ACE2-dependent and -independent SARS-CoV-2 entries dictate viral replication and inflammatory response during infection.

Excessive inflammation is the primary cause of mortality in patients with severe COVID-19, yet the underlying mechanisms remain poorly understood. Our study reveals that ACE2-dependent and -independent entries of SARS-CoV-2 in epithelial cells versus myeloid cells dictate viral replication and inflammatory responses. Mechanistically, SARS-CoV-2 NSP14 potently enhances NF-κB signalling by promoting IKK phosphorylation, while SARS-CoV-2 ORF6 exerts an opposing effect. In epithelial cells, ACE2-dependent SARS-CoV-2 entry enables viral replication, with translated ORF6 suppressing NF-κB signalling. In contrast, in myeloid cells, ACE2-independent entry blocks the translation of ORF6 and other viral structural proteins due to inefficient subgenomic RNA transcription, but NSP14 could be directly translated from genomic RNA, resulting in an abortive replication but hyperactivation of the NF-κB signalling pathway for proinflammatory cytokine production. Importantly, we identified TLR1 as a critical factor responsible for viral entry and subsequent inflammatory response through interaction with E and M proteins, which could be blocked by the small-molecule inhibitor Cu-CPT22. Collectively, our findings provide molecular insights into the mechanisms by which strong viral replication but scarce inflammatory response during the early (ACE2-dependent) infection stage, followed by low viral replication and potent inflammatory response in the late (ACE2-independent) infection stage, may contribute to COVID-19 progression.

USP3 plays a critical role in the induction of innate immune tolerance.

Microbial products, such as lipopolysaccharide (LPS), can elicit efficient innate immune responses against invading pathogens. However, priming with LPS can induce a form of innate immune memory, termed innate immune "tolerance", which blunts subsequent NF-κB signaling. Although epigenetic and transcriptional reprogramming has been shown to play a role in innate immune memory, the involvement of post-translational regulation remains unclear. Here, we report that ubiquitin-specific protease 3 (USP3) participates in establishing "tolerance" innate immune memory through non-transcriptional feedback. Upon NF-κB signaling activation, USP3 is stabilized and exits the nucleus. The cytoplasmic USP3 specifically removes the K63-linked polyubiquitin chains on MyD88, thus negatively regulating TLR/IL1ß-induced inflammatory signaling activation. Importantly, cytoplasmic translocation is a prerequisite step for USP3 to deubiquitinate MyD88. Additionally, LPS priming could induce cytoplasmic retention and faster and stronger cytoplasmic translocation of USP3, enabling it to quickly shut down NF-κB signaling upon the second LPS challenge. This work identifies a previously unrecognized post-translational feedback loop in the MyD88-USP3 axis, which is critical for inducing normal "tolerance" innate immune memory.

Development of a TCR-like antibody and chimeric antigen receptor against NY-ESO-1/HLA-A2 for cancer immunotherapy.

BACKGROUND: The current therapeutic antibodies and chimeric antigen receptor (CAR) T cells are capable of recognizing surface antigens, but not of intracellular proteins, thus limiting the target coverage for drug development. To mimic the feature of T-cell receptor (TCR) that recognizes the complex of major histocompatibility class I and peptide on the cell surface derived from the processed intracellular antigen, we used NY-ESO-1, a cancer-testis antigen, to develop a TCR-like fully human IgG1 antibody and its derivative, CAR-T cells, for cancer immunotherapy. METHODS: Human single-chain variable antibody fragment (scFv) phage library (~10∧11) was screened against HLA-A2/NY-ESO-1 (peptide 157-165) complex to obtain target-specific antibodies. The specificity and affinity of those antibodies were characterized by flow cytometry, ELISA, biolayer interferometry, and confocal imaging. The biological functions of CAR-T cells were evaluated against target tumor cells in vitro. In vivo antitumor activity was investigated in a triple-negative breast cancer (TNBC) model and primary melanoma tumor model in immunocompromised mice. RESULTS: Monoclonal antibody 2D2 identified from phage-displayed library specifically bound to NY-ESO-1157-165 in the context of human leukocyte antigen HLA-A*02:01 but not to non-A2 or NY-ESO-1 negative cells. The second-generation CAR-T cells engineered from 2D2 specifically recognized and eliminated A2+/NY-ESO-1+tumor cells in vitro, inhibited tumor growth, and prolonged the overall survival of mice in TNBC and primary melanoma tumor model in vivo. CONCLUSIONS: This study showed the specificity of the antibody identified from human scFv phage library and demonstrated the potential antitumor activity by TCR-like CAR-T cells both in vitro and in vivo, warranting further preclinical and clinical evaluation of the TCR-like antibody in patients. The generation of TCR-like antibody and its CAR-T cells provides the state-of-the-art platform and proof-of-concept validation to broaden the scope of target antigen recognition and sheds light on the development of novel therapeutics for cancer immunotherapy.

