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Standard-of-care for resectable gastric/gastroesophageal junction cancer includes surgery and neoadjuvant-adjuvant 5-fluorouracil-leucovorin-oxaliplatin-docetaxel (FLOT) chemotherapy. Early-phase clinical studies support further clinical development of the immune checkpoint inhibitor (ICI); durvalumab, an anti-PD-L1 antibody, in patients with gastric/gastroesophageal junction cancer. Accumulating evidence indicates that ICIs combined with FLOT chemotherapy improve clinical outcomes in patients with advanced or metastatic cancer. We describe the rationale for and the design of MATTERHORN, a randomized, double-blind, placebo-controlled, phase III study investigating the efficacy and safety of neoadjuvant-adjuvant durvalumab and FLOT chemotherapy followed by adjuvant durvalumab monotherapy in patients with resectable gastric/gastroesophageal junction cancer. The planned sample size is 900 patients, the primary end point is event-free survival and safety and tolerability will be evaluated. Clinical trial registration: NCT04592913 (ClinicalTrials.gov). ispartof: FUTURE ONCOLOGY vol:18 issue:20 pages:2465-2473 ispartof: location:England status: published

We have developed an oncolytic Newcastle disease virus (NDV) that has potent in vitro and in vivo anti-tumor activities and attenuated pathogenicity in chickens. In this ex vivo study using the same recombinant NDV backbone with GFP transgene (NDV-GFP, designated as rNDV), we found that rNDV induces maturation of monocyte-derived immature dendritic cells (iDCs) by both direct and indirect mechanisms, which promote development of antigen-specific T cell responses. Addition of rNDV directly to iDCs culture induced DC maturation, as demonstrated by the increased expression of costimulatory and antigen-presenting molecules as well as the production of type I interferons (IFNs). rNDV infection of the HER-2 positive human breast cancer cell line (SKBR3) resulted in apoptotic cell death, release of proinflammatory cytokines, and danger-associated molecular pattern molecules (DAMPs) including high-mobility group protein B1 (HMGB1) and heat shock protein 70 (HSP70). Addition of rNDV-infected SKBR3 cells to iDC culture resulted in greatly enhanced upregulation of the maturation markers and release of type I IFNs by DCs than rNDV-infected DCs only. When co-cultured with autologous T cells, DCs pre-treated with rNDV-infected SKBR3 cells cross-primed T cells in an antigen-specific manner. Altogether, our data strongly support the potential of oncolytic NDV as efficient therapeutic agent for cancer treatment.

TPS373 Background: Esophageal cancer is the eighth most common cancer type and the sixth leading cause of cancer-related death worldwide, and esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer. For patients with locally advanced, unresectable ESCC (AJCC 8th Stage II–IVA), definitive chemoradiotherapy (dCRT) is the current standard of care; however, up to half of patients will experience disease progression within two years of dCRT, and overall survival rates remain suboptimal. The combination of immune checkpoint inhibitors with CRT has demonstrated synergistic antitumor activity in pre-clinical models, and recent clinical data has demonstrated clinical benefit of combined programmed cell death-1 inhibition and preoperative CRT in patients with locally advanced ESCC. KUNLUN (NCT04550260) is a Phase 3, multicenter, global study evaluating the efficacy and safety of durvalumab, a programmed cell death ligand-1 (PD-L1) inhibitor, given concurrently with, and after, dCRT in patients with locally advanced, unresectable ESCC. Methods: Approximately 600 patients will be randomized 2:1 to receive either durvalumab with dCRT (cisplatin plus fluorouracil or cisplatin plus capecitabine, with a total dose of 50–64 Gy radiation), followed by durvalumab for up to approximately 24 months, or placebo with dCRT, followed by placebo for up to approximately 24 months. Eligible patients will have histologically or cytologically confirmed ESCC, and present with locally advanced, unresectable ESCC that is deemed suitable for dCRT. Patients must have an Eastern Cooperative Oncology Group performance status of 0 or 1, and have not received prior anti-cancer treatment. The co-primary endpoint of the study is progression-free survival according to RECIST v1.1 as assessed by blinded independent central review in all randomized patients and in patients with PD-L1-high tumors. Additional endpoints include overall survival and safety. Study enrollment is ongoing. Funding: This study was sponsored by AstraZeneca. References: Hong MH, et al. J Clin Oncol 2019;37(15 suppl). Abs 4027. Clinical trial information: NCT04550260.

