cancer patients and its blockade enhances antitumor adoptive T cell therapy efficacy. Thus, we uncovered a mechanism of PD-1 upregulation with potential tumor immunotherapeutic applications.

Regulatory T cells (Tregs) expressing forkhead/winged-helix transcription factor Foxp3 represent a distinct lineage of lymphocytes which play a central role in protecting the host from autoimmune diseases. However, Tregs also pose a major problem to anti-tumor immunity. Growing body of evidence from both laboratory and clinical investigations has demonstrated that expansion and accumulation of these immunosuppressive cells correlates with advanced tumor growth and predicts poor disease prognosis. How tumor development subverts normal self-tolerance function of Tregs thereby thwarts host anti-tumor immunity remains elusive. This review will discuss our current knowledge in understanding the dynamics and plasticity of Foxp3+ Treg activation and induction in tumor bearing hosts and their interaction with various antigen presenting cells (APCs) in tumor microenvironment leading to the establishment of active local and systemic immune suppression.

SummaryPrevious studies suggest that thymus produces a homogenous population of natural regulatory T (Treg) cells that express a transcriptional factor FOXP3 and control autoimmunity through a cell-contact-dependent mechanism. We found two subsets of FOXP3+ natural Treg cells defined by the expression of the costimulatory molecule ICOS in the human thymus and periphery. Whereas the ICOS+FOXP3+ Treg cells used interleukin-10 to suppress dendritic cell function and transforming growth factor (TGF)-β to suppress T cell function, the ICOS−FOXP3+ Treg cells used TGF-β only. The survival and proliferation of the two subsets of Treg cells were differentially regulated by signaling through ICOS or CD28, respectively. We suggest that the selection of natural Treg cells in thymus is coupled with Treg cell differentiation into two subsets imprinted with different cytokine expression potentials and use both cell-contact-dependent and independent mechanisms for immunosuppression in periphery.

The sirtuin family of nicotinamide adenine dinucleotide-dependent (NAD) deacetylases plays an important role in aging and metabolic regulation. In yeast, the Sir2 gene and its homolog Hst2 independently mediate the action of caloric restriction on lifespan extension. The mammalian Sir2 ortholog, SIRT1, is up-regulated by caloric restriction and deacetylates a variety of substrates, including histones and the forkhead box O (FOXO) transcription factors. The mammalian ortholog of Hst2, SIRT2, was shown to co-localize with microtubules and functions as alpha-tubulin deacetylase. During G2/M phase, SIRT2 proteins enter nuclei and deacetylate histones. We report here that the expression of SIRT2 is elevated in the white adipose tissue and kidney of caloric-restricted mice. Oxidative stress, such as hydrogen peroxide treatment, also increases SIRT2 expression in cells. We have demonstrated that SIRT2 binds to FOXO3a and reduces its acetylation level. SIRT2 hence increases FOXO DNA binding and elevates the expression of FOXO target genes, p27(Kip1), manganese superoxide dismutase and Bim. As a consequence, SIRT2 decreases cellular levels of reactive oxygen species. Furthermore, as Bim is a pro-apoptotic factor, SIRT2 promotes cell death when cells are under severe stress. Therefore, mammalian SIRT2 responds to caloric restriction and oxidative stress to deacetylate FOXO transcription factors.

Although there is evidence for distinct roles of myeloid dendritic cells (DCs [mDCs]) and plasmacytoid pre-DCs (pDCs) in regulating T cell–mediated adaptive immunity, the concept of functional DC subsets has been questioned because of the lack of a molecular mechanism to explain these differences. In this study, we provide direct evidence that maturing mDCs and pDCs express different sets of molecules for T cell priming. Although both maturing mDCs and pDCs upregulate the expression of CD80 and CD86, only pDCs upregulate the expression of inducible costimulator ligand (ICOS-L) and maintain high expression levels upon differentiation into mature DCs. High ICOS-L expression endows maturing pDCs with the ability to induce the differentiation of naive CD4 T cells to produce interleukin-10 (IL-10) but not the T helper (Th)2 cytokines IL-4, -5, and -13. These IL-10–producing T cells are T regulatory cells, and their generation by ICOS-L is independent of pDC-driven Th1 and Th2 differentiation, although, in the later condition, some contribution from endogenous IL-4 cannot be completely ruled out. Thus, in contrast to mDCs, pDCs are poised to express ICOS-L upon maturation, which leads to the generation of IL-10–producing T regulatory cells. Our findings demonstrate that mDC and pDCs are intrinsically different in the expression of costimulatory molecules that drive distinct types of T cell responses.