Pharmacological inhibition of fatty acid synthesis blocks SARS-CoV-2 replication.

Caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 is a virus-induced inflammatory disease of the airways and lungs that leads to severe multi-organ damage and death. Here we show that cellular lipid synthesis is required for SARS-CoV-2 replication and offers an opportunity for pharmacological intervention. Screening a short-hairpin RNA sublibrary that targets metabolic genes, we identified genes that either inhibit or promote SARS-CoV-2 viral infection, including two key candidate genes, ACACA and FASN, which operate in the same lipid synthesis pathway. We further screened and identified several potent inhibitors of fatty acid synthase (encoded by FASN), including the US Food and Drug Administration-approved anti-obesity drug orlistat, and found that it inhibits in vitro replication of SARS-CoV-2 variants, including more contagious new variants, such as Delta. In a mouse model of SARS-CoV-2 infection (K18-hACE2 transgenic mice), injections of orlistat resulted in lower SARS-CoV-2 viral levels in the lung, reduced lung pathology and increased mouse survival. Our findings identify fatty acid synthase inhibitors as drug candidates for the prevention and treatment of COVID-19 by inhibiting SARS-CoV-2 replication. Clinical trials are needed to evaluate the efficacy of repurposing fatty acid synthase inhibitors for severe COVID-19 in humans.

Microbiota regulate innate immune signaling and protective immunity against cancer.

Microbiota play critical roles in regulating colitis and colorectal cancer (CRC). However, it is unclear how the microbiota generate protective immunity against these disease states. Here, we find that loss of the innate and adaptive immune signaling molecule, TAK1, in myeloid cells (Tak1ΔM/ΔM) yields complete resistance to chemical-induced colitis and CRC through microbiome alterations that drive protective immunity. Tak1ΔM/ΔM mice exhibit altered microbiota that are critical for resistance, with antibiotic-mediated disruption ablating protection and Tak1ΔM/ΔM microbiota transfer conferring protection against colitis or CRC. The altered microbiota of Tak1ΔM/ΔM mice promote IL-1ß and IL-6 signaling pathways, which are required for induction of protective intestinal Th17 cells and resistance. Specifically, Odoribacter splanchnicus is abundant in Tak1ΔM/ΔM mice and sufficient to induce intestinal Th17 cell development and confer resistance against colitis and CRC in wild-type mice. These findings identify specific microbiota strains and immune mechanisms that protect against colitis and CRC.

Beclin 2 negatively regulates innate immune signaling and tumor development.

