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Target identification for chimeric antigen receptor (CAR) T-cell therapies remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through T-cell receptor mimic antibodies. Mass spectrometry (MS) of HLA ligands from 8 hematologic and nonhematologic cancer cell lines identified a shared, non-immunogenic, HLA-A*02–restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene. CAR T cells directed against the ALNEQIARL:HLA-A*02 complex exhibited high sensitivity and specificity for recognition and killing of multiple cancer types, especially those of hematologic origin, and were efficacious in mouse models against a human leukemia and a solid tumor. In contrast, no toxicities toward resting or activated healthy leukocytes as well as hematopoietic stem cells were observed. This shows how MS can inform the design of broadly reactive therapeutic T-cell receptor mimic CAR T-cell therapies that can target multiple cancer types currently not druggable by small molecules, conventional CAR T cells, T cells, or antibodies.

Chimeric antigen receptor (CAR) T cell therapy has shown success in the treatment of hematopoietic malignancies; however, relapse remains a significant issue. To overcome this, we engineered "Orexi" CAR T cells to locally secrete a high affinity CD47-blocker, CV1, at the tumor, and treated tumors in combination with an orthogonally targeted monoclonal antibody. Traditional CAR T cells plus antibody were additive in effect in xenograft models and this effect was potentiated by CAR T cell local CV1 secretion. Furthermore, OrexiCAR-secreted CV1 reversed immunosuppression of myelomonocytoid cells, both in vitro and within the tumor microenvironment. Local secretion of the CD47 inhibitor bypasses the CD47 sink found on all cells in the body and may prevent systemic toxicities. This combination of CAR T cell therapy, local CD47 blockade, and orthogonal antibody may be a combinatorial strategy to overcome the limitations of each individual monotherapy.

T cell exhaustion resulting from chronic antigen stimulation and immunosuppression in the tumor microenvironment (TME) limits CAR T efficacy in the solid tumor setting. We have previously shown that engineering therapeutic T cell products using CRISPR-based gene insertion of a dual shRNA cassette targeting Fas and PTPN2 significantly increased antitumor efficacy of Integrated Circuit T cells (ICTs) in ovarian cancer models. We sought to build on this finding to induce additional gene perturbations that improve the efficacy of ICTs. Transforming growth factor (TGF)-b is an immunosuppressive cytokine that potently inhibits T cell responses and is present at high levels in numerous solid tumors, including renal cell carcinoma (RCC). In order to render ICT cells less susceptible to TGF-b-mediated suppression, we developed a quadruple shRNA cassette that simultaneously targets Fas, PTPN2, and TGFBR. Candidate TGFBR-targeting shRNAs were selected for their ability to reduce surface TGFBR receptor expression and impair proximal (pSMAD) or distal (CD103, PD-1) signaling through TGFBR. While single shRNAs against TGFBR did not rescue ICT cell activity in the presence of TGF-b, likely due to partial knockdown of TGFBR signaling, a cassette encoding two shRNAs against TGFBR restored ICT function to similar levels observed in the absence of TGF-b. The quadruple shRNA cassettes targeting Fas/PTPN2/TGFBR significantly enhanced antitumor activity of ICT cells in multiple xenograft tumor models relative to Fas/PTPN2 cassettes. These results demonstrate the utility of multiplexed shRNA strategies to render therapeutic T cells resistant to orthogonal suppressive pathways in solid tumors. Citation Format: Thomas J. Gardner, Adam Litterman, Brenal K. Singh, Luisa Silva, Mukund Hari, Stanley Zhou, Colin Tang, Sahil Joshi, John Gagnon, Oliver Takacsi-Nagy, Jason Hall, Hans Pope, James Zhang, Alma Gomez, Jeremy Chen, Suchismita Mohanty, Vince Thomas, Nicholas Quant, Beatriz Millare, Amy-Jo Casbon, Natalie Bezman, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Multiplexed shRNA cassettes targeting orthogonal pathways (FAS/PTPN2/TGFBR) enhance the potency of integrated circuit T cells (ICTs) in multiple solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1768.