Induction of type I IFN (IFN-I) is essential for host antiviral immune responses. However, IFN-I also plays divergent roles in antibacterial immunity, persistent viral infections, autoimmune diseases, and tumorigenesis. IFN regulatory factor 3 (IRF3) is the master transcription factor that controls IFN-I production via phosphorylation-dependent dimerization in most cell types in response to viral infections and various innate stimuli by pathogen-associated molecular patterns (PAMPs). To monitor the dynamic process of IRF3 activation, we developed a novel IRF3 dimerization reporter based on bimolecular luminescence complementation (BiLC) techniques, termed the IRF3-BiLC reporter. Robust induction of luciferase activity of the IRF3-BiLC reporter was observed upon viral infection and PAMP stimulation with a broad dynamic range. Knockout of TANK-binding kinase 1, the critical upstream kinase of IRF3, as well as the mutation of serine 386, the essential phosphorylation site of IRF3, completely abolished the luciferase activity of IRF3-BiLC reporter, confirming the authenticity of IRF3 activation. Taken together, these results demonstrated that the IRF3-BiLC reporter is a highly specific, reliable, and sensitive system to measure IRF3 activity. Using this reporter system, we further observed that the temporal pattern and magnitude of IRF3 activation induced by various PAMPs are highly complex with distinct cell type–specific characteristics, and IRF3 dimerization is a direct regulatory node for IFN-α/β receptor–mediated feed-forward regulation and crosstalk with other pathways. Therefore, the IRF3-BiLC reporter has multiple potential applications, including mechanistic studies as well as the identification of novel compounds that can modulate IRF3 activation.

埃博拉病毒病是由纤丝病毒科(filoviridae)的埃博拉病毒(Ebola virus，EBOV)所引起的一种急性出血性传染病，该病病死率极高，达50%-90%, 是人类最致命的病毒性传染病之一。世界卫生组织统计数据显示，在2013年底至2015年西非埃博拉疫情大爆发中超过22500人感染埃博拉病毒，因感染丧生的人数达9000人。不论是感染病例数量、死亡人数，还是受影响地区范围，都达到了该病毒1976年被发现以来的最大规模。2014年8 月8 日，EBOV 的爆发被正式做为国际高度关注的突发公共卫生事件。目前，世界各国都没有有效控制埃博拉疫情的药物和治疗方法。 该文结合埃博拉病毒生命周期，从病毒入侵、宿主释放可溶性蛋白等角度分析了25-羟基胆固醇（25-hydroxyl cholesterol，25HC）等天然免疫抗病毒小分子化合物对埃博拉病毒的潜在抑制作用，从预防埃博拉病毒感染角度阐述了新的药物靶点的研究战略进展，运用系统生物学方法阐述了病原体-宿主之间的相互作用。 文章首先介绍了靶向埃博拉病毒入侵宿主细胞过程的小分子抑制剂的研究进展。不同于其他包膜病毒，埃博拉病毒是通过诱导细胞启动微胞饮（Macropinocytosis）作用而侵入细胞内部。最近研究显示，two-pore channel(TPC)蛋白家族在埃博拉病毒入侵宿主细胞的过程中扮演着重要角色，而小分子化合物Tetrandrine能够潜在特异性地抑制TPC通道，从而阻碍病毒的入侵过程。因此，类似于TPCs，靶向病毒入侵的其他小分子抑制药物还有待进一步研究发现。同时，宿主编码的干扰素诱导ISG蛋白具有强烈的抗病毒感染天然免疫效应功能。作者实验室研究结果显示胆固醇-25 羟化酶（Cholesterol-25-hydroxylase，CH25H）能够将胆固醇转化成一种可溶性因子-25 羟基胆固醇（25HC）。在P-4生物安全等级研究中证明25HC可显著降低野生型EBOV（Zaire Strain，扎伊尔株）对人体细胞的感染，显示其具有作为新型抗埃博拉病毒感染的紧急治疗药物的巨大潜力。 除了靶向病毒入侵过程，文章还描述了靶向病毒出芽从宿主细胞脱落、感染细胞释放可溶性蛋白过程的药物研究进展。其中，埃博拉病毒GP蛋白，无论是覆盖在病毒表面，还是在感染过程中从受感染细胞脱落，都可以参与多种免疫应答反应，在病毒生命周期以及宿主-病原体相互作用过程中扮演重要角色，是抑制埃博拉病毒侵染、复制、发病过程的潜在有效靶点。 目前，为国家抗击EBOV 病毒侵染、传播的小分子药物应急战略储备，研制和制备高效、安全且能快速效治疗埃博拉病毒的药物迫在眉睫。针对不同靶点的小分子抑制药物，进一步阐明埃博拉病毒病的发病机理还有待进一步研究。（