Beclin 2 plays a critical role in metabolic regulation and obesity, but its functions in innate immune signaling and cancer development remain largely unknown. Here, we identified Beclin 2 as a critical negative regulator of inflammation and lymphoma development. Mice with homozygous ablation of BCL2-interacting protein 2 (Becn2) developed splenomegaly and lymphadenopathy and markedly increased ERK1/2 and NF-κB signaling for proinflammatory cytokine production. Beclin 2 targeted the key signaling kinases MEKK3 and TAK1 for degradation through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway. Mechanistically, Beclin 2 recruited MEKK3 or TAK1 through ATG9A to form a complex (Beclin 2-ATG9A-MEKK3) on ATG9A+ vesicles upon ULK1 activation. Beclin 2 further interacted with STX5 and STX6 to promote the fusion of MEKK3- or TAK1-associated ATG9A+ vesicles to phagophores for subsequent degradation. Importantly, Becn2-deficient mice had a markedly increased incidence of lymphoma development, with persistent STAT3 activation. Myeloid-specific ablation of MEKK3 (Map3k3) completely rescued the phenotypes (splenomegaly, higher amounts of proinflammatory cytokines, and cancer incidence) of Becn2-deficient mice. Hence, our findings have identified an important role of Beclin 2 in the negative regulation of innate immune signaling and tumor development through an ATG9A-dependent, but ATG16L/Beclin 1/LC3-independent, autophagic pathway, thus providing a potential target for the treatment of inflammatory diseases and cancer.

JMJD3 regulates CD4 T cell trafficking by targeting actin cytoskeleton regulatory gene Pdlim4.

Histone H3K27 demethylase, JMJD3 plays a critical role in gene expression and T-cell differentiation. However, the role and mechanisms of JMJD3 in T cell trafficking remain poorly understood. Here we show that JMJD3 deficiency in CD4+ T cells resulted in an accumulation of T cells in the thymus, and reduction of T cell number in the secondary lymphoid organs. We identified PDLIM4 as a significantly down-regulated target gene in JMJD3-deficient CD4+ T cells by gene profiling and ChIP-seq analyses. We further showed that PDLIM4 functioned as an adaptor protein to interact with S1P1 and filamentous actin (F-actin), thus serving as a key regulator of T cell trafficking. Mechanistically, JMJD3 bound to the promoter and gene body regions of Pdlim4 gene and regulated its expression by interacting with zinc finger transcription factor KLF2. Our findings have identified Pdlim4 as a JMJD3 target gene that affects T-cell trafficking by cooperating with S1P1, and provided insights into the molecular mechanisms by which JMJD3 regulates genes involved in T cell trafficking.

Downregulation of Human Endogenous Retrovirus Type K (HERV-K) Viral env RNA in Pancreatic Cancer Cells Decreases Cell Proliferation and Tumor Growth.

Purpose: We investigated the role of the human endogenous retrovirus type K (HERV-K) envelope (env) gene in pancreatic cancer.Experimental Design: shRNA was employed to knockdown (KD) the expression of HERV-K in pancreatic cancer cells.Results: HERV-K env expression was detected in seven pancreatic cancer cell lines and in 80% of pancreatic cancer patient biopsies, but not in two normal pancreatic cell lines or uninvolved normal tissues. A new HERV-K splice variant was discovered in several pancreatic cancer cell lines. Reverse transcriptase activity and virus-like particles were observed in culture media supernatant obtained from Panc-1 and Panc-2 cells. HERV-K viral RNA levels and anti-HERV-K antibody titers were significantly higher in pancreatic cancer patient sera (N = 106) than in normal donor sera (N = 40). Importantly, the in vitro and in vivo growth rates of three pancreatic cancer cell lines were significantly reduced after HERV-K KD by shRNA targeting HERV-K env, and there was reduced metastasis to lung after treatment. RNA-Seq results revealed changes in gene expression after HERV-K env KD, including RAS and TP53. Furthermore, downregulation of HERV-K Env protein expression by shRNA also resulted in decreased expression of RAS, p-ERK, p-RSK, and p-AKT in several pancreatic cancer cells or tumors.Conclusions: These results demonstrate that HERV-K influences signal transduction via the RAS-ERK-RSK pathway in pancreatic cancer. Our data highlight the potentially important role of HERV-K in tumorigenesis and progression of pancreatic cancer, and indicate that HERV-K viral proteins may be attractive biomarkers and/or tumor-associated antigens, as well as potentially useful targets for detection, diagnosis, and immunotherapy of pancreatic cancer. Clin Cancer Res; 23(19); 5892-911. ©2017 AACR.

