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Viruses are the second leading cause of cancer worldwide, and human papillomavirus (HPV)-associated head and neck cancers are increasing in incidence in the United States. HPV preferentially infects the crypts of the tonsils rather than the surface epithelium. The present study sought to characterize the unique microenvironment within the crypts to better understand the viral tropism of HPV to a lymphoid-rich organ. Laser-capture microdissection of distinct anatomic areas (crypts, surface epithelium, and germinal centers) of the tonsil, coupled with transcriptional analysis and multiparameter immunofluorescence staining demonstrated that the tonsillar crypts are enriched with myeloid populations that co-express multiple canonical and noncanonical immune checkpoints, including PD-L1, CTLA-4, HAVCR2 (TIM-3), ADORA2A, IDO1, BTLA, LGALS3, CDH1, CEACAM1, PVR, and C10orf54 (VISTA). The resident monocytes may foster a permissive microenvironment that facilitates HPV infection and persistence. Furthermore, the myeloid populations within HPV-associated tonsil cancers co-express the same immune checkpoints, providing insight into potential novel immunotherapeutic targets for HPV-associated head and neck cancers.

Generating responses to tumor antigens poses a challenge for immunotherapy. This phase II trial (NCT02129075) tested fms-like tyrosine kinase 3 (Flt3) ligand pre-treatment enhancement of responses to dendritic cell (DC)-targeting vaccines. We evaluated a regimen of Flt3L (CDX-301) to increase DCs and other antigen-presenting cells, poly-ICLC (TLR3 agonist that activates DCs) and a vaccine comprising anti-DEC-205-NY-ESO-1, a fusion antibody targeting CD205, linked to NY-ESO-1. High-risk melanoma patients were randomized to vaccine, with and without CDX-301. The end point was immune response to NY-ESO-1. Flt3L increased peripheral monocytes and conventional DCs (cDCs), including cross-presenting cDC1 and cDC2 and plasmacytoid DCs. Significant increases in humoral and T-cell responses and activation of DCs, natural killer cells and T cells were elicited. Transcriptional analyses revealed gene signatures associated with CDX-301 induction of an early, durable immune response. This study reveals in vivo effects of Flt3L on innate immune cells in the setting of vaccination, leading to an immunogenic vaccine regimen. Bhardwaj et al. report that adding Flt3 ligand to the treatment strategy effectively increased DC populations and increased T-cell responses in a randomized phase II trial of a DC-targeted vaccine for the melanoma antigen NY-ESO-1.

International audience; Down syndrome (DS) is characterized by the occurrence of three copies of human chromosome 21 (HSA21). HSA21 contains a cluster of four interferon receptor (IFN-R) genes: IFNAR1, IFNAR2, IFNGR2, and IL10RB. DS patients often develop mucocutaneous infections and autoimmune diseases, mimicking patients with heterozygous gain-of-function (GOF) STAT1 mutations, which enhance cellular responses to three types of interferon (IFN). A gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with DS. We report high levels of IFN-αR1, IFN-αR2, and IFN-γR2 expression on the surface of monocytes and EBV-transformed-B (EBV-B) cells from studying 45 DS patients. Total and phosphorylated STAT1 (STAT1 and pSTAT1) levels were constitutively high in unstimulated and IFN-α- and IFN-γ-stimulated monocytes from DS patients but lower than those in patients with GOF STAT1 mutations. Following stimulation with IFN-α or -γ, but not with IL-6 or IL-21, pSTAT1 and IFN-γ activation factor (GAF) DNA-binding activities were significantly higher in the EBV-B cells of DS patients than in controls. These responses resemble the dysregulated responses observed in patients with STAT1 GOF mutations. Concentrations of plasma type I IFNs were high in 12% of the DS patients tested (1.8% in the healthy controls). Levels of type I IFNs, IFN-Rs, and STAT1 were similar in DS patients with and without recurrent skin infections. We performed a genome-wide transcriptomic analysis based on principal component analysis and interferon modules on circulating monocytes. We found that DS monocytes had levels of both IFN-α- and IFN-γ-inducible ISGs intermediate to those of monocytes from healthy controls and from patients with GOF STAT1 mutations. Unlike patients with GOF STAT1 mutations, patients with DS had normal circulating Th17 counts and a high proportion of terminally differentiated CD8+ T cells with low levels of STAT1 expression. We conclude a mild interferonopathy in Down syndrome leads to an incomplete penetrance at both cellular and clinical level, which is not correlate with recurrent skin bacterial or fungal infections. The constitutive upregulation of type I and type II IFN-R, at least in monocytes of DS patients, may contribute to the autoimmune diseases observed in these individuals.