Background Despite considerable efforts at developing therapeutic vaccines for cancer, clinical translation of preclinical successes has been challenging, largely due to the difficulty of inducing strong and sustained cytotoxic T lymphocyte (CTL) responses in patients. Several peptide-based cancer vaccines have failed to show sustainable tumor regression in the clinic, possibly because of a lack of optimization of both the adjuvant and antigen components of the preparations. Here, we aimed to develop and optimize a vaccine format utilizing a synthetic long peptide (SLP) containing the human papilloma virus 16 (HPV16) E7 antigen, with a centrally located defined MHC class I epitope, and evaluate its immunogenicity and efficacy in combination with various adjuvant formulations. Methods E731–73 SLP was tested alone or in combination with toll-like receptor (TLR)3, TLR4, TLR7/8 and TLR9 agonists and formulated in oil-in-water (o/w) or water-in-oil (w/o) emulsions to determine a vaccine format inducing a robust CD8 T cell response in murine models. Once a lead vaccine format was determined, we examined its ability to inhibit tumor growth in the murine TC-1 model that expresses HPV16 E7 antigen. Results We identified the TLR9 agonist CpG formulated in a squalene-based o/w emulsion as the most potent adjuvant, inducing the expansion of multifunctional antigen specific CD8 T cells with cytolytic potential. We also demonstrated that SLP E731–73 + CpG + o/w emulsion vaccine can provide prophylactic and more importantly, therapeutic benefit in the TC-1 murine tumor model. Conclusions Our results demonstrate that the novel vaccine format E7 SLP + CpG delivered in an o/w emulsion holds potential for the promotion of strong CTL responses and tumor eradication and encourages further development of peptide/adjuvant vaccines in cancer immunotherapy strategies. Electronic supplementary material The online version of this article (10.1186/s12885-019-5725-y) contains supplementary material, which is available to authorized users.

Background & Aims Current therapies for chronic hepatitis B virus (HBV) infection control viral replication but do not eliminate the risk of progression to hepatocellular carcinoma. HBV-specific CD8 T cells are necessary for viral control, but they are rare and exhausted during chronic infection. Preclinical studies have shown that blockade of the PD-1:PD-L1 axis can restore HBV-specific T cell functionality. The aim of this study was to analyze how the clinical and treatment status of patients impacts the ability of HBV-specific T cells to respond to PD-L1 blockade. Methods Expression patterns of the PD-1:PD-L1/PD-L2 axis were analyzed in healthy donors and chronically infected patients in different clinical phases of disease. A functional assay was performed to quantify baseline HBV-specific T cell responses in chronically infected patients. Baseline responses were then compared to those attained in the presence of an anti-PD-L1 monoclonal antibody (MEDI2790). Results Chronically infected patients were characterized by the upregulation of PD-1 within the T cell compartment and a concomitant upregulation of PD-L1 on myeloid dendritic cells. The upregulation was maximal in HBV e antigen (HBeAg)-positive patients but persisted after HBeAg negativization and was not restored by long-term treatment. HBV reactivity, measured as frequency of HBV-specific T cells, was significantly higher in HBeAg-negative patients with lower HBV DNA levels, independently of HBV surface antigen or alanine aminotransferase levels. Anti-PD-L1 blockade with MEDI2790 increased both the number of IFN-γ-producing T cells and the amount of IFN-γ produced per cell in 97% of patients with detectable HBV reactivity, independently of patients’ clinical or treatment status. Conclusion Patients with lower levels of HBV DNA and the absence of HBeAg have more intact HBV-specific T cell immunity and may benefit the most from PD-L1 blockade as a monotherapy. Lay summary Hepatitis B virus (HBV)-specific T cell responses during chronic infection are weak due to the upregulation of inhibitor molecules on the immune cells. In this study we show that the inhibitory PD-1:PD-L1 axis is upregulated during chronic HBV infection and successful antiretroviral therapy does not restore normal levels of PD-1 and PD-L1 expression. However, in HBV e antigen-negative patients, treatment with an anti-PD-L1 antibody can increase the functionality of HBV-specific T cell responses by an average of 2-fold and is a promising new therapy for patients with chronic HBV infection., Graphical abstract Unlabelled Image, Highlights • Upregulation of the PD-1:PD-L1 axis is more profound in HBeAg-positive samples. • This upregulation does not normalize in HBeAg-negative patients, or patients under antiviral therapy. • HBV-specific T cell reactivity is higher in HBeAg-negative patients with low HBV DNA levels. • 97% of HBV-reactive patients respond to anti-PD-L1 blockade with MEDI2790 irrespective of their clinical status.

Background/objectives There is currently no licensed prophylactic or therapeutic vaccine for HSV-2 infection. Methods We developed a novel preclinical vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with the potent synthetic TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. The vaccine was tested for immunogenicity and efficacy in pre-clinical models for preventative and therapeutic vaccination. Results Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. Supporting the use of G103 therapeutically, the vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score. Conclusions Taken together, the data show that G103 is a viable candidate for development of a novel prophylactic and therapeutic HSV-2 vaccine.