Cytotoxicity of human endogenous retrovirus K-specific T cells toward autologous ovarian cancer cells.

PURPOSE: To determine whether HERV-K envelope (ENV) protein could function as a tumor-associated antigen and elicit specific T-cell responses against autologous ovarian cancer cells. EXPERIMENTAL DESIGN: The expression of HERV-K transcripts and ENV protein, the presence of serum antibodies against HERV-K, reverse transcriptase (RT) activities, and cellular immune responses in primary ovarian cancer tissues and patient blood samples were analyzed and compared with samples from patients with benign ovarian diseases and normal female donors. RESULTS: Ovarian cancer cells in primary tumors and ascites expressed markers of cancer stem cells and markers of both mesenchymal and epithelial cells. Expression of HERV transcripts and HERV-K ENV protein and reverse transcriptase activities were higher in ovarian cancer compared with adjacent normal and benign tissues. The ovarian cancer patient plasma also had high reverse transcriptase activities and the ovarian cancer patient sera contained HERV-K immunoreactive antibodies. HERV-K-specific T cells generated from autologous dendritic cells pulsed with HERV-K ENV antigens exhibited phenotypes and functions consistent with a cellular immune response including T-cell proliferation, IFNγ production, and HERV-K-specific cytotoxic T lymphocyte (CTL) activity. Significantly higher CTL lysis of autologous tumor cells than of uninvolved normal cells was demonstrated in patients with ovarian cancer than patients with benign diseases and further enhanced lysis was observed if T regulatory cells were depleted. CONCLUSION: Endogenous retroviral gene products in ovarian cancer may represent a potentially valuable new pool of tumor-associated antigens for targeting of therapeutic vaccines to ovarian cancer. Clin Cancer Res; 21(2); 471-83. ©2014 AACR.

Current advances in T-cell-based cancer immunotherapy.

Cancer is a leading cause of death worldwide; due to the lack of ideal cancer biomarkers for early detection or diagnosis, most patients present with late-stage disease at the time of diagnosis, thus limiting the potential for successful treatment. Traditional cancer treatments, including surgery, chemotherapy and radiation therapy, have demonstrated very limited efficacy for patients with late-stage disease. Therefore, innovative and effective cancer treatments are urgently needed for cancer patients with late-stage and refractory disease. Cancer immunotherapy, particularly adoptive cell transfer, has shown great promise in the treatment of patients with late-stage disease, including those who are refractory to standard therapies. In this review, we will highlight recent advances and discuss future directions in adoptive cell transfer based cancer immunotherapy.

Human endogenous retrovirus type K antibodies and mRNA as serum biomarkers of early-stage breast cancer.

A simple and accurate test to detect early-stage breast cancer has not been developed. Previous studies indicate that the level of human endogenous retrovirus type K (group HERV-K(HML-2)) transcription may be increased in human breast tumors. We hypothesized that HERV-K(HML-2) reactivation can serve as a biomarker for early detection of breast cancer. Serum samples were collected from women without cancer (controls) and patients with ductal carcinoma in situ (DCIS) and invasive breast cancer. ELISA assays were used to detect serum anti-HERV-K(HML-2) antibody titers. RNA was extracted from sera and analyzed by real-time RT-PCR to quantitate the level of HERV-K(HML-2) mRNA. We measured significantly higher serum mRNA and serum antibody titers against HERV-K(HML-2) proteins in women with DCIS and stage I disease than in women without cancer. At optimized cutoffs for the antibody titers, the assay produced an area under the receiver operating characteristic curve (AUC) of 0.89 (95% confidence interval 0.77-1.00) for DCIS and of 0.95 (95% confidence interval 0.89-1.00) for invasive breast cancer. These AUCs are comparable to those observed for mammograms. We also found that serum HERV-K(HML-2) mRNA tended to be higher in breast cancer patients with a primary tumor who later on developed the metastatic disease than in patients who did not develop cancer metastasis. Our results show that HERV-K(HML-2) antibodies and mRNA are already elevated in the blood at an early stage of breast cancer, and further increase in patients who are at risk of developing a metastatic disease.