BackgroundIt is becoming clear that tumor immune T cell infiltration and its functional orientation have substantial effect on cancer progression, influencing both response to therapy and prognosis. In this pan-cancer study, the previously described Immunologic Constant of Rejection (ICR) signature is used to define opposing immune phenotypes (i.e., immuneactive and immune-silent) across 31 different histologies. We systematically analyze the interconnections between the genetic programming of neoplasms and their immune orientation across different histologies, and the prognostic impact of such interplay. Moreover, we investigated the predictive value of ICR classification across various public datasets of immune checkpoint inhibition therapy.MethodsRNA-seq data of samples from a total of 9,282 patient tumor samples representing 31 cancer types were obtained from The Cancer Genome Atlas (TCGA). We classified each cancer type based on the expression of the ICR gene signature. Oncogenic pathway gene set enrichment and mutational status were analyzed in relation to ICR phenotypes. To explore whether tumorintrinsic attributes associate with the prognostic value of ICR across cancers, we compared mutational load, oncogenic alterations and expression of oncogenic pathways between cancer types using an integrative bioinformatic pipeline.ResultsOur analyses identified a distinct prognostic connotation of ICR depending on cancer histology. We identified several oncogenic pathways whose enrichment inversely correlated with ICR in multiple tumor types. We found several cancer specific pathways that were differentially enriched between tumors in which ICR had a prognostic impact versus the ones in which ICR did not bear any prognostic connotation such as proliferation and TGF-beta signaling. Importantly, this conditional impact of ICR was also validated in the context of immune checkpoint inhibition treatment.ConclusionsWe identified tumor-intrinsic attributes that correlate with immune phenotypes and potentially influence their development. In addition, a relationship was observed between the enrichment of oncogenic pathways and the prognostic significance of the ICR and its predictive value for patients treated with anti-CTLA4 immune checkpoint inhibition. Such information can be used to prioritize potential candidates for therapies aimed at converting immune-silent into immuneactive tumors and to refine stratification algorithms.

We recently found by single-cell mass cytometry that ex vivo human B cells internalize graphene oxide (GO). The functional impact of such uptake on B cells remains unexplored. Here, we disclosed the effects of GO and amino-functionalized GO (GONH2) interacting with human B cells in vitro and ex vivo at the protein and gene expression levels. Moreover, our study considered three different subpopulations of B cells and their functionality in terms of: (i) cytokine production, (ii) activation markers, (iii) killing activity towards cancer cells. Single-cell mass cytometry screening revealed the higher impact of GO on cell viability towards naive, memory, and plasma B cell subsets. Different cytokines such as granzyme B (GrB) and activation markers, like CD69, CD80, CD138, and CD38, were differently regulated by GONH2 compared to GO, supporting possible diverse B cell activation paths. Moreover, co-culture experiments also suggest the functional ability of both GOs to activate B cells and therefore enhance the toxicity towards HeLa cancer cell line. Complete transcriptomic analysis on a B cell line highlighted the distinctive GO and GONH2 elicited responses, inducing pathways such as B cell receptor and CD40 signaling pathways, key players for GrB secretion. B cells were regularly left behind the scenes in graphene biological studies; our results may open new horizons in the development of GO-based immune-modulatory strategies having B cell as main actors.

