Type I interferon induced during chronic viral infection favors B-cell development in the thymus.

Chronic viral infections cause thymic involution yet the potential for broader, longer-term impact on thymic composition remains unexplored. Here we show that chronic, but not acute, lymphocytic choriomeningitis virus infection promotes a unique population of immature B cells in the thymus. We show that chronic viral infection promotes signals within the thymus, including the expression of B-cell activating factor (BAFF), that favor the maturation of this population as these cells acquire expression of CD19 and immunoglobulin M. Mechanistically, type I interferon (IFN-I), predominantly IFNß, signals to thymic hematopoietic cells, strongly delaying T-cell development at the earliest precursor stage. Furthermore, IFN-I signaling to the nonhematopoietic compartment provides a second signal essential to favor B-cell differentiation and maturation within the thymus. Importantly, chronic infection yields changes in the B-cell population for at least 50 days following infection, long after thymic atrophy has subsided. Thus, the inflammatory milieu induced by chronic viral infection has a profound, and long-lasting, effect on thymic composition leading to the generation of a novel population of thymic B cells.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

An epidemic Zika virus isolate suppresses antiviral immunity by disrupting antigen presentation pathways.

Zika virus (ZIKV) has emerged as an important global health threat, with the recently acquired capacity to cause severe neurological symptoms and to persist within host tissues. We previously demonstrated that an early Asian lineage ZIKV isolate induces a highly activated CD8 T cell response specific for an immunodominant epitope in the ZIKV envelope protein in wild-type mice. Here we show that a contemporary ZIKV isolate from the Brazilian outbreak severely limits CD8 T cell immunity in mice and blocks generation of the immunodominant CD8 T cell response. This is associated with a more sustained infection that is cleared between 7- and 14-days post-infection. Mechanistically, we demonstrate that infection with the Brazilian ZIKV isolate reduces the cross-presentation capacity of dendritic cells and fails to fully activate the immunoproteasome. Thus, our study provides an isolate-specific mechanism of host immune evasion by one Brazilian ZIKV isolate, which differs from the early Asian lineage isolate and provides potential insight into viral persistence associated with recent ZIKV outbreaks.

Point-activated ESR1Y541S has a dramatic effect on the development of sexually dimorphic organs.

Mutations in the estrogen receptor α (ERα) occur in endocrine-resistant metastatic breast cancer. However, a major gap persists with the lack of genetically tractable immune competent mouse models to study disease. Hence, we developed a Cre-inducible murine model expressing a point-activated ESR1Y541S (ESR1Y537S in humans) driven by its endogenous promoter. Germline expression of mutant ESR1Y541S reveals dramatic developmental defects in the reproductive organs, mammary glands, and bones of the mice. These observations provide critical insights into the tissue-specific roles of ERα during development and highlights the potential use of our model in further developmental and cancer studies.

Cyclophilin D Regulates Antiviral CD8+ T Cell Survival in a Cell-Extrinsic Manner.

CD8+ T cell-mediated immunity is critical for host defense against viruses and requires mitochondria-mediated type I IFN (IFN-I) signaling for optimal protection. Cyclophilin D (CypD) is a mitochondrial matrix protein that modulates the mitochondrial permeability transition pore, but its role in IFN-I signaling and CD8+ T cell responses to viral infection has not been previously explored. In this study, we demonstrate that CypD plays a critical extrinsic role in the survival of Ag-specific CD8+ T cell following acute viral infection with lymphocytic choriomeningitis virus in mice. CypD deficiency resulted in reduced IFN-I and increased CD8+ T cell death, resulting in a reduced antiviral CD8+ T cell response. In addition, CypD deficiency was associated with an increase in pathogen burden at an early time-point following infection. Furthermore, our data demonstrate that transfer of wild-type macrophages (expressing CypD) to CypD-deficient mice can partially restore CD8+ T cell responses. These results establish that CypD plays an extrinsic role in regulating optimal effector CD8+ T cell responses to viral infection. Furthermore, this suggests that, under certain circumstances, inhibition of CypD function may have a detrimental impact on the host's ability to respond to viral infection.

Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.

Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism.

Human CD4 T cells are constantly exposed to IL-12 during infections and certain autoimmune disorders. The current paradigm is that IL-12 promotes the differentiation of naïve CD4 T cells into Th1 cells, but recent studies suggest IL-12 may play a more complex role in T cell biology. We examined if exposure to IL-12 alters human CD4 T cell responses to subsequent TCR stimulation. We found that IL-12 pretreatment increased TCR-induced IFN-γ, TNF-α, IL-13, IL-4 and IL-10 production. This suggests that prior exposure to IL-12 potentiates the TCR-induced release of a range of cytokines. We observed that IL-12 mediated its effects through both transcriptional and post-transcriptional mechanisms. IL-12 pretreatment increased the phosphorylation of AKT, p38 and LCK following TCR stimulation without altering other TCR signaling molecules, potentially mediating the increase in transcription of cytokines. In addition, the IL-12-mediated enhancement of cytokines that are not transcriptionally regulated was partially driven by increased oxidative metabolism. Our data uncover a novel function of IL-12 in human CD4 T cells; specifically, it enhances the release of a range of cytokines potentially by altering TCR signaling pathways and by enhancing oxidative metabolism.

Enhancing Dendritic Cell-based Immunotherapy with IL-2/Monoclonal Antibody Complexes for Control of Established Tumors.

U.S. Food and Drug Administration-approved high-dose IL-2 therapy and dendritic cell (DC) immunization offer time-tested treatments for malignancy, but with defined issues of short in vivo t1/2, toxicity, and modest clinical benefit. Complexes of IL-2 with specific mAbs (IL-2c) exhibit improved stability in vivo with reduced toxicity and are capable of stimulating NK cell and memory phenotype CD8 T cell proliferation. In this study, we demonstrate that IL-2c treatment in tumor-bearing mice can enhance NK cell and tumor-specific CD8 T cell numbers. Importantly, DC immunization coupled with stabilized IL-2c infusion drastically improves the tumor-specific effector CD8 T cell response. DC + IL-2c treatment enhances number, 41BB and GITR expression, granzyme B production, CTL/regulatory T cell ratio, and per-cell killing capacity of CD8 T cells without increasing inhibitory molecule expression. Notably, IL-2c treatment of anti-CD3-stimulated human CD8 T cells resulted in higher number and granzyme B production, supporting the translational potential of this immunotherapy strategy for human malignancy. DC + IL-2c treatment enhances both endogenous NK cell and tumor Ag-specific CD8 T cell immunity to provide a marked reduction in tumor burden in multiple models of pre-existing malignancy in B6 and BALB/c mice. Depletion studies reveal contributions from both tumor-specific CD8 T cells and NK cells in control of tumor burden after DC + IL-2c treatment. Together, these data suggest that combination therapy with DC and IL-2c may be a potent treatment for malignancy.

Pathogen-specific inflammatory milieux tune the antigen sensitivity of CD8(+) T cells by enhancing T cell receptor signaling.

CD8(+) T cells confer host protection through T-cell-receptor (TCR)-mediated recognition of foreign antigens presented by infected cells. Thus, generation of CD8(+) T cell populations with high antigen sensitivity is critical for efficient pathogen clearance. Besides selection of high-affinity TCRs, the molecular mechanisms regulating the antigen sensitivity of CD8(+) T cells remain poorly defined. Herein, we have demonstrated that the antigen sensitivity of effector and memory CD8(+) T cells is dynamically regulated and can be tuned by pathogen-induced inflammatory milieux independently of the selection of cells with higher TCR affinity. Mechanistically, we have demonstrated that the signal-transduction capacity of key TCR proximal molecules is enhanced by inflammatory cytokines, which reduced the antigen density required to trigger antimicrobial functions. Dynamic tuning of CD8(+) T cell antigen sensitivity by inflammatory cytokines most likely optimizes immunity to specific pathogens while minimizing the risk of immunopathology at steady state.