Identification of special AT-rich sequence binding protein 1 as a novel tumor antigen recognized by CD8+ T cells: implication for cancer immunotherapy.

BACKGROUND: A large number of human tumor-associated antigens that are recognized by CD8(+) T cells in a human leukocyte antigen class I (HLA-I)-restricted fashion have been identified. Special AT-rich sequence binding protein 1 (SATB1) is highly expressed in many types of human cancers as part of their neoplastic phenotype, and up-regulation of SATB1 expression is essential for tumor survival and metastasis, thus this protein may serve as a rational target for cancer vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Twelve SATB1-derived peptides were predicted by an immuno-informatics approach based on the HLA-A*02 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from HLA-A*02(+) healthy donors and/or HLA-A*02(+) cancer patients. The recognition of HLA-A*02(+) SATB1-expressing cancer cells was also tested. Among the twelve SATB1-derived peptides, SATB1(565-574) frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and cancer patients. Importantly, SATB1(565-574)-specific T cells recognized and killed HLA-A*02(+) SATB1(+) cancer cells in an HLA-I-restricted manner. CONCLUSIONS/SIGNIFICANCE: We have identified a novel HLA-A*02-restricted SATB1-derived peptide epitope recognized by CD8(+) T cells, which, in turn, recognizes and kills HLA-A*02(+) SATB1(+) tumor cells. The SATB1-derived epitope identified may be used as a diagnostic marker as well as an immune target for development of cancer vaccines.

Identification of prostate-specific G-protein coupled receptor as a tumor antigen recognized by CD8(+) T cells for cancer immunotherapy.

BACKGROUND: Prostate cancer is the most common cancer among elderly men in the US, and immunotherapy has been shown to be a promising strategy to treat patients with metastatic castration-resistant prostate cancer. Efforts to identify novel prostate specific tumor antigens will facilitate the development of effective cancer vaccines against prostate cancer. Prostate-specific G-protein coupled receptor (PSGR) is a novel antigen that has been shown to be specifically over-expressed in human prostate cancer tissues. In this study, we describe the identification of PSGR-derived peptide epitopes recognized by CD8(+) T cells in an HLA-A2 dependent manner. METHODOLOGY/PRINCIPAL FINDINGS: Twenty-one PSGR-derived peptides were predicted by an immuno-informatics approach based on the HLA-A2 binding motif. These peptides were examined for their ability to induce peptide-specific T cell responses in peripheral blood mononuclear cells (PBMCs) obtained from either HLA-A2(+) healthy donors or HLA-A2(+) prostate cancer patients. The recognition of HLA-A2 positive and PSGR expressing LNCaP cells was also tested. Among the 21 PSGR-derived peptides, three peptides, PSGR3, PSGR4 and PSGR14 frequently induced peptide-specific T cell responses in PBMCs from both healthy donors and prostate cancer patients. Importantly, these peptide-specific T cells recognized and killed LNCaP prostate cancer cells in an HLA class I-restricted manner. CONCLUSIONS/SIGNIFICANCE: We have identified three novel HLA-A2-restricted PSGR-derived peptides recognized by CD8(+) T cells, which, in turn, recognize HLA-A2(+) and PSGR(+) tumor cells. The PSGR-derived peptides identified may be used as diagnostic markers as well as immune targets for development of anticancer vaccines.

Immunotherapeutic potential of anti-human endogenous retrovirus-K envelope protein antibodies in targeting breast tumors.