Sepsis develops after a dysregulated host inflammatory response to a systemic infection. Identification of sepsis biomarkers has been challenging because of the multifactorial causes of disease susceptibility and progression. Public transcriptomic data are a valuable resource for mechanistic discoveries and cross-studies concordance of heterogeneous diseases. Nonetheless, the approach requires structured methodologies and effective visualization tools for meaningful data interpretation. Currently, no such database exists for sepsis or systemic inflammatory diseases in human. Hence we curated SysInflam HuDB (http://sepsis.gxbsidra.org/dm3/geneBrowser/list), a unique collection of human blood transcriptomic datasets associated with systemic inflammatory responses to sepsis. The transcriptome collection and the associated clinical metadata are integrated onto a user-friendly and Web-based interface that allows the simultaneous exploration, visualization, and interpretation of multiple datasets stemming from different study designs. To date, the collection encompasses 62 datasets and 5719 individual profiles. Concordance of gene expression changes with the associated literature was assessed, and additional analyses are presented to showcase database utility. Combined with custom data visualization at the group and individual levels, SysInflam HuDB facilitates the identification of specific human blood gene signatures in response to infection (e.g., patients with sepsis versus healthy control subjects) and the delineation of major genetic drivers associated with inflammation onset and progression under various conditions.

A cancer immune phenotype characterized by an active T-helper 1 (Th1)/cytotoxic response is associated with responsiveness to immunotherapy and favorable prognosis across different tumors. However, in some cancers, such an intratumoral immune activation does not confer protection from progression or relapse. Defining mechanisms associated with immune evasion is imperative to refine stratification algorithms, to guide treatment decisions and to identify candidates for immune-targeted therapy. Molecular alterations governing mechanisms for immune exclusion are still largely unknown. The availability of large genomic datasets offers an opportunity to ascertain key determinants of differential intratumoral immune response. We follow a network-based protocol to identify transcription regulators (TRs) associated with poor immunologic antitumor activity. We use a consensus of four different pipelines consisting of two state-of-the-art gene regulatory network inference techniques, regularized gradient boosting machines and ARACNE to determine TR regulons, and three separate enrichment techniques, including fast gene set enrichment analysis, gene set variation analysis and virtual inference of protein activity by enriched regulon analysis to identify the most important TRs affecting immunologic antitumor activity. These TRs, referred to as master regulators (MRs), are unique to immune-silent and immune-active tumors, respectively. We validated the MRs coherently associated with the immune-silent phenotype across cancers in The Cancer Genome Atlas and a series of additional datasets in the Prediction of Clinical Outcomes from Genomic Profiles repository. A downstream analysis of MRs specific to the immune-silent phenotype resulted in the identification of several enriched candidate pathways, including NOTCH1, TGF-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\beta $\end{document}, Interleukin-1 and TNF-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\alpha $\end{document} signaling pathways. TGFB1I1 emerged as one of the main negative immune modulators preventing the favorable effects of a Th1/cytotoxic response.

In tumor-bearing mice, cyclic fasting or fasting-mimicking diets (FMD) enhance the activity of antineoplastic treatments by modulating systemic metabolism and boosting antitumor immunity. Here we conducted a clinical trial to investigate the safety and biological effects of cyclic, five-day FMD in combination with standard antitumor therapies. In 101 patients, the FMD was safe, feasible, and resulted in a consistent decrease of blood glucose and growth factor concentration, thus recapitulating metabolic changes that mediate fasting/FMD anticancer effects in preclinical experiments. Integrated transcriptomic and deep-phenotyping analyses revealed that FMD profoundly reshapes anticancer immunity by inducing the contraction of peripheral blood immunosuppressive myeloid and regulatory T-cell compartments, paralleled by enhanced intratumor Th1/cytotoxic responses and an enrichment of IFNγ and other immune signatures associated with better clinical outcomes in patients with cancer. Our findings lay the foundations for phase II/III clinical trials aimed at investigating FMD antitumor efficacy in combination with standard antineoplastic treatments. Significance: Cyclic FMD is well tolerated and causes remarkable systemic metabolic changes in patients with different tumor types and treated with concomitant antitumor therapies. In addition, the FMD reshapes systemic and intratumor immunity, finally activating several antitumor immune programs. Phase II/III clinical trials are needed to investigate FMD antitumor activity/efficacy. This article is highlighted in the In This Issue feature, p. 1