Clinically effective adoptive T cell therapy for the treatment of solid tumors will require robust T cell expansion, persistence, and potency. The Janus-kinase signal transducer and activator of transcription (JAK-STAT) pathway governs T cell activation and differentiation, and can thereby serve as a critical regulator of these properties. To take advantage of the benefits of STAT signaling in programming an antitumor T-cell response, we used synthetic biology to create a library of proteins, termed Synthetic Pathway Activators (SPAs) which constitutively drive STAT signaling without the need for external cytokine input. SPAs can be designed to engage activity of multiple STAT family transcription factors at variable levels through rational design. We have developed several classes of SPAs, including but not limited to Class I SPAs, which primarily increase pSTAT3 activity, and Class II SPAs, which increase pSTAT5 activity. When constitutively expressed in ArsenalBio Integrated Circuit T (ICT) cells, SPAs result in significant enhancements in T-cell potency and expansion. Repetitive stimulation assays, wherein T cells are challenged with tumor cells every 2 days, reveal that Class I SPAs result in 6-log or higher improved tumor cell clearance over a 2-week assay period. Across various mouse xenograft models, SPA-expressing ICTs reach at least 6-fold improved tumor growth inhibition. RNAseq and ATACseq analysis indicate dramatic changes to gene expression profiles in T cells expressing Class I SPAs, with maintenance of T cell stem-like phenotypes, and restricted accessibility of various exhaustion marker genes. Importantly, despite significantly increased levels of expansion, ICTs equipped with SPAs are not immortalized, showing no signs of cytokine-independent outgrowth. In addition, SPA-expressing ICT cells rapidly contract following tumor clearance in-vivo. The SPA platform represents a novel, tunable, and T cell intrinsic approach for engineering cell fates that result in potent anti-tumor properties. Citation Format: Thomas J. Gardner, Beatriz Millare, Anzhi Yao, Ashley Cass, Suchismita Mohanty, Jeremy Chen, Alma Gomez, David DeTomaso, Manching Ku, Lionel Berthoin, Meng Lim, Azalea Ong, Vince Thomas, Nicholas Quant, Brian Hsu, Amy-Jo Casbon, Natalie Bezman, Aaron Cooper, Levi Gray-Rupp, Angela C. Boroughs, W. Nicholas Haining. Tunable STAT activation by synthetic pathway activators (SPAs) increases engineered T-cell potency and persistence. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4073.

Human cytomegalovirus (CMV) is a ubiquitous β-herpesvirus that establishes latent asymptomatic infections in healthy individuals but can cause serious infections in immunocompromised people, resulting in increased risk of morbidity and mortality. The current FDA-approved CMV drugs target late stages of the CMV life-cycle. While these drugs are effective in most cases, they have serious drawbacks, including poor oral bioavailability, dose-limiting toxicity, and a low barrier to resistance. Given the clinical relevance of CMV-associated diseases, novel therapies are needed. Thus, a novel class of compounds that inhibits the early stages of the CMV life-cycle was identified and found to block infection of different strains in physiologically relevant cell types. This class of compounds, N-arylpyrimidinamine (NAPA), demonstrated potent anti-CMV activity against ganciclovir-sensitive and -resistant strains in in vitro replication assays, a selectivity index30, and favorable in vitro ADME properties. Mechanism of action studies demonstrated that NAPA compounds inhibit an early step of virus infection. NAPA compounds are specific inhibitors of cytomegaloviruses and exhibited limited anti-viral activity against other herpesviruses. Collectively, we have identified a novel class of CMV inhibitor that effectively limits viral infection and proliferation.

Chimeric Antigen Receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient’s own T cells are engineered to recognize a tumor antigen, resulting in activation of a local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B-cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T-cell immunologic functions, and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically-administered small-molecule prodrug in situ at a tumor site. We show that these Synthetic Enzyme-Armed KillER (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.

The recent emergence of engineered cellular therapies, such as Chimeric antigen receptor (CAR) CAR T and T cell receptor (TCR) engineered T cells, has shown great promise in the treatment of various cancers. These agents aggregate and expand exponentially at the tumor site, resulting in potent immune activation and tumor clearance. Moreover, the ability to elaborate these cells with therapeutic agents, such as antibodies, enzymes, and immunostimulatory molecules, presents an unprecedented opportunity to specifically modulate the tumor microenvironment through cell-mediated drug delivery. This unique pharmacology, combined with significant advances in synthetic biology and cell engineering, has established a new paradigm for cells as vectors for drug delivery. Targeted cellular micropharmacies (TCMs) are a revolutionary new class of living drugs, which we envision will play an important role in cancer medicine and beyond. Here, we review important advances and considerations underway in developing this promising advancement in biological therapeutics.