Supplemental Digital Content is available in the text., Dendritic cells (DCs) are essential antigen-presenting cells for the initiation of cytotoxic T-cell responses and therefore attractive targets for cancer immunotherapy. We have developed an integration-deficient lentiviral vector termed ID-VP02 that is designed to deliver antigen-encoding nucleic acids selectively to human DCs in vivo. ID-VP02 utilizes a genetically and glycobiologically engineered Sindbis virus glycoprotein to target human DCs through the C-type lectin DC-SIGN (CD209) and also binds to the homologue murine receptor SIGNR1. Specificity of ID-VP02 for antigen-presenting cells in the mouse was confirmed through biodistribution studies showing that following subcutaneous administration, transgene expression was only detectable at the injection site and the draining lymph node. A single immunization with ID-VP02 induced a high level of antigen-specific, polyfunctional effector and memory CD8 T-cell responses that fully protected against vaccinia virus challenge. Upon homologous readministration, ID-VP02 induced a level of high-quality secondary effector and memory cells characterized by stable polyfunctionality and expression of IL-7Rα. Importantly, a single injection of ID-VP02 also induced robust cytotoxic responses against an endogenous rejection antigen of CT26 colon carcinoma cells and conferred both prophylactic and therapeutic antitumor efficacy. ID-VP02 is the first lentiviral vector which combines integration deficiency with DC targeting and is currently being investigated in a phase I trial in cancer patients.

Background The expansion of antigen-specific CD8 T cells is important in generating an effective and long-lasting immune response to tumors and viruses. Glucocorticoid-induced tumor necrosis factor receptor family-related receptor (GITR) is a co-stimulatory receptor that binds the GITR ligand (GITRL). Agonism of GITR can produce important signals that drive expansion of effector T cell populations. Methods We explored two separate murine tumor models, CT26 and TC-1, for responsiveness to GITR Ligand Fusion Protein(GITRL-FP) monotherapy. In TC-1, GITRL-FP was also combined with concurrent administration of an E7-SLP vaccine. We evaluated tumor growth inhibition by tumor volume measurements as well as changes in CD8 T cell populations and function including cytokine production using flow cytometry. Additionally, we interrogated how these therapies resulted in tumor antigen-specific responses using MHC-I dextramer staining and antigen-specific restimulations. Results In this study, we demonstrate that a GITR ligand fusion protein (GITRL-FP) is an effective modulator of antigen-specific CD8 T cells. In a CT26 mouse tumor model, GITRL-FP promoted expansion of antigen-specific T cells, depletion of regulatory T cells (Tregs), and generation of long-lasting CD8 T cell memory. This memory expansion was dependent on the dose of GITRL-FP and resulted in complete tumor clearance and protection from tumor rechallenge. In contrast, in TC-1 tumor–bearing mice, GITRL-FP monotherapy could not prime an antigen-specific CD8 T cell response and was unable to deplete Tregs. However, when combined with a vaccine targeting E7, treatment with GITRL-FP resulted in an augmentation of the vaccine-induced antigen-specific CD8 T cells, the depletion of Tregs, and a potent antitumor immune response. In both model systems, GITR levels on antigen-specific CD8 T cells were higher than on all other CD8 T cells, and GITRL-FP interacted directly with primed antigen-specific CD8 T cells. Conclusions When taken together, our results demonstrate that the delivery of GITRL-FP as a therapeutic can promote anti-tumor responses in the presence of tumor-specific CD8 T cells. These findings support further study into combination partners for GITRL-FP that may augment CD8 T-cell priming as well as provide hypotheses that can be tested in human clinical trials exploring GITR agonists including GITRL-FP. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0247-0) contains supplementary material, which is available to authorized users.

Lentiviral vectors (LVs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. A safety concern for use of LVs in humans is the generation of replication-competent lentivirus (RCL), which may arise due to recombination between the split genomes of third-generation LVs. Although no RCL has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may further reduce the risk of RCL formation. Here we describe design elements introduced to the gag/pol plasmid with the intention of eliminating psi-gag recombination between the vector genome and gag/pol. These design changes, consisting of codon optimization of the gag/pol sequence and the deletion of the Rev-responsive element, abrogate the requirement for Rev in expression of Gag protein, thus the resulting gag/pol construct being Rev independent (RI gag/pol). We show that generating vector using the RI gag/pol construct has no effect on particle production or transduction titers. The RI and wild-type gag/pol vectors function equivalently as antigen-specific immunotherapy, potently inducing antigen-specific CD8 T cells that protect against challenge with vaccinia virus. Most importantly, the designed RI gag/pol eliminated detectable psi-gag recombination. Interestingly, we detected recombination between the vector genome and gag/pol from regions without sequence homology. Our findings imply that although unpredictable recombination events may still occur, the RI gag/pol design is sufficient to prevent psi-gag recombination.