IntroductionAccurate triage is an important first step to effectively manage the clinical treatment of severe cases in a pandemic outbreak. In the current COVID-19 global pandemic, there is a lack of reliable clinical tools to assist clinicians to perform accurate triage. Host response biomarkers have recently shown promise in risk stratification of disease progression; however, the role of these biomarkers in predicting disease progression in patients with COVID-19 is unknown. Here, we present a protocol outlining a prospective validation study to evaluate the biomarkers’ performance in predicting clinical outcomes of patients with COVID-19.Methods and analysisThis prospective validation study assesses patients infected with COVID-19, in whom blood samples are prospectively collected. Recruited patients include a range of infection severity from asymptomatic to critically ill patients, recruited from the community, outpatient clinics, emergency departments and hospitals. Study samples consist of peripheral blood samples collected into RNA-preserving (PAXgene/Tempus) tubes on patient presentation or immediately on study enrolment. Real-time PCR (RT-PCR) will be performed on total RNA extracted from collected blood samples using primers specific to host response gene expression biomarkers that have been previously identified in studies of respiratory viral infections. The RT-PCR data will be analysed to assess the diagnostic performance of individual biomarkers in predicting COVID-19-related outcomes, such as viral pneumonia, acute respiratory distress syndrome or bacterial pneumonia. Biomarker performance will be evaluated using sensitivity, specificity, positive and negative predictive values, likelihood ratios and area under the receiver operating characteristic curve.Ethics and disseminationThis research protocol aims to study the host response gene expression biomarkers in severe respiratory viral infections with a pandemic potential (COVID-19). It has been approved by the local ethics committee with approval number 2020/ETH00886. The results of this project will be disseminated in international peer-reviewed scientific journals.

Large independent analyses on cancer cell lines followed by functional studies have identified Schlafen 11 (SLFN11), a putative helicase, as the strongest predictor of sensitivity to DNA-damaging agents (DDAs), including platinum. However, its role as a prognostic biomarker is undefined, partially due to the lack of validated methods to score SLFN11 in human tissues. Here, we implemented a pipeline to quantify SLFN11 in human cancer samples. By analyzing a cohort of high-grade serous ovarian carcinoma (HGSOC) specimens before platinum-based chemotherapy treatment, we show, for the first time to our knowledge, that SLFN11 density in both the neoplastic and microenvironmental components was independently associated with favorable outcome. We observed SLFN11 expression in both infiltrating innate and adaptive immune cells, and analyses in a second, independent, cohort revealed that SLFN11 was associated with immune activation in HGSOC. We found that platinum treatments activated immune-related pathways in ovarian cancer cells in an SLFN11-dependent manner, representative of tumor-immune transactivation. Moreover, SLFN11 expression was induced in activated, isolated immune cell subpopulations, hinting that SLFN11 in the immune compartment may be an indicator of immune transactivation. In summary, we propose SLFN11 is a dual biomarker capturing simultaneously interconnected immunological and cancer cell-intrinsic functional dispositions associated with sensitivity to DDA treatment.

Transcriptome profiling approaches have been widely used to investigate the mechanisms underlying psoriasis pathogenesis. Most researchers have measured changes in transcript abundance in skin biopsies; relatively few have examined transcriptome changes in the blood. Although less relevant to the study of psoriasis pathogenesis, blood transcriptome profiles can be readily compared across various diseases. Here, we used a pre-established set of 382 transcriptional modules as a common framework to compare changes in blood transcript abundance in two independent public psoriasis datasets. We then compared the resulting “transcriptional fingerprints” to those obtained for a reference set of 16 pathological or physiological states. The perturbations in blood transcript abundance in psoriasis were relatively subtle compared to the changes we observed in other autoimmune and auto-inflammatory diseases. However, we did observe a consistent pattern of changes for a set of modules associated with neutrophil activation and inflammation; interestingly, this pattern resembled that observed in patients with Kawasaki disease. This similarity between the blood-transcriptome signatures in psoriasis and Kawasaki disease suggests that the immune mechanisms driving their pathogenesis might be partially shared.