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.

Context Target identification for CAR-T cell therapies, especially shared targets among different malignancies, remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through TCR-mimic antibodies. Objective and Design We hypothesized that mass spectrometry (MS)-based analysis of the presented HLA ligands of multiple cancer cell lines can be utilized to identify a shared, tumor-associated HLA ligand, which could then be targeted by TCR-mimic CAR-T cells specific for this particular HLA ligand. Results MS of HLA ligands from eight hematological and non-hematological cancer cell lines identified a shared, non-immunogenic, HLA-A*02 restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene, which was later identified in >90% (20/22) of all A*02-positive cell lines tested. A TCR-mimic scFv was prepared against this epitope, termed “NDC80-C.” CAR-T cells transduced with the NDC80-C, directed against the ALNEQIARL:HLA-A*02 complex on cancer cells, demonstrated high sensitivity and specificity for recognizing and killing multiple cancer types with a high preference for hematological malignancies (e.g., AML, ALL, DLBCL, and ALCL). In contrast, healthy leukocytes, activated B and T-cells, or hematopoietic stem cells from A*02-positive and -negative donors were not lysed. NDC80-C CAR-T cells suppressed colony formation of primary AML cells but not of healthy stem cells. Additionally, NDC80-C CAR-T cells were efficacious in mouse models against human mesothelioma and leukemia. Conclusions Our study demonstrates how MS can inform the design of tumor-agnostic TCR-mimic therapeutic platforms that target structures that are currently not druggable by small molecules, conventional CAR-T cells, T-cells, or antibodies. This strategy lays the groundwork for a potential antibody platform therapy or CAR-T cell with efficacy against highly proliferative A*02-positive cancer cells, independent of the respective cancer type.

Chimeric antigen receptor (CAR) T cells represent a novel class of FDA-approved drugs with high efficacy against refractory B cell derived malignancies and potentially other cancer types. However, target selection for CAR T cell therapy remains challenging as cell surface proteins are not cancer-specific and therefore often not adaptable for CAR T cell therapy. In contrast, many intracellular proteins can be highly tumor specific and are targetable after proteasomal degradation and presentation on human leukocyte antigen (HLA) complexes recognized by T cell receptor mimic antibodies. This class of antibodies recognizes peptide:HLA complexes with a similar mode of recognition as a TCR, but with the clinical versatility and applicability of an antibody. To identify a tumor specific target that is presented as a peptide in conjunction with the highly prevalent HLA allele A*02:01, we immunopurified peptide:HLA complexes from various cancer cell lines of different origins, separated HLA ligands from complexes and identified their peptide sequences via mass spectrometry. Network analysis of the resulting HLA ligand datasets identified shared biological processes among the tumor cell lines that were not present in network analyses of published datasets of healthy human tissue HLA ligandomes. Through this filtering process several potential targets were identified and an HLA ligand derived from kinetochore NDC80 protein homolog (NDC80) was selected as a target. The NDC80 derived peptide was detected in over 90% of the A*02 positive cell lines tested and never reported to be present in HLA ligand datasets of healthy human tissues. Furthermore, NDC80 has been shown to be differentially expressed in malignant compared to adjacent non-malignant tissues and is associated with poor prognosis in many cancer types. After utilizing E-ALPHA®phage library screening, one clone (NDC80-L1) was selected as the lead TCR mimic antibody. Overall, NDC80-L1 showed high specificity for the target HLA:peptide complex in both antibody and CAR T cell format in vitro and demonstrated binding primarily to the central region of the HLA ligand as determined by alanine screening assays. The exquisite specificity of NDC80-L1 was further illustrated by NDC80 knockdown experiments as well as successful immunopurification of the target peptide together with no relevant off-targets from BV173 ALL cells in mass spectrometry assays. Given the high specificity, sensitivity was assessed primarily in a potent CAR T cell format: Multiple tumor cell lines of different origin (e.g. ALL, AML, lymphoma, melanoma, mesothelioma, pancreatic and thyroid cancer) were successfully killed in vitro by NDC80-L1 CAR T cells, but no toxicity towards A*02:01 positive CAR T cells, healthy PBMCs or NDC80 target negative cell lines was observed. Interestingly, NDC80-L1 CAR T cells demonstrated highest efficacy in hematological malignancies most likely correlating with elevated expression of antigen presentation machinery and rapid cell division which leads to strong surface expression of NDC80 peptides. In summary, CAR T cells directed against peptide/HLA-A*02 derived from the NDC80 protein effectively kill multiple cancer cell lines in vitro without evidence of relevant off-target killing. However, the improved killing especially against ALL, AML and lymphomas highlights the potential of these CAR T cells to preferentially eliminate cancer cells with high proliferative capacity. Future in vivo studies with CAR T cell and antibody format will further investigate this TCR mimic antibody's potential as a tumor-agnostic therapeutic agent. Disclosures Klatt: MSKCC/EUREKA: Patents & Royalties: MSKCC AND EUREKA THERAPUETICS HAVE FILED A PATENT FOR THIS ANTIBODY/SCFV. Yang:Eureka Therapuetics: Current Employment, Current equity holder in private company, Patents & Royalties: MSKCC and Eureka have filed patent for this TCRm and ScvF. Liu:Eureka Therapue: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Dao:Eureka Therapeutics: Consultancy. Liu:Eureka Therapeutics: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Scheinberg:Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Arvenas: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Oncopep: Consultancy; Pfizer: Consultancy, Current equity holder in private company; Lantheus: Current equity holder in private company; Enscyse: Current equity holder in private company.