SummaryType I interferons (IFNs) are central to antiviral defense, but how they orchestrate immune cell function is incompletely understood. We determined that IFNs produced during murine cytomegalovirus (MCMV) infection differentially affect dendritic cells (DCs) and natural killer (NK) cells. IFNs induce cell-intrinsic responses in DCs, activating antiproliferative, antiviral, and lymphocyte-activating gene networks, consistent with high activity of the transcription factor STAT1 in these cells. By comparison, NK cells exhibit lower STAT1 expression and reduced IFN responsiveness. Rather, IFNs indirectly affect NK cells by inducing IL-15, which activates the transcription factor E2F and stimulates genes promoting cell expansion. IFN cell-intrinsic responses are necessary in DCs, but not NK cells, for MCMV resistance. Thus, sensitivity to IFN-induced cytokines and differences in IFN receptor signaling program immune cells to mount distinct responses that promote viral control.

NK cells recognize virus-infected cells with germline-encoded activating and inhibitory receptors that do not undergo genetic recombination or mutation. Accordingly, NK cells are often considered part of the innate immune response. The innate response comprises rapid early defenders that do not form immune memory. However, there is increasing evidence that experienced NK cells provide increased protection to secondary infection, a hallmark of the adaptive response. In this study, we compare the dynamics of the innate and adaptive immune responses by examining the kinetic profiles of the NK and T cell response to murine CMV infection. We find that, unexpectedly, the kinetics of NK cell proliferation is neither earlier nor faster than the CD4 or CD8 T cell response. Furthermore, early NK cell contraction after the peak of the response is slower than that of T cells. Finally, unlike T cells, experienced NK cells do not experience biphasic decay after the response peak, a trait associated with memory formation. Rather, NK cell contraction is continuous, constant, and returns to below endogenous preinfection levels. This indicates that the reason why Ag-experienced NK cells remain detectable for a prolonged period after adoptive transfer and infection is in part due to the high precursor frequency, slow decay rate, and low background levels of Ly49H+ NK cells in recipient DAP12-deficient mice. Thus, the quantitative contribution of Ag-experienced NK cells in an endogenous secondary response, with higher background levels of Ly49H+ NK cells, may be not be as robust as the secondary response observed in T cells.

Mouse CMV (MCMV) infection rapidly induces the proliferation of NK cells, which correlates with immunological protection. Whether NK cells primed during acute response against MCMV are maintained for the long term is not known. In this study, we used TcrdH2BeGFP mice in which maturing NK cells are genetically labeled with a pulse of very stable histone-2B–eGFP. In this system, we found that the reporter protein was diluted out upon NK cell division during acute MCMV infection. At the same time, mature NK cells in uninfected mice showed only very limited turnover in vivo. Three months after primary infection when MCMV latency was established, the majority of peripheral NK cells still displayed a higher record of proliferation than NK cells in mock-infected controls. This observation included both Ly49H+ and Ly49H– NK cells. Conversely, naive NK cells did not show more proliferation after transfer into latently MCMV-infected mice than that after transfer into mock-infected control mice. This indicated that the observed alterations of the NK cell compartment in MCMV latency were “legacy” (i.e., resulting from prior events during the initial immune response). Together, these results suggest that antiviral immune responses induce sustained alterations of innate lymphocyte populations that extend far beyond the first days of acute infection.

NK cells and gammadelta T cells are distinct subsets of lymphocytes that contextually share multiple phenotypic and functional characteristics. However, the acquisition and the extent of these similarities remain poorly understood. Here, using T cell receptor delta locus-histone 2B-enhanced GFP (Tcrd-H2BEGFP) reporter mice, we show that germ-line transcription of Tcrd occurs in all maturing NK cells. We also describe a population of mouse NK-like cells that are indistinguishable from "bona fide" NK cells using standard protocols. Requirements for V(D)J recombination and a functional thymus, along with very low-level expression of surface TCRgammadelta but high intracellular CD3, define these cells as gammadelta T cells. "NK-like gammadelta T cells" are CD127+, have a memory-activated phenotype, express multiple NK cell receptors and readily produce interferon-gamma in response to IL-12/IL-18 stimulation. The close phenotypic resemblance between NK cells and NK-like gammadelta T cells is a source of experimental ambiguity in studies bridging NK and T cell biology, such as those on thymic NK cell development. Instead, it ascribes chronic TCRgammadelta engagement as a means of acquiring NK-like function.

Activated NK T cells are known to rapidly stimulate NK cells and, subsequently, CD8+ T cells and B cells. In this report, we first demonstrate that the downstream effects induced by α-galactosylceramide activated NK T cells on NK cells are mainly dependent on IFN-γ. We found that NK T cell activation of NK cells requires a functional IFN-γ signaling in macrophages and dendritic cells but not in B cells, NK cells, or NK T cells. NK T cell activation is dendritic cell-dependent whereas NK T cell activation of NK cells is indirect and in part mediated by macrophages. Interestingly, in this context, macrophage participation in the CD1d Ag presentation of α-galactosylceramide to NK T cells is not necessary. These data indicate that NK T cell-dependent activation of macrophages is required for optimal NK T cell-induced stimulation of NK cells.