Background It is now well established that the immune system has a substantial role in controlling cancer growth and progression. Immunotherapy is quickly coming to the forefront of cancer treatment,however the implementation of immunotherapy in pediatric solid cancers, which classically display a low mutational load, is hindered by insufficient understanding of the determinants of cancer immune responsiveness in children. In order to better understand tumor-host interplay, we sought to characterize solid pediatric cancers based on immunological parameters(1)using analytes extracted from gene expression data. Methods We performed single sample GeneSet Enrichment Analysis for 105 immune signatures previously described on 5pediatric tumors (410patients) from TARGET dataset(1) to identify coherent signature modules. Then we clustered samples according to representative signatures (1)and compared survival across clusters. We completed the analysis by analyzing the enrichment of immune subpopulations and the expression of the immune checkpoints. The degree of dysregulation of oncogenic pathways was also assessed. The performance of previously identified immune signatures as the Immunologic Constant of Rejection(2,3),which captures an active Th1/cytotoxic response associated with favorable prognosis and responsiveness to immunotherapy, was also checked within each tumor subtype. Results We found 5main modules, in agreement with results obtained in adult solid tumors:Wound Healing,TGF-B signaling,IFN-G signaling, Macrophages and Lymphocytes (figure 1). These 5 modules clustered pediatric patients into 6 immune subtypes S1-S6 with distinct survival (S2vsS4,p=0.0044, adjusted for cancer type),S2 cluster has the best overall survival and characterized by low enrichment of wound healing signature, high Th1, low Th2 and high expression of HLA 1 and HLA2, while the opposite holds true for cluster S4 with the worst survival and highest enrichment of wound healing signature, high Th2, and low Th1.The S6 cluster is characterized by highest enrichment of lymphocyte signature, the highest expression of immune checkpoints accompanied by elevated expression of exhaustion markers, and an unpolarized immune response with high abundance of macrophages. Additionally, pan-cancer, the upregulation of WNT-Beta catenin pathway is associated with adverse outcome and lack of T-cell infiltration. In the per-cancer analysis, ICR is associated with better survival in osteosarcoma and with worse survival in Wilms’ tumors,similarly with what observed in adult kidney’s cancer despite the different embryological origin. Conclusions We demonstrated that pediatric solid cancers can be classified according to their immune disposition, unveiling unexpected similarity with adults’ tumors.Immunological parameters might be explored to refine diagnostic and prognostic biomarkers and to identify potential immune responsive tumors. This is the first pan-cancer immunogenomic analysis in children. References Thorsson V, Gibbs DL, Brown SD, et al. The immune landscape of cancer. Immunity 2018. 48(4):812–830. Roelands J, Hendrickx W, et al. ‘Oncogenic states dictate the prognostic and predictive connotations of intratumoral immune response.’ Journal for immunotherapy of cancer 2020;vol. 8:1. Galon J, Angell HK, Bedognetti D, Marincola FM. The continuum of cancer immunosurveillance: prognostic, predictive, and mechanistic signatures. Immunity 2013;39:11–26.

BackgroundTranscriptome profiling approaches have been widely used in the investigation of mechanisms underlying psoriasis pathogenesis. In most instances, changes in transcript abundance have been measured in skin biopsies. Fewer studies have examined changes in the blood samples from patients with psoriasis. While changes in the periphery may be less relevant, the blood cell transcriptome analysis presents the distinct advantage of being amenable to comparison across diseases.MethodsTwo public psoriasis blood transcriptome datasets were reanalyzed and compared against reference datasets encompassing 16 immune states and pathologies, employing a recently established modular repertoire framework.ResultsAlthough perturbations in psoriasis were relatively subtle in comparison to other auto-immune or auto-inflammatory diseases, consistent changes were observed for a signature associated with neutrophil activation/inflammation. This transcriptional signature most resembled that of subjects with Kawasaki disease.ConclusionsThe similarities observed between psoriasis and Kawasaki disease blood transcriptome signatures suggest that immune mechanisms driving the pathogenesis of these diseases may be at least partially shared. This notion is reinforced by case reports describing the development of psoriasis disease in patients with Kawasaki disease. Potential implications for novel therapeutic approaches, including the repurposing of biologic drugs targeting IL17 or its receptor for the treatment of Kawasaki disease are discussed.