Selective pharmacological inhibition of phosphoinositide 3-kinase p110delta opposes the progression of autoimmune diabetes in non-obese diabetic (NOD) mice.

During the progression of autoimmune (type 1) diabetes, T cells and macrophages infiltrate the pancreas, disrupt islet function, and destroy insulin-producing beta cells. B-lymphocytes, particularly innate like B-cell populations such as marginal zone B cells and B-1 cells, have been implicated in many autoimmune diseases, and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. Hence, inhibitors of B cell signaling pathways could be useful for limiting the autoimmune processes that contribute to type 1 diabetes. Signaling via phosphoinositide 3-kinase (PI3K) regulates many cellular processes. The p110δ isoform of PI3K is expressed primarily in cells of hematopoietic origin and the catalytic activity of p110δ is important for B cell migration, activation, proliferation, and antigen presentation. Because innate-like B cells are particularly sensitive to inhibition of p110δ activity, and p110δ inhibitors also suppress pro-inflammatory functions of other cell types that contribute to autoimmunity, we tested whether a p110δ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that long-term preventative treatment of pre-diabetic NOD mice with IC87114, a highly selective small molecule inhibitor of p110δ, reduced the infiltration of inflammatory cells into the pancreatic islets and, accordingly, delayed and reduced the loss of glucose homeostasis. Moreover in a therapeutic treatment mode, IC87114 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. These findings suggest that PI3Kδ inhibitors could be useful for managing type 1 diabetes.

Toll-like receptor 3 signaling on macrophages is required for survival following coxsackievirus B4 infection.

Toll-like receptor 3 (TLR3) has been proposed to play a central role in the early recognition of viruses by sensing double stranded RNA, a common intermediate of viral replication. However, several reports have demonstrated that TLR3 signaling is either dispensable or even harmful following infection with certain viruses. Here, we asked whether TLR3 plays a role in the response to coxsackievirus B4 (CB4), a prevalent human pathogen that has been associated with pancreatitis, myocarditis and diabetes. We demonstrate that TLR3 signaling on macrophages is critical to establish protective immunity to CB4. TLR3 deficient mice produced reduced pro-inflammatory mediators and are unable to control viral replication at the early stages of infection resulting in severe cardiac damage. Intriguingly, the absence of TLR3 did not affect the activation of several key innate and adaptive cellular effectors. This suggests that in the absence of TLR3 signaling on macrophages, viral replication outpaces the developing adaptive immune response. We further demonstrate that the MyD88-dependent signaling pathways are not only unable to compensate for the loss of TLR3, they are also dispensable in the response to this RNA virus. Our results demonstrate that TLR3 is not simply part of a redundant system of viral recognition, but rather TLR3 plays an essential role in recognizing the molecular signatures associated with specific viruses including CB4.

Development of a red-shifted fluorescence-based assay for SARS-coronavirus 3CL protease: identification of a novel class of anti-SARS agents from the tropical marine sponge Axinella corrugata.

SARS-coronavirus (SARS-CoV) encodes a main protease, 3CLpro, which plays an essential role in the viral life cycle and is currently the prime target for discovering new anti-coronavirus agents. In this article, we report our success in developing a novel red-shifted (RS) fluorescence-based assay for 3CLpro and its application for identifying small-molecule anti-SARS agents from marine organisms. We have synthesised and characterised the first generation of a red-shifted internally quenched fluorogenic substrate (RS-IQFS) for 3CLpro based on resonance energy transfer between the donor and acceptor pair CAL Fluor Red 610 and Black Hole Quencher-1 (Km and kcat values of 14 microM and 0.65 min-1). The RS-IQFS primary sequence was selected based on the results of our screening analysis of 3CLpro performed using a series of blue-shifted (BS)-IQFSs corresponding to the 3CLpro-mediated cleavage junctions of the SARS-CoV polyproteins. In contrast to BS-IQFSs, the RS-IQFS was not susceptible to fluorescence interference from coloured samples and allowed for successful screening of marine natural products and identification of a coumarin derivative, esculetin-4-carboxylic acid ethyl ester, a novel 3CLpro inhibitor (IC50=46 microM) and anti-SARS agent (EC50=112 microM; median toxic concentration>800 microM) from the tropical marine sponge Axinella corrugata.