JC virus (JCV), a member of the polyomavirus family, causes a demyelinating disease of the central nervous system (CNS) in humans known as progressive multifocal leukoencephalopathy. Although glial cells are the principal target of JCV productive infection in progressive multifocal leukoencephalopathy patients, little is known regarding the site of JCV persistence and the mechanisms by which the virus spreads to the CNS to cause disease. Previous work has demonstrated the presence of replicating JCV DNA in B lymphocytes from peripheral blood, tonsil, and spleen and it has been hypothesized that lymphocytes may be one site of JCV persistence. Detection of viral gene products in renal tubules and excretion of JC virions in the urine suggests JCV persistence in the kidney. A respiratory route of viral transmission has also been hypothesized implicating the lung as another possible site of persistent JCV infection. Earlier studies from our laboratory have shown that terminal alpha 2,6-linked sialic acid is a critical component of the JCV receptor. In this report we examined the tissue distribution of this JCV receptor-type sialic acid in a panel of normal human tissues. Our results demonstrate that in normal brain JCV receptor-type sialic acids are expressed on oligodendrocytes and astrocytes, but not on cortical neurons. The receptor-type sialic acid is also more highly expressed on B lymphocytes than on T lymphocytes in normal human spleen and tonsil. In addition, both the kidney and lung express abundant levels of alpha 2-6-linked sialic acids. Our data show a striking correlation between the expression of the JCV receptor-type sialic acid on cells and their susceptibility to infection by the virus. These findings also support the hypothesis of JCV persistence in lymphoid tissue and B-cell-facilitated viral dissemination to the CNS.

Natural killer (NK) cells have the ability to become activated under the appropriate conditions by utilizing one or more cell surface receptors that are capable of inducing NK cell cytokine production and/or cytotoxicity. The expression of a variable array of inhibitory receptors on the surface of NK cells acts to counterbalance the positive signals initiated through activating receptors. Increasing evidence suggests an important role for both activating and inhibitory NK cell receptors in an appropriate and controlled NK response to infectious agents.

The killer cell lectin-like receptor G1 (KLRG1) is the mouse homolog of the rat mast cell function-associated Ag and contains an immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. In this study we demonstrate that both pathogenic and nonpathogenic in vivo activation of NK cells induces the expression of KLRG1 on their cell surface. Upon infection with murine CMV, this induction peaks between days 5 and 7 with ∼90% of the NK cells expressing KLRG1. On day 1.5 post-murine CMV infection of C57BL/6 mice, the main producers of IFN-γ are the KLRG1-negative NK cells. This effect has been recapitulated in vitro as we show that engagement of KLRG1 on a transfected NK cell line inhibits both cytokine production and NK cell-mediated cytotoxicity. Taken together, these data illustrate the crucial role played by KLRG1 during the termination of mouse NK cell activation.

As sentinels of the immune system, dendritic cells (DCs) play an essential role in regulating cellular immune responses. One of the main challenges of developing DC-targeted therapies includes the delivery of antigen to DCs in order to promote the activation of antigen-specific effector CD8 T cells. With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to human DCs. This platform, termed ID-VP02, utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with posttranslational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of human DCs. In addition, ID-VP02 incorporates safety features in its design that include two redundant mechanisms to render ID-VP02 integration-deficient. Here, we describe the characteristics that allow ID-VP02 to specifically transduce human DCs, and the advances that ID-VP02 brings to conventional third-generation lentiviral vector design as well as demonstrate upstream production yields that will enable manufacturing feasibility studies to be conducted.

A cell-based phenotypic screen for inhibitors of biofilm formation in mycobacteria identified the small molecule TCA1, which has bactericidal activity against both drug-susceptible and -resistant Mycobacterium tuberculosis (Mtb) and sterilizes Mtb in vitro combined with rifampicin or isoniazid. In addition, TCA1 has bactericidal activity against nonreplicating Mtb in vitro and is efficacious in acute and chronic Mtb infection mouse models both alone and combined with rifampicin or isoniazid. Transcriptional analysis revealed that TCA1 down-regulates genes known to be involved in Mtb persistence. Genetic and affinity-based methods identified decaprenyl-phosphoryl-β-D-ribofuranose oxidoreductase DprE1 and MoeW, enzymes involved in cell wall and molybdenum cofactor biosynthesis, respectively, as targets responsible for the activity of TCA1. These in vitro and in vivo results indicate that this compound functions by a unique mechanism and suggest that TCA1 may lead to the development of a class of antituberculosis agents.