Autoantibodies targeting the H+/K+-ATPase proton pump of the gastric parietal cell (parietal cell antibodies [PCA]) are diagnostic of atrophic body gastritis (ABG) leading to pernicious anemia (PA). PCA, ABG, and PA occur in increased frequency in patients with type 1 diabetes and their relatives and are considered “minor” components of forms of autoimmune polyglandular syndrome (APS). A customized radioimmunoprecipitation assay was applied to 6,749 samples from the Type 1 Diabetes Genetics Consortium to measure ATP4A autoreactivity. Autoantibody prevalence was correlated with variants in HLA class II, PTPN22, and CTLA4 genes. With an ATP4A radioimmunoprecipitation assay, PCA were detected in sera from 20.9% of affected individuals. PCA prevalence increased with age and was greater in females (25.3%) than males (16.5%) and among Hispanics (36.3%) and blacks (26.2%) compared with non-Hispanic whites (20.8%) and Asians (16.7%). PCA and other organ-specific autoantibodies GAD65, IA-2, thyroid peroxidase (TPO), 21-hydroxylase (21-OH), and transglutaminase (TG) clustered within families with heritability estimates from 71 to 95%. PCA clustered with TPO, 21-OH, and persistent GAD65 autoantibodies but not with celiac (TG) or IA-2 autoantibodies. PCA-positive subjects showed an increased frequency of DRB1*0404, DPB1*0201, and PTPN22 R620W (rs2476601-T) and a decreased frequency of DRB1*0101, DPB1*0301, and CTLA4 CT60 (rs3087243-T). Genetic variants accounted for 4–5% of the heritable risk for PCA. The same alleles were associated with other autoantibody phenotypes in a consistent pattern. Whereas most of the heritable risk for PCA and other antibodies reflects genetic effects that are tissue specific, parietal cell autoimmunity is a major pathogenetic contributor in APS2.

Ricin toxin, an A-B toxin from Ricinus communis, induces cell death through the inhibition of protein synthesis. The toxin binds to the cell surface via its B chain (RTB) followed by its retrograde trafficking through intracellular compartments to the ER where the A chain (RTA) is transported across the membrane and into the cytosol. Ricin A chain is transported across the ER membrane utilizing cellular proteins involved in the disposal of aberrant ER proteins by a process referred to as retrograde translocation. Given the current lack of therapeutics against ricin intoxication, we developed a high-content screen using an enzymatically attenuated RTA chimera engineered with a carboxy-terminal enhanced green fluorescent protein (RTA(E177Q)egfp) to identify compounds that target RTA retrograde translocation. Stabilizing RTA(E177Q)egfp through the inclusion of proteasome inhibitor produced fluorescent peri-nuclear granules. Quantitative analysis of the fluorescent granules provided the basis to discover compounds from a small chemical library (2080 compounds) with known bioactive properties. Strikingly, the screen found compounds that stabilized RTA molecules within the cell and several compounds limited the ability of wild type RTA to suppress protein synthesis. Collectively, a robust high-content screen was developed to discover novel compounds that stabilize intracellular ricin and limit ricin intoxication.

Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro . In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169 IE2-YFP ) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169 IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.

The prototypic β-herpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. The CMV envelope consists of various protein complexes that enable wide viral tropism. More specifically, the glycoprotein complex gH/gL/gO (gH-trimer) is required for infection of all cell types, while the gH/gL/UL128/130/131a (gH-pentamer) complex imparts specificity in infecting epithelial, endothelial and myeloid cells. Here we utilize state-of-the-art robotics and a high-throughput neutralization assay to screen and identify monoclonal antibodies (mAbs) targeting the gH glycoproteins that display broad-spectrum properties to inhibit virus infection and dissemination. Subsequent biochemical characterization reveals that the mAbs bind to gH-trimer and gH-pentamer complexes and identify the antibodies' epitope as an ‘antigenic hot spot' critical for virus entry. The mAbs inhibit CMV infection at a post-attachment step by interacting with a highly conserved central alpha helix-rich domain. The platform described here provides the framework for development of effective CMV biologics and vaccine design strategies., Human cytomegalovirus (CMV) poses a risk for immunosuppressed patients and newborns, with limited treatment options available. Here, Gardner et al. use a high-throughput approach and identify monoclonal antibodies that bind a highly conserved domain in the viral glycoprotein gH as potent inhibitors of CMV infection.

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease.

ISG15 is an interferon (IFN)-α/β-induced ubiquitin-like protein. It exists as a free molecule, intracellularly and extracellularly, and conjugated to target proteins. Studies in mice have demonstrated a role for Isg15 in antiviral immunity. By contrast, human ISG15 was shown to have critical immune functions, but not in antiviral immunity. Namely, free extracellular ISG15 is crucial in IFN-γ-dependent antimycobacterial immunity, while free intracellular ISG15 is crucial for USP18-mediated downregulation of IFN-α/β signalling. Here we describe ISG15-deficient patients who display no enhanced susceptibility to viruses in vivo, in stark contrast to Isg15-deficient mice. Furthermore, fibroblasts derived from ISG15-deficient patients display enhanced antiviral protection, and expression of ISG15 attenuates viral resistance to WT control levels. The species-specific gain-of-function in antiviral immunity observed in ISG15 deficiency is explained by the requirement of ISG15 to sustain USP18 levels in humans, a mechanism not operating in mice., ISG15 is a ubiquitin-like protein which has important immune-related functions in mice and humans. Here the authors demonstrate that, unlike in mice, human ISG15 stabilizes UPS18 and that ISG15-deficient human cells are more resistant to viral infection.

Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is proficient at establishing lifelong persistence within the host in part due to immune modulating genes that limit immune recognition. HCMV encodes at least five glycoproteins within its unique short (US) genomic region that interfere with MHC class I antigen presentation, thus hindering viral clearance by cytotoxic T lymphocytes (CTL). Specifically, US3 retains class I within the endoplasmic reticulum (ER), while US2 and US11 induce class I heavy chain destruction. A cooperative effect on class I down-regulation during stable expression of HCMV US2 and US3 has been established. To address the impact of US3 on US11-mediated MHC class I down-regulation, the fate of class I molecules was examined in US3/US11-expressing cells and virus infection studies. Co-expression of US3 and US11 resulted in a decrease of surface expression of class I molecules. However, the class I molecules in US3/US11 cells were mostly retained in the ER with an attenuated rate of proteasome destruction. Analysis of class I levels from virus-infected cells using HCMV variants either expressing US3 or US11 revealed efficient surface class I down-regulation upon expression of both viral proteins. Cells infected with both US3 and US11 expressing viruses demonstrate enhanced retention of MHC class I complexes within the ER. Collectively, the data suggests a paradigm where HCMV-induced surface class I down-regulation occurs by diverse mechanisms dependent on the expression of specific US genes. These results validate the commitment of HCMV to limiting the surface expression of class I levels during infection.