BackgroundAn immune active cancer phenotype typified by a T helper 1 (Th-1) immune response has been associated with increased responsiveness to immunotherapy and favorable prognosis in some but not all cancer types. The reason of this differential prognostic connotation remains unknown.MethodsTo explore the contextual prognostic value of cancer immune phenotypes, we applied a multimodal pan-cancer analysis among 31 different histologies (9282 patients), encompassing immune and oncogenic transcriptomic analysis, mutational and neoantigen load and copy number variations.ResultsWe demonstrated that the favorable prognostic connotation conferred by the presence of a Th-1 immune response was abolished in tumors displaying specific tumor-cell intrinsic attributes such as high transforming growth factor-beta (TGF-β) signaling and low proliferation capacity. This observation was independent of mutation rate. We validated this observation in the context of immune checkpoint inhibition. WNT-βcatenin, barrier molecules, Notch, hedgehog, mismatch repair, telomerase activity and AMPK signaling were the pathways most coherently associated with an immune silent phenotype together with mutations of driver genes includingIDH1/2, FOXA2, HDAC3, PSIP1, MAP3K1, KRAS, NRAS, EGFR, FGFR3, WNT5AandIRF7.ConclusionsThis is the first systematic study demonstrating that the prognostic and predictive role of a bona fide favorable intratumoral immune response is dependent on the disposition of specific oncogenic pathways. This information could be used to refine stratification algorithms and prioritize hierarchically relevant targets for combination therapies.

A potential role for the long-chain acyl-CoA synthetase family member 1 (ACSL1) in the immunobiology of sepsis was explored during a hands-on training workshop. Participants first assessed the robustness of the potential gap in biomedical knowledge identified via an initial screen of public transcriptome data and of the literature associated with ACSL1. Increase in ACSL1 transcript abundance during sepsis was confirmed in several independent datasets. Querying the ACSL1 literature also confirmed the absence of reports associating ACSL1 with sepsis. Inferences drawn from both the literature (via indirect associations) and public transcriptome data (via correlation) point to the likely participation of ACSL1 and ACSL4, another family member, in inflammasome activation in neutrophils during sepsis. Furthermore, available clinical data indicate that levels of ACSL1 and ACSL4 induction was significantly higher in fatal cases of sepsis. This denotes potential translational relevance and is consistent with involvement in pathways driving potentially deleterious systemic inflammation. Finally, while ACSL1 expression was induced in blood in vitro by a wide range of pathogen-derived factors as well as TNF, induction of ACSL4 appeared restricted to flagellated bacteria and pathogen-derived TLR5 agonists and IFNG. Taken together, this joint review of public literature and omics data records points to two members of the acyl-CoA synthetase family potentially playing a role in inflammasome activation in neutrophils. Translational relevance of these observations in the context of sepsis and other inflammatory conditions remain to be investigated.

Steps involved in reductionist investigation approaches can be imitated using public transcriptome datasets as source of training material. In the present report trainees explored an apparent gap in biological knowledge for FAM129A (family with sequence similarity 129 member A). Elevated abundance of FAM129A transcripts were observed in a transcriptome dataset where neutrophils were exposed in vitro to plasma of patients with sepsis. However, no literature linking FAM129A and either neutrophils, sepsis or inflammation could be identified. Additional datasets were selected to independently validate this initial observation and further explore differential expression of FAM129A in the context of sepsis studies. Follow on investigations carried out at the bench confirmed restriction of the expression of FAM129A protein at the surface of circulating blood neutrophils and monocytes. A potential role for FAM129A in neutrophil survival was inferred from profiling of literature associated with FAM129A, which remains to be investigated in further follow on investigations.

Biomarkers to assess the severity of acute respiratory syncytial virus (RSV) infection are needed. We conducted a meta-analysis of 490 unique profiles from six public RSV blood transcriptome datasets. A repertoire of 382 well-characterized transcriptional modules was used to define dominant host responses to RSV infection. The consolidated RSV cohort was stratified according to four traits: “interferon response” (IFN), “neutrophil-driven inflammation” (Infl), “cell cycle” (CC), and “erythrocytes” (Ery). Eight prevalent blood transcriptome phenotypes were thus identified. Among those three Ery+ phenotypes comprised higher proportions of patients requiring intensive care. We posit that the erythrocyte module is linked to an overabundance of immunosuppressive erythroid cells that might underlie progression to severe RSV infection. These findings outline potential priority areas for biomarker development and investigations into the immune biology of RSV infection. The approach that was employed here will also permit to delineate prevalent blood transcriptome phenotypes in other settings.