New tools are needed to study the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), to facilitate new drug discovery and vaccine development. We have developed methodology to genetically incorporate unnatural amino acids into proteins in Mycobacterium smegmatis, BCG and Mtb, grown both extracellularly in culture and inside host cells. Orthogonal mutant tRNATyr/tyrosyl-tRNA synthetase pairs derived from Methanococcus jannaschii and evolved in Escherichia coli incorporate a variety of unnatural amino acids (including photocrosslinking, chemically reactive, heavy atom containing, and immunogenic amino acids) into proteins in response to the amber nonsense codon. By taking advantage of the fidelity and suppression efficiency of the MjtRNA/pIpaRS pair in mycobacteria, we are also able to use p-iodophenylalanine to induce the expression of proteins in mycobacteria both extracellularly in culture and inside of mammalian host cells. This provides a new approach to regulate the expression of reporter genes or mycobacteria endogenous genes of interest. The establishment of the unnatural amino acid expression system in Mtb, an intracellular pathogen, should facilitate studies of TB biology and vaccine development.

Following the discovery of innate immune receptors, the topics of innate immunity and its role in defense against infective agents have recently blossomed into very active research fields, after several decades of neglect. Among innate immune cells, natural killer (NK) cells are endowed with the unique ability to recognize and kill cells infected with a variety of pathogens, irrespective of prior sensitization to these microbes. NK cells have a number of other functions, including cytokine production and immunoregulatory activities. Major advances have recently been made in the understanding of the role of NK cells in the physiopathology of infectious diseases. The cellular and molecular mechanisms regulating the acquisition of effector functions by NK cells and their triggering upon pathogenic encounters are being unraveled. The possibility that the power of NK cells could be harnessed for the design of innovative treatments against infections is a major incentive for biologists to further explore NK cell subset complexity and to identify the ligands that activate NK cell receptors.

Genome-wide expression profiling of mouse and human leukocytes reveal conserved transcriptional programs of plasmacytoid or conventional dendritic cell subsets., Background Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8α in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes. Results We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively. Conclusion Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.

International audience; Plasmacytoid dendritic cells (pDCs) are an important source of IFN-alpha/beta in response to a variety of viruses in vivo, including murine cytomegalovirus (MCMV). However, the respective contributions of various infected organs, and within these of pDCs, conventional dendritic cells and other cells, to the systemic production of IFN-alpha/beta or other innate cytokines during viral infections in vivo is largely unknown. Whether a specialization of pDC subsets in the production of different patterns of innate cytokines exists in vivo in response to a viral infection has not been investigated. Here, by analyzing for the first time directly ex vivo, at the single-cell level, the simultaneous production of up to three cytokines in pDCs isolated from MCMV-infected mice, we show that (i) pDCs are the quasi-exclusive source of IFN-alpha/beta, IL-12 and tumor necrosis factor (TNF)-alpha, early during MCMV infection, in two immunocompetent mouse lines and with two viral strains, (ii) pDC activation for IFN-alpha/beta production is organ specific and (iii) a significant proportion of pDCs simultaneously produce IFN-alpha/beta, TNF-alpha and IL-12, although TNF-alpha and IFN-alpha/beta appear more often co-expressed with one another than each of them with IL-12. Altogether, these results show a broad and non-redundant role of pDCs in early innate detection of, and defense against, viral infection. The data also show differences in the responsiveness of pDCs from different tissues and suggest distinct molecular requirements for pDC production of various cytokines. These observations must be taken into account when designing new antiviral vaccination strategies aimed at harnessing pDC responses.

NK cells and gammadelta T cells are distinct subsets of lymphocytes that contextually share multiple phenotypic and functional characteristics. However, the acquisition and the extent of these similarities remain poorly understood. Here, using T cell receptor delta locus-histone 2B-enhanced GFP (Tcrd-H2BEGFP) reporter mice, we show that germ-line transcription of Tcrd occurs in all maturing NK cells. We also describe a population of mouse NK-like cells that are indistinguishable from "bona fide" NK cells using standard protocols. Requirements for V(D)J recombination and a functional thymus, along with very low-level expression of surface TCRgammadelta but high intracellular CD3, define these cells as gammadelta T cells. "NK-like gammadelta T cells" are CD127+, have a memory-activated phenotype, express multiple NK cell receptors and readily produce interferon-gamma in response to IL-12/IL-18 stimulation. The close phenotypic resemblance between NK cells and NK-like gammadelta T cells is a source of experimental ambiguity in studies bridging NK and T cell biology, such as those on thymic NK cell development. Instead, it ascribes chronic TCRgammadelta engagement as a means of acquiring NK-like function.

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ , and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ . NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122 + NK1.1 + DX5 − stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo . Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.