Background Autoimmune gastritis (ABG) and pernicious anemia (PA) are prototypical, organ-specific autoimmune diseases whose prevalence in the general population are 2 and 0.15-1%, respectively. The incidence of disease increases with age and with is frequently associated with other autoimmune disorders such as type 1 diabetes (T1D). Early diagnosis of ABG/PA is essential for the prevention and/or treatment before manifestations of chronic disease are irreversible. Parietal cell autoantibody (PCA) detection via ELISA is currently the most widely used biomarker of disease with diagnosis confirmed by subsequent immunohistochemistry via biopsy.

SummaryConsiderable details about microRNA (miRNA) biogenesis and regulation have been uncovered, but little is known about the fate of the miRNA subsequent to target regulation. To gain insight into this process, we carried out kinetic analysis of a miRNA's turnover following termination of its biogenesis and during regulation of a target that is not subject to Ago2-mediated catalytic cleavage. By quantitating the number of molecules of the miRNA and its target in steady state and in the course of its decay, we found that each miRNA molecule was able to regulate at least two target transcripts, providing in vivo evidence that the miRNA is not irreversibly sequestered with its target and that the nonslicing pathway of miRNA regulation is multiple-turnover. Using deep sequencing, we further show that miRNA recycling is limited by target regulation, which promotes posttranscriptional modifications to the 3′ end of the miRNA and accelerates the miRNA's rate of decay. These studies provide new insight into the efficiency of miRNA regulation that help to explain how a miRNA can regulate a vast number of transcripts and that identify one of the mechanisms that impart specificity to miRNA decay in mammalian cells.

Zinc transporter 8 (ZnT8) is a newly discovered islet autoantigen in human type 1A diabetes (T1D).The objective was to document changes in ZnT8 autoantibody (ZnT8A) titer and prevalence after onset of disease in relationship to 65 kDa glutamate decarboxylase antibody (GADA) and islet cell antigen antibody (IA2A).Autoantibody radioimmunoprecipitation assays were performed on sera from three groups: 21 individuals monitored every 3 months from diagnosis for 2.5 yr; 61 individuals monitored at six monthly intervals for 5-12 yr; and a cross-sectional study of 424 patients with T1D of 20-57 yr duration. Circulating C-peptide was determined as an index of residual β-cell function.ZnT8A titers declined exponentially from clinical onset of T1D with a t(1/2) ranging from 26 to 530 wk, similar to C-peptide (23-300 wk). Life-table analysis of antibody prevalence to 12 yr indicated that ZnT8A measured with either Arg325 or Trp325 probes persisted for a shorter interval than IA2A. Although prevalence of ZnT8A, IA2A, and GADA were comparable at disease onset (70.4 vs. 73.4 vs. 64%), only 6.7% of individuals remained ZnT8A positive after 25 yr compared with 19.5% for IA2A and 25.9% for GADA. Titers of ZnT8A and IA2A in seropositive individuals decreased progressively, whereas GADA remained elevated consistent with periodic reactivation of GADA humoral autoimmunity.ZnT8 humoral autoreactivity declines rapidly in the first years after disease onset and is less persistent than IA2A or GADA in the longer term. ZnT8A determination may be a useful measure of therapeutic efficacy in the context of immune-based clinical interventions.