The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin.

Proprotein convertases (PCs) are an important class of host-cell serine endoproteases implicated in many physiological and pathological processes. Owing to their expanding roles in the proteolytic events required for generating infectious microbial pathogens and for tumor growth and invasiveness, there is increasing interest in identifying endogenous PC inhibitors. Here we report the identification of Spn4A, a previously uncharacterized secretory pathway serine protease inhibitor (serpin) from Drosophila melanogaster that contains a consensus furin cleavage site, -Arg(P4)-Arg-Lys-Arg(P1) downsream-, in its reactive site loop (RSL). Our biochemical and kinetics analysis revealed that recombinant Spn4A inhibits human furin (K(i), 13 pM; k(ass), 3.2 x 10(7) M(-1) x s(-1)) and Drosophila PC2 (K(i), 3.5 nM; k(ass), 9.2 x 10(4) M(-1) x s(-1)) by a slow-binding mechanism characteristic of serpin molecules and forms a kinetically trapped SDS-stable complex with each enzyme. For both PCs, the stoichiometry of inhibition by Spn4A is nearly 1, which is characteristic of known physiological serpin-protease interactions. Mass analysis of furin-Spn4A reaction products identified the actual reactive site center of Spn4A to be -Arg(P4)-Arg-Lys-Arg(P1)-downstream-. Moreover, we demonstrate that Spn4A's highly effective PC inhibition properties are critically dependent on the unusual length of its RSL, which is composed of 18 aa instead of the typical 17-residue RSL found in most other inhibitory serpins. The identification of Spn4A, the most potent and effective natural serpin of PCs identified to date, suggests that Spn4A could be a prototype of endogenous serpins involved in the precise regulation of PC-dependent proteolytic cleavage events in the secretory pathway of eukaryotic cells.

Serpin mechanism of hepatitis C virus nonstructural 3 (NS3) protease inhibition: induced fit as a mechanism for narrow specificity.

Hepatitis C virus (HCV) nonstructural 3 (NS3) serine protease disrupts important cellular antiviral signaling pathways and plays a pivotal role in the proteolytic maturation of the HCV polyprotein precursor. This recent discovery has fostered the search for NS3 protease inhibitors. However, the enzyme's unusual induced fit behavior and peculiar molecular architecture have imposed considerable obstacles to the development of small molecule inhibitors. In this article, we demonstrate that such unique induced fit behavior and the chymotrypsin-like catalytic domain can provide the structural plasticity necessary to generate protein-based inhibitors of the NS3 protease. We took advantage of the macromolecular scaffold of a Drosophila serpin, SP6, which intrinsically supports chymotrypsin-like enzyme inhibition, to design a novel class of potent and selective inhibitors. We show that altering the SP6 reactive site loop (RSL) resulted in the development of the first effective (K(i) of 34 nm) and selective serpin, SP6(EVC/S), directed at the NS3 protease. SP6(EVC/S) operates as a suicide substrate inhibitor, and its partitioning between the complex-forming and proteolytic pathways for the NS3 protease is HCV NS4A cofactor-dependent and -specific. Once bound to the protease active site, SP6(EVC/S) partitions with equal probability to undergo proteolysis by NS3 at the C-terminal site of the engineered RSL, (P(6))Glu-Ile-(P(4))Val-Met-Thr-(P(1))Cys- downward arrow -(P(1)')Ser, or to form a covalent acyl-enzyme complex characteristic of cognate protease-serpin pairs. Our results also reveal a novel cofactor-induced serpin mechanism of enzyme inhibition that could be explored for developing effective and selective inhibitors of other important induced fit viral proteases of the Flaviviridae family such as the West Nile virus NS3 endoprotease.