ZVex™ and GLAAS™ are two dendritic-cell (DC) targeting platform technologies designed to enhance immune responses through the in vivo induction of antigen specific CD8 and CD4 T-cells, respectively. ZVex™ is a lentiviral vector pseudotyped with a modified Sindbis virus envelope engineered to deliver tumor antigen-encoding nucleic acids to dendritic cells in vivo. GLAAS™(Glucopyranosyl Lipid A Adjuvant System) activates DC by binding to the TLR-4 receptor and inducing strong Th1 type CD4 responses against co-delivered recombinant proteins. Currently both platform technologies are being investigated in phase I clinical trials in cancer patients. Human carbonic anhydrase 9 (hCAIX) is a tumor-associated transmembrane antigen that is over-expressed on various cancer cell types. We mapped hCAIX specific, multi-functional CD8 and CD4 T-cell epitopes within the extracellular and transmembrane regions of the protein for the mouse haplotype H-2b by intracellular cytokine staining. Mice lethally challenged s.c. on the flank with a B16-F10 tumor cell line expressing the hCAIX protein (designated BC.12) fully controlled large tumors (>100 mm2) when therapeutically immunized (subcutaneously at the base of tail) with ZVex™ encoding hCAIX or the recombinant hCAIX protein with GLAAS™ (either s.c. or i.m.). In both models, tumor control was dose-dependent. Additionally, the presence of a strong transmembrane H-2b restricted CD8 T-cell epitope was required for tumor control and regression. hCAIX-specific CD8 T-cell responses were detectable as far out as day 67 post challenge in mice displaying full regression of tumor. These results demonstrate proof of concept for ZVex™ and GLAAS™ platform technologies in an aggressive murine melanoma model. Citation Format: David J. Campbell, Rebecca S. Reeves, Patrick A. Flynn, Scott H. Robbins, Peter Berglund, Jan H. ter Meulen. Therapeutic efficacy of the ZVex™ and GLAAS™ platforms in a B16-F10/hCAIX melanoma mouse model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2505. doi:10.1158/1538-7445.AM2015-2505

Plasmacytoid dendritic cells (pDCs) are specialized DCs that produce high levels of type I IFN upon viral infection. Despite their key immunoregulatory role, little is known about pDC ontogeny or how developmental events regulate their function. We show that mice expressing low levels of the transcription factor Ikaros (Ik(L/L)) lack peripheral pDCs, but not other DC subsets. Loss of pDCs is associated with an inability to produce type I IFN after challenge with Toll-like receptor-7 and -9 ligands, or murine cytomegalovirus (MCMV) infection. In contrast, conventional DCs are present in normal numbers and exhibit normal responses in vivo after challenge with MCMV or inactivated toxoplasma antigen. Interestingly, Ik(L/L) bone marrow (BM) cells contain a pDC population that appears blocked at the Ly-49Q- stage of differentiation and fails to terminally differentiate in response to Flt-3L, a cytokine required for pDC differentiation. This differentiation block is strictly dependent on a cell-intrinsic requirement for Ikaros in pDC-committed precursors. Global gene expression profiling of Ik(L/L) BM pDCs reveals an up-regulation of genes not normally expressed, or expressed at low levels, in WT pDCs. These studies suggest that Ikaros controls pDC differentiation by silencing a large array of genes.

DAP12 is an ITAM-containing adaptor molecule conveying activating properties to surface receptors on many cell types. We show here that DAP12 paradoxically down-modulates plasmacytoid dendritic cell (pDC) cytokine production in vivo during murine CMV (MCMV) infection. Higher levels of IFN-αβ and IL-12 were detected upon MCMV infection or CpG treatment in DAP12-deficient (DAP12°) mice as compared with wild-type (WT) mice. This resulted from altered homeostasis and enhanced responsiveness of pDCs in DAP12° animals. Increased numbers of pDCs were observed in the periphery of both naive and MCMV-infected DAP12° mice. A higher proportion of pDCs was activated in infected DAP12° mice, as demonstrated by intracellular staining using an optimized protocol for simultaneous detection of IFN-α and IFN-β. The homeostasis of WT and DAP12° pDCs did not differ in mixed bone marrow chimeric mice. In addition, a similar efficiency of pDC differentiation was observed in vitro in Fms-like tyrosine kinase receptor 3 ligand cultures of WT and DAP12° bone marrow cells. This suggests that DAP12 signaling effects on pDC homeostasis are indirect. In contrast, in response to CpG, DAP12-mediated effects on both IL-12 and IFN-αβ production were intrinsic to the pDCs. However, in response to MCMV, only IL-12 but not IFN-αβ production was affected by pDC-intrinsic DAP12 signaling. Thus, DAP12 signaling in pDCs can mediate different regulatory effects on their functions, depending on the mechanisms of pDC activation. The potential implications of the regulation of pDC functions by DAP12 for promoting health over disease are discussed.

Several recent publications have focused on the newly described interactions between natural-killer (NK) cells and dendritic cells (DCs). Activated NK cells induce DC maturation either directly or in synergy with suboptimal levels of microbial signals. Immature DCs appear susceptible to autologous NK-cell-mediated cytolysis while mature DCs are protected. NK-cell-induced DC activation is dependent on both tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) secretion and a cell-cell contact involving NKp30. In vitro, interleukin-12 (IL-12)/IL-18, IL-15, and IFN-α/β production by activated DCs enhance, in turn, NK-cell IFN-γ production, proliferation, and cytotoxic potential, respectively. In vivo, NK-cell/DC interactions may occur in lymphoid organs as well as in nonlymphoid tissues, and their consequences are multiple. By inducing DC activation, NK-cell activation induced by tumor cells can indirectly promote antitumoral T-cell responses. Reciprocally, DCs activated through Toll-like receptors (TLRs) induce potent NK-cell activation in antiviral responses. Thus, DCs and NK cells are equipped with complementary sets of receptors that allow the recognition of various pathogenic agents, emphasizing the role of NK-cell/DC crosstalk in the coordination of innate and adaptive immune responses.

