Cutting Edge: Critical Role of Glycolysis in Human Plasmacytoid Dendritic Cell Antiviral Responses.

Plasmacytoid dendritic cells (pDCs) are vital to antiviral defense, directing immune responses via secretion of huge concentrations of IFN-α. These cells are critical in protecting the lung against clinically relevant respiratory viruses, particularly influenza (Flu), a virus responsible for substantial worldwide morbidity and mortality. How pDC responses to such viral pathogens are regulated, however, is poorly understood in humans. Using an unbiased approach of gene chip analysis, we discovered that Flu significantly affects metabolism in primary human pDCs. We demonstrate that Flu and RV, another common respiratory virus, induce glycolysis in pDCs and that this metabolic pathway regulates pDC antiviral functions, including IFN-α production and phenotypic maturation. Intranasal vaccination of human volunteers with live influenza virus also increases glycolysis in circulating pDCs, highlighting a previously unrecognized potential role for metabolism in regulating pDC immune responses to viral infections in humans.

Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.

Counterregulation between the FcepsilonRI pathway and antiviral responses in human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play essential roles in directing immune responses. These cells may be particularly important in determining the nature of immune responses to viral infections in patients with allergic asthma as well those with other atopic diseases. The purposes of this study were 1) to compare the functional capacity of pDCs in patients with one type of allergic disorder, allergic asthma, and controls; 2) to determine whether IgE cross-linking affects antiviral responses of influenza-exposed pDCs; and 3) to determine whether evidence of counterregulation of FcepsilonRIalpha and IFN-alpha pathways exists in these cells. pDC function was assessed in a subset of asthma patients and in controls by measuring IFN-alpha production after exposure of purified pDCs to influenza viruses. FcepsilonRIalpha expression on pDCs was determined by flow cytometry in blood samples from patients with allergic asthma and controls. pDCs from patients with asthma secreted significantly less IFN-alpha upon exposure to influenza A (572 versus 2815; p = 0.03), and secretion was inversely correlated with serum IgE levels. Moreover, IgE cross-linking prior to viral challenge resulted in 1) abrogation of the influenza-induced pDC IFN-alpha response; 2) diminished influenza and gardiquimod-induced TLR-7 upregulation in pDCs; and 3) interruption of influenza-induced upregulation of pDC maturation/costimulatory molecules. In addition, exposure to influenza and gardiquimod resulted in upregulation of TLR-7, with concomitant downregulation of FcepsilonRIalpha expression in pDCs. These data suggest that counterregulation of FcepsilonRI and TLR-7 pathways exists in pDCs, and that IgE cross-linking impairs pDC antiviral responses.

Rapid Generation of TCR and CD8αß Transgenic Virus Specific T Cells for Immunotherapy of Leukemia.

Background: Virus-specific T cells (VSTs) are an attractive cell therapy platform for the delivery of tumor-targeted transgenic receptors. However, manufacturing with conventional methods may require several weeks and intensive handling. Here we evaluated the feasibility and timelines when combining IFN-γ cytokine capture (CC) with retroviral transduction for the generation of T cell receptor (TCR) and CD8αß (TCR8) transgenic VSTs to simultaneously target several viral and tumor antigens in a single product. Methods: Healthy donor peripheral blood mononuclear cells were stimulated with cytomegalovirus (CMV) and Epstein-Barr-Virus (EBV) peptide mixtures derived from immunogenic viral proteins, followed by CC bead selection. After 3 days in culture, cells were transduced with a retroviral vector encoding four genes (a survivin-specific αßTCR and CD8αß). TCR8-transgenic or control VSTs were expanded and characterized for their phenotype, specificity and anti-viral and anti-tumor functions. Results: CC selected cells were efficiently transduced with TCR8. Average fold expansion was 269-fold in 10 days, and cells contained a high proportion of CD8+ T central memory cells. TCR8+ VSTs simultaneously expressed native anti-viral and transgenic anti-survivin TCRs on their cell surface. Both control and TCR8+ VSTs produced cytokines to and killed viral targets, while tumor targets were only recognized and killed by TCR8+ VSTs. Conclusions: IFN-γ cytokine capture selects and activates CMV and EBV-specific memory precursor CD8+ T cells that can be efficiently gene-modified by retroviral transduction and rapidly ex vivo expanded. Our multi-specific T cells are polyfunctional and recognize and kill viral and leukemic targets expressing the cognate antigens.

Transgenic CD8αß co-receptor rescues endogenous TCR function in TCR-transgenic virus-specific T cells.