Background Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to graft versus tumor effect (GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD have been identified to date. To study the mechanisms of GVHD and GVT and to identify potential GVHD markers we applied a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcriptome profiling. The TFA is a multiplex microfluidics q-PCR based assay, linked to a computational model for functional analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures (Chaussabel et a 2014, Speake et a 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases. Herein we report the discovery of an association between the expression abundance of a set of genes pre-transplant and the development of acute or chronic GVHD. Methods Seventy-eight patients candidate to allo-HCT were enrolled on the study. Patients donated micro-quantities of blood (50-600 microliters) prior to transplant conditioning, weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high-throughput qPCR system. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Here we report the univariate analysis of gene expression data and its association with GVHD in 31 patients for whom data are available until at least day 169 post-transplant (169-672, median 513). SIgnificant genes were tested in a longitudinal analysis with 4 combined linear-mixed models, measuring the parameters: 1) groups (GVHD NO-GVHD) 2) time 3) interaction of groups over time. Results Patients' characteristics are summarized in Fig 1; 26 patients developed GVHD (12 acute GVHD only, 3 late acute GVHD, 1 overlap syndrome, 5 acute and chronic GVHD and 5 chronic GVHD only). By TFA analysis of pre-transplant samples, we identified 3 genes significantly downregulated among patients who developed GVHD (GPSM3, LRG1 and EPHX4), and 2 genes borderline (1.5 fold upregulated in the GVHD group, but with a non-significant p-value (Volcano-plot Fig.1). Of note, 12 of them were >4 fold upregulated, and their transcriptome pertains to B cells, T lymphocytes, monocytes, IFN, TNF, other cytokines and neutrophil activation. Moreover, among these genes IFN-genes were consistently represented by multiple IFN-modules, emerging as promising candidates. Longitudinal analyses confirmed that EPHX4 and GPSM3 distinguished the cohorts for all the measured parameters in the post-transplant time. EPHX4 increased over time in both groups but significantly more in the NO-GVHD group (with a fixed effect estimate of 2.05); GPSM3 instead decreased over time, again significantly more in the NO-GVHD group (with a fixed effect estimate of -0.95). LRG3 showed significance only for the parameter "time", resulting after transplant more abundant in the GVHD group, thus confirming previous literature data. IFN modules were also tested longitudinally, showing very distinct patterns in the cohorts, and several significant genes (IFI27, NT5C3, CCR1, LBA1, Fig.1) Conclusion Our data demonstrate that TFA may be used to assess expression of immune related genes in transplant patients and to identify specific pathways that may be predictive for GVHD development. Additional work is required to validate our data in a larger cohort and for better understanding of the role of those genes in triggering or protecting against GVHD. Upon completion of the study and the longitudinal analyses, we aim to define in depth transcriptome signatures of GVHD and relapse. Disclosures No relevant conflicts of interest to declare.

A hands-on training workshop was devised with omics data used as source material to emulate reductionist investigation approaches. NUDT16, a member of the Nudix hydrolase family was selected as a candidate gene on the basis of: 1) it being upregulated in neutrophils exposed in vitro to serum of patients with sepsis, AND 2) the absence of overlap between the NUDT16 and sepsis, inflammation or neutrophil literature. We next sought to corroborate the initial finding in five public transcriptome sepsis datasets in which NUDT16 transcript levels were measured. In each of these dataset NUDT16 transcript abundance was found to be significantly increased in septic patients in vivo in comparison to uninfected controls. Next, biological concepts were extracted from the NUDT16 literature. The main concepts to emerge from profiling this literature were RNA decapping, inosine triphosphate, and inosine diphosphate. Through these concepts, indirect links could be established between NUDT16 and the sepsis/inflammation/neutrophil literature. A potential role for NUDT16 could, in turn, be inferred in the degradation of mRNAs in activated neutrophils. Follow on experiments that would be necessary in order to further explore such inference are discussed.