Reference EPFL-ARTICLE-190193doi:10.1073/pnas.1315914110View record in Web of Science Record created on 2013-11-04, modified on 2017-05-12

MHC class I-specific Ly49 inhibitory receptors regulate NK cell activation, thereby preventing autologous damage to normal cells. Ly49 receptors are also expressed on a subset of CD8+ T cells whose origin and function remain unknown. We report here that, despite their phenotypic and cytolytic similarities, Ly49+CD8+ T cells and conventional Ly49−CD44high memory-phenotype CD8+ T cells present strikingly distinct features. First, under steady state conditions Ly49+CD8+ T cells are poor cytokine producers (TNF-α and IFN-γ) upon TCR triggering. Second, Ly49+CD8+ T cells are not induced upon various settings of Ag immunization or microbial challenge. However, Ly49 can be induced on a fraction of self-specific CD8+ T cells if CD4+ T cells are present. Finally, the size of the Ly49+CD8+ T cell subset is selectively reduced in the absence of STAT1. These results indicate that Ly49 expression is associated with a differentiation program of cytolytic CD8+ T cells triggered upon chronic antigenic exposure. They further suggest that the size of the Ly49+CD8+ T cell subset marks a history of CD8+ T cell activation that might preferentially result from endogenous inducers of inflammation rather than from microbial infections.

NK cells are capable of responding quickly to infectious challenge and contribute to the early defense against a wide variety of pathogens. Although the innate NK cell response to murine CMV (MCMV) has been extensively characterized, its resolution and the fate of the activated NK cell population remains unexplored. Herein, we characterize both the expansion and contraction phases of the NK cell response to MCMV. We demonstrate that NK cell recruitment into the immune response to MCMV infection is restricted to the first 3 days of infection and as the peripheral NK cell compartment expands, NK cells undergo accelerated phenotypic maturation. During the resolution of the immune response, NK cell compartmental contraction is marked by the selective death of responding NK cells. Additionally, throughout the infection, a naive NK cell pool that remains responsive to additional stimuli is actively maintained. These findings illustrate the plasticity of the NK cell compartment in response to pathogens and underscore the homeostatic maintenance of the resting peripheral NK cell pool.

With the goal of creating antigen-directed immunotherapeutics that can be safely administered directly to patients, Immune Design has developed a platform of novel integration-deficient lentiviral vectors that target and deliver antigen-encoding nucleic acids to dendritic cells (DCs) in order to promote the activation of antigen-specific effector CD8 T cells. This platform, termed DCVex(TM), utilizes a novel genetic variant of a Sindbis virus envelope glycoprotein with post-translational carbohydrate modifications in combination with Vpx, a SIVmac viral accessory protein, to achieve efficient targeting and transduction of DCs. In addition, DCVex(TM) incorporates safety features in its design that include redundant mechanisms to render DCVex(TM) integration-deficient, as well as genetic modifications that eliminate psi-gag recombination between split genome components. The characteristics that allow DCVex(TM) to specifically transduce human DCs and the advances that DCVex(TM) brings to conventional third-generation lentiviral vector design demonstrate its potential as a vaccine designed to utilize DCs for cancer immunotherapy. Citation Format: Semih U. Tareen, Brenna Kelley-Clarke, Christopher J. Nicolai, Megan M. Slough, Chintan D. Vin, Neal Van Hoeven, Scott H. Robbins, Jan H. ter Meulen, Peter Berglund. DCVex(TM): A novel integration-deficient lentivector technology that incorporates genetic and post-translational elements to target dendritic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 702. doi:10.1158/1538-7445.AM2014-702

Yields were evaluated three years after applied treatments to determine if responses that were not evident during earlier years eventually occurred. Potassium sulfate was applied to established, non‐irrigated, K deficient trees on fine textured soil by banding, placing in augered holes, adding to the backfilled trenches, and by injecting into the soil. Trenches were dug in the fall beside trees to break roots and ammended during backfilling with K2SO4, dolomite lime or combinations of the two. Additional trees received a heavy compost mulch in the early fall. Trenching treatments were generally detrimental. Trenching alone reduced yield and leaf Ca but increased fruit soluble solids content. Trenching plus K2SO4, trenching and lime, all soil amendments, and mushroom compost elevated leaf K from deficient or below normal to the normal range, but decreased leaf Mg. Most K application techniques eventually increased yield, but simple surface applications of K2SO4 in a narrow band were as effective a...