BACKGROUND: Genetically engineered virus-specific T cells (VSTs) are a platform for adoptive cell therapy after allogeneic hematopoietic stem cell transplantation. However, redirection to a tumor-associated antigen by the introduction of a transgenic T-cell receptor (TCR) reduces anti-viral activity, thereby impeding the possibility of preventing or treating two distinct complications-malignant relapse and viral infection-with a single cell therapy product. Availability of CD8αß co-receptor molecules can significantly impact class I restricted T-cell activation, and thus, we interrogated whether transgenic CD8αß improves anti-viral activity mediated by native VSTs with or without a co-expressed transgenic TCR (TCR8). METHODS: Our existing clinical VST manufacturing platform was adapted and validated to engineer TCR+ or TCR8+ VSTs targeting cytomegalovirus and Epstein-Barr virus. Simultaneous anti-viral and anti-tumor function of engineered VSTs was assessed in vitro and in vivo. We used pentamer staining, interferon (IFN)-γ enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), cytotoxicity assays, co-cultures, and cytokine secretion assays for the in vitro characterization. The in vivo anti-tumor function was assessed in a leukemia xenograft mouse model. RESULTS: Both transgenic CD8αß alone and TCR8 had significant impact on the anti-viral function of engineered VSTs, and TCR8+ VSTs had comparable anti-viral activity as non-engineered VSTs as determined by IFN-γ ELISpot, ICS and cytotoxicity assays. TCR8-engineered VSTs had improved anti-tumor function and greater effector cytokine production in vitro, as well as enhanced anti-tumor function against leukemia xenografts in mice. CONCLUSION: Incorporation of transgenic CD8αß into vectors for TCR-targetable antigens preserves anti-viral activity of TCR transgenic VSTs while simultaneously supporting tumor-directed activity mediated by a transgenic TCR. Our approach may provide clinical benefit in preventing and treating viral infections and malignant relapse post-transplant.

Single-cell transcriptomics identifies multiple pathways underlying antitumor function of TCR- and CD8αß-engineered human CD4+ T cells.

Transgenic coexpression of a class I-restricted tumor antigen-specific T cell receptor (TCR) and CD8αß (TCR8) redirects antigen specificity of CD4+ T cells. Reinforcement of biophysical properties and early TCR signaling explain how redirected CD4+ T cells recognize target cells, but the transcriptional basis for their acquired antitumor function remains elusive. We, therefore, interrogated redirected human CD4+ and CD8+ T cells by single-cell RNA sequencing and characterized them experimentally in bulk and single-cell assays and a mouse xenograft model. TCR8 expression enhanced CD8+ T cell function and preserved less differentiated CD4+ and CD8+ T cells after tumor challenge. TCR8+CD4+ T cells were most potent by activating multiple transcriptional programs associated with enhanced antitumor function. We found sustained activation of cytotoxicity, costimulation, oxidative phosphorylation- and proliferation-related genes, and simultaneously reduced differentiation and exhaustion. Our study identifies molecular features of TCR8 expression that can guide the development of enhanced immunotherapies.

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity.

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.

A conserved role for Hox paralog group 4 in regulation of hematopoietic progenitors.

Regulatory circuits that control stem cell fate decisions can be identified and understood by manipulating individual regulatory elements genetically. While impractical in the rare somatic stem cells of primary tissue, this approach is feasible in embryonic stem cells differentiated in vitro into the somatic stem cell type of interest. We present an improved highly efficient targeting system allowing genes to be integrated into a predetermined, doxycycline-inducible locus, and corresponding inducible embryonic stem cell lines to be generated rapidly. We apply this system to evaluate a key hematopoietic progenitor cell regulatory element, HoxB4, and its mammalian paralogs, whose effects have not yet been tested in this context. We show that all Hox paralog group 4 members, A4, B4, C4, and D4, have similar effects on hematopoietic stem and progenitor self-renewal in vitro, and thus classify Hox paralog group 4 as promoting self-renewal. Each paralog group 4 member both promotes proliferation and inhibits differentiation, enabling the exponential expansion of hematopoietic progenitors from the c-kit(+)/CD41(+) cell fraction of day 6 murine embryoid bodies. By evaluating a set of deletion mutants we show that sequences in addition to the homeodomain and hexapeptide motif are required for this activity. These results highlight the utility of this expression system to perform functional and structural analyses of genetic regulators of cell fate decisions.

Blockade of virus infection by human CD4+ T cells via a cytokine relay network.

CD4(+) T cells directly participate in bacterial clearance through secretion of proinflammatory cytokines. Although viral clearance relies heavily on CD8(+) T cell functions, we sought to determine whether human CD4(+) T cells could also directly influence viral clearance through cytokine secretion. We found that IFN-gamma and TNF-alpha, secreted by IL-12-polarized Th1 cells, displayed potent antiviral effects against a variety of viruses. IFN-gamma and TNF-alpha acted directly to inhibit hepatitis C virus replication in an in vitro replicon system, and neutralization of both cytokines was required to block the antiviral activity that was secreted by Th1 cells. IFN-gamma and TNF-alpha also exerted antiviral effects against vesicular stomatitis virus infection, but in this case, functional type I IFN receptor activity was required. Thus, in cases of vesicular stomatitis virus infection, the combination of IFN-gamma and TNF-alpha secreted by human Th1 cells acted indirectly through the IFN-alpha/beta receptor. These results highlight the importance of CD4(+) T cells in directly regulating antiviral responses through proinflammatory cytokines acting in both a direct and indirect manner.