BACKGROUND: The envelope (env) protein of the human endogenous retrovirus type K (HERV-K) family is commonly expressed on the surface of breast cancer cells. We assessed whether HERV-K env is a potential target for antibody-based immunotherapy of breast cancer. METHODS: We examined the expression of HERV-K env protein in various malignant (MDA-MB-231, MCF-7, SKBR3, MDA-MB-453, T47D, and ZR-75-1) and nonmalignant (MCF-10A and MCF-10AT) human breast cell lines by immunoblot, enzyme-linked immunosorbent assay, immunofluorescence staining, and flow cytometry. Anti-HERV-K env monoclonal antibodies (mAbs; 6H5, 4D1, 4E11, 6E11, and 4E6) were used to target expression of HERV-K, and antitumor effects were assessed by quantifying growth and apoptosis of breast cancer cells in vitro, and tumor growth in vivo in mice (n = 5 per group) bearing xenograft tumors. The mechanisms responsible for 6H5 mAb-mediated effects were investigated by microarray assays, flow cytometry, immunoblot, and immunofluorescence staining. The expression of HERV-K env protein was assessed in primary breast tumors (n = 223) by immunohistochemistry. All statistical tests were two-sided. RESULTS: The expression of HERV-K env protein in malignant breast cancer cell lines was substantially higher than nonmalignant breast cells. Anti-HERV-K-specific mAbs inhibited growth and induced apoptosis of breast cancer cells in vitro. Mice treated with 6H5 mAb showed statistically significantly reduced growth of xenograft tumors compared with mice treated with control immunoglobulin (control [mIgG] vs 6H5 mAb, for tumors originating from MDA-MB-231 cells, mean size = 1448.33 vs 475.44 mm(3); difference = 972.89 mm(3), 95% CI = 470.17 to 1475.61 mm(3); P < .001). Several proteins involved in the apoptotic signaling pathways were overexpressed in vitro in 6H5 mAb-treated malignant breast cells compared with mIgG-treated control. HERV-K expression was detected in 148 (66%) of 223 primary breast tumors, and a higher rate of lymph node metastasis was associated with HERV-K-positive compared with HERV-K-negative tumors (43% vs 23%, P = .003). CONCLUSION: Monoclonal antibodies against HERV-K env protein show potential as novel immunotherapeutic agents for breast cancer therapy.

Expression of Human Endogenous Retrovirus Type K Envelope Protein is a Novel Candidate Prognostic Marker for Human Breast Cancer.

We previously observed that the HERV type K (HERV-K) envelope (env) protein was expressed in the majority of human breast tumors from a U.S. cohort of women from Texas. We also made the preliminary observation that the expression of HERV-K env transcripts was associated with markers of disease progression. In this follow-up study, env protein expression was evaluated immunohistochemically in an additional 195 paraffin-embedded breast tumors from a second U.S. patient cohort (Baltimore, Maryland) and in 110 tumors from Chinese patients. Moreover, we compared env transcript expression between fresh-frozen normal and cancerous breast tissues. We observed that while env mRNA and protein expression was undetectable in normal breast tissue and in a subset of uninvolved normal-appearing tissue adjacent to the tumor epithelium, it was overexpressed in most tumors. Furthermore, env expression was associated with breast cancer progression. In Baltimore cohort women, HERV-K tumor positivity was significantly associated with disease stage and lymph node metastasis. In Chinese women, HERV-K env positivity was significantly associated with tumor size, TNM stage, and lymph node metastases, which is consistent with the observations in the U.S. cohort. We also found that Chinese breast cancer patients with a high expression of HERV-K had a decreased overall survival compared with patients who had either a moderate or low HERV-K expression in their tumors (P = 0.049, χ(2) log rank test). In conclusion, the HERV-K env gene is expressed in the majority of breast cancers from U.S. or Chinese women but not in normal breast tissue. High expression of HERV-K env protein in breast cancer patients is associated with markers of disease progression and poor disease outcome, indicating that HERV-K env protein is a novel candidate prognostic marker for breast cancer.