The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

Human cytomegalovirus (CMV) is a latent and persistent virus whose proliferation increases morbidity and mortality of immune-compromised individuals. The current anti-CMV therapeutics targeting the viral DNA polymerase or the major immediate-early (MIE) gene locus are somewhat effective at limiting CMV-associated disease. However, due to low bioavailability, severe toxicity, and the development of drug resistant CMV strains following prolonged treatment, current anti-CMV therapeutics are insufficient. To help address this shortfall, we established a high-content assay to identify inhibitors targeting CMV entry and the early steps of infection. The infection of primary human fibroblasts with a variant of the CMV laboratory strain AD169 expressing a chimeric IE2-yellow fluorescence protein (YFP) (AD169 IE2-YFP ) provided the basis for the high-content assay. The localization of IE2-YFP to the nucleus shortly following an AD169 IE2-YFP infection induced a robust fluorescent signal that was quantified using confocal microscopy. The assay was optimized to achieve outstanding assay fitness and high Z ’ scores. We then screened a bioactive chemical library consisting of 2080 compounds and identified hit compounds based on the decrease of fluorescence signal from IE2-YFP nuclear expression. The hit compounds likely target various cellular processes involved in the early steps of infection including capsid transport, chromatin remodeling, and viral gene expression. Extensive secondary assays confirmed the ability of a hit compound, convallatoxin, to inhibit infection of both laboratory and clinical CMV strains and limit virus proliferation. Collectively, the data demonstrate that we have established a robust high-content screen to identify compounds that limit the early steps of the CMV life cycle, and that novel inhibitors of early infection events may serve as viable CMV therapeutics.

Antigenic diversity shapes immunity in distinct and unexpected ways. This is particularly true of the humoral response generated against influenza A viruses. Although it is known that immunological memory developed against previously encountered influenza A virus strains affects the outcome of subsequent infections, exactly how sequential exposures to antigenically variant viruses shape the humoral immune response in humans remains poorly understood. To address this important question, we performed a longitudinal analysis of antibody titers against various pandemic and seasonal strains of influenza virus spanning a 20-year period (1987 to 2008) with samples from 40 individuals (birth dates, 1917 to 1952) obtained from the Framingham Heart Study. Longitudinal increases in neutralizing antibody titers were observed against previously encountered pandemic H2N2, H3N2, and H1N1 influenza A virus strains. Antibody titers against seasonal strains encountered later in life also increased longitudinally at a rate similar to that against their pandemic predecessors. Titers of cross-reactive antibodies specific to the hemagglutinin stalk domain were also investigated because they are influenced by exposure to antigenically diverse influenza A viruses. These titers rose modestly over time, even in the absence of major antigenic shifts. No sustained increase in neutralizing antibody titers against an antigenically more stable virus (human cytomegalovirus) was observed. The results herein describe a role for antigenic variation in shaping the humoral immune compartment and provide a rational basis for the hierarchical nature of antibody titers against influenza A viruses in humans.

Members of the Herpesviridae family have the capacity to undergo both lytic and latent infection to establish a lifelong relationship with their host. Following primary infection, human cytomegalovirus (HCMV) can persist as a subclinical, recurrent infection for the lifetime of an individual. This quiescent portion of its life cycle is termed latency and is associated with periodic bouts of reactivation during times of immunosuppression, inflammation, or stress. In order to exist indefinitely and establish infection, HCMV encodes a multitude of immune modulatory mechanisms devoted to escaping the host antiviral response. HCMV has become a paradigm for studies of viral immune evasion of antigen presentation by both major histocompatibility complex (MHC) class I and II molecules. By restricting the presentation of viral antigens during both productive and latent infection, HCMV limits elimination by the human immune system. This review will focus on understanding how the virus manipulates the pathways of antigen presentation in order to modulate the host response to infection.

Recent research has shown that grazing by the algivorous minnows of the genus Campostoma can have a significant effect on both structural and functional parameters in stream ecosystems, influencing algal height and type, primary productivity, carbon dynamics, bacterial biomass and the size fractionation of benthic organic matter. This study was undertaken to evaluate the effect of minnow grazing on benthic particulate organic matter (BPOM) under the controlled conditions available in experimental streams. For this study, four grazed and four ungrazed artificial streams were sampled for BPOM on two dates. The samples were partitioned into large, medium, fine and ultrafine fractions via wet filtration. Fish grazed systems had significantly larger percentages of the fine fraction and significantly smaller percentages of the ultrafine fraction. The ability of grazers to alter particle size distribution is important to overall stream organic matter dynamics because recent studies have shown the importance of particle size in determining bacterial numbers and activity, which in turn influence fundamental stream process like respiration and organic carbon dynamics.

Viruses are obligate parasites and thus require the machinery of the host cell to replicate. Inhibition of host factors co-opted during active infection is a strategy hosts use to suppress viral replication and a potential pan-antiviral therapy. To define the cellular proteins and processes required for a virus during infection is thus crucial to understanding the mechanisms of virally induced disease. In this report, we generated fully infectious tagged influenza viruses and used infection-based proteomics to identify pivotal arms of cellular signaling required for influenza virus growth and infectivity. Using mathematical modeling and genetic and pharmacologic approaches, we revealed that modulation of Sec61-mediated cotranslational translocation selectively impaired glycoprotein proteostasis of influenza as well as HIV and dengue viruses and led to inhibition of viral growth and infectivity. Thus, by studying virus-human protein-protein interactions in the context of active replication, we have identified targetable host factors for broad-spectrum antiviral therapies. Viruses are obligate parasites dependent on the host cell machinery. Using infection-based proteomics, biochemistry, and mathematical modeling, Marazzi and colleagues reveal that targeting host factors controlling essential cellular functions can provide broad-spectrum antiviral effects. Loss-of-function and chemical inhibition of one such factor, Sec61, inhibited influenza, HIV, and dengue virus replication. Fil: Heaton, Nicholas S.. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Moshkina, Natasha. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Fenouil, Romain. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Gardner, Thomas J.. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Aguirre, Sebastian. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Shah, Priya S.. University of California; Estados Unidos Fil: Zhao, Nan. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Manganaro, Lara. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Hultquist, Judd F.. University of California; Estados Unidos Fil: Noel, Justine. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Sachs, David H.. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Hamilton, Jennifer. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Leon, Paul E.. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Chawdury, Amit. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Tripathi, Shashank. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Melegari, Camilla. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Campisi, Laura. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Hai, Rong. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Metreveli, Giorgi. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Gamarnik, Andrea Vanesa. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: García Sastre, Adolfo. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Greenbaum, Benjamin. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Simon, Viviana. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Fernandez Sesma, Ana. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Krogan, Nevan J.. University of California; Estados Unidos Fil: Mulder, Lubbertus C.F.. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: van Bakel, Harm. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Tortorella, Domenico. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Taunton, Jack. University of California; Estados Unidos Fil: Palese, Peter. Icahn School Of Medicine At Mount Sinai; Estados Unidos Fil: Marazzi, Ivan. Icahn School Of Medicine At Mount Sinai; Estados Unidos

A system of recirculating artificial streams is described that has low initial cost and operating expense and operates efficiently over long periods of time. Although individual stream units are large enough to accommodate minnows and crayfish, they are sufficiently compact that replication with up to 12 streams is possible in a relatively small laboratory. The system is simple in design, with no moving parts (except pumps) and no costly flow meters or regulating devices. Replication of stream units is facilitated by depending on inlet ports of fixed size, full-capacity operation of pumps, and uniform diameters of distribution systems. A basic stream tank unit is described in detail, with techniques for combining up to 12 units for water-exchange and inflow-outflow of water in different experimental applications. We have operated the system for almost a year with very little cost or repair. Substrates incubated in the laboratory streams readily grow an algal flora similar to that of nearby natura...

Water column and stream measurements of eight trace metals are presented for the Lake Hoare system. With the exception of Cd, metals showed little tendency to accumulate in the upper reservoir (> 24 m) of this closed-basin lake. Residence time trends for trace and major elements in this system were comparable to those in the oceans. It is concluded that particle reactive elements will behave in a similar manner from one closed aquatic system to another, and will tend to undergo rapid removal in comparison with the major elements. Of the eight metals studied, the adjacent transition series elements Mn, Fe, and Co had the shortest residence times.

Major ion, nutrient, transition metal, and cadmium concentrations are presented for nine meltwater streams flowing into Lake Fryxell, a permanently stratified lake with an anoxic hypolimnion in Taylor Valley, Antarctica. For the major ions, stream compositions are considered in terms of dissolution of marine-derived salts and chemical weathering of local rocks. Although Lake Fryxell has undergone significant evaporative concentration, only calcite, of the simple salts, is predicted to precipitate. Geochemical budgets indicate, however, that large quantities of K, Mg, and SO4 have also been removed from the lake. Reverse weathering may be an important sink for K and Mg, although magnesium removal with calcium carbonate phases is also likely. Assuming constancy of composition over recent geologic time, all of the salts in the Fryxell water column could have been delivered under present flows in about three thousand years (chloride age).