Polymorphonuclear neutrophils (PMNs) are terminally differentiated cells that are involved in innate immune responses and form an early line of defense against pathogens. More recently, it has been shown that PMNs have immunosuppressive abilities on other immune cells. However, the effect of PMNs on T cell responses during bacterial infection remains to be determined. In this report, we examined the interaction of PMNs and T cells in response to infection with Burkholderia pseudomallei, the causative agent of human melioidosis. We observed that CD4+ T cell proliferation and IFN-γ production in response to polyclonal activators is significantly inhibited by uninfected PMNs, and to a greater extent B. pseudomallei–infected PMNs. Programmed death ligand 1 (PD-L1), a known regulator of T cell activation, is increased in mRNA expression in the blood of patients and upon infection of PMNs in vitro. The increased expression of PD-L1 was correlated with the degree of T cell inhibition in individuals with type 2 diabetes, a major risk factor of melioidosis. In vitro, addition of anti–PD-L1 Abs blocked this inhibitory activity and restored proliferation of CD4+ T cells and IFN-γ production, suggesting that PD-L1 on B. pseudomallei–infected PMNs is a regulatory molecule for the functions of T cells and may be involved in pathogenesis versus control of melioidosis.

The increased application of high-throughput approaches in translational research has expanded the number of publicly available data repositories. Gathering additional valuable information contained in the datasets represents a crucial opportunity in the biomedical field. To facilitate and stimulate utilization of these datasets, we have recently developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB). In this note, we describe a curated compendium of 13 public datasets on human breast cancer, representing a total of 2142 transcriptome profiles. We classified the samples according to different immune based classification systems and integrated this information into the datasets. Annotated and harmonized datasets were uploaded to GXB. Study samples were categorized in different groups based on their immunologic tumor response profiles, intrinsic molecular subtypes and multiple clinical parameters. Ranked gene lists were generated based on relevant group comparisons. In this data note, we demonstrate the utility of GXB to evaluate the expression of a gene of interest, find differential gene expression between groups and investigate potential associations between variables with a specific focus on immunologic classification in breast cancer. This interactive resource is publicly available online at: http://breastcancer.gxbsidra.org/dm3/geneBrowser/list.

Melioidosis, caused by Burkholderia pseudomallei, is endemic in northeastern Thailand and Northern Australia. Severe septicemic melioidosis is associated with high levels of pro-inflammatory cytokines and is correlated with poor clinical outcomes. IL-10 is an immunoregulatory cytokine, which in other infections can control the expression of pro-inflammatory cytokines, but its role in melioidosis has not been addressed. Here, whole blood of healthy seropositive individuals (n = 75), living in N. E. Thailand was co-cultured with B. pseudomallei and production of IL-10 and IFN-γ detected and the cellular sources identified. CD3− CD14+ monocytes were the main source of IL-10. Neutralization of IL-10 increased IFN-γ, IL-6 and TNF-α production and improved bacteria killing. IFN-γ production and microbicidal activity were impaired in individuals with diabetes mellitus (DM). In contrast, IL-10 production was unimpaired in individuals with DM, resulting in an IL-10 dominant cytokine balance. Neutralization of IL-10 restored the IFN-γ response of individuals with DM to similar levels observed in healthy individuals and improved killing of B. pseudomallei in vitro. These results demonstrate that monocyte derived IL-10 acts to inhibit potentially protective cell mediated immune responses against B. pseudomallei, but may also moderate the pathological effects of excessive cytokine production during sepsis.

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei–exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.

Introduction Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to the immune response of the donor cells against the malignant cells (graft versus tumor effect; GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD and GVT have been identified to date. Additionally, clinical GVHD diagnosis can be challenging due to chemotherapy-related or infection-related organ toxicity manifestations, which further complicate prediction and treatment stratification algorithms. In order to study the mechanisms of GVHD and GVT and to identify potential GVHD markers we apply a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcript profiling. The TFA is a multiplex microfluidics q-PCR based assay linked with a computational model for modular functional transcriptome analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures from < 1 ml of blood (Chaussabel and Baldwin. Nat Rev Immunol 2014, Speake et al. Clin Exp Immunol 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases (Banchereau R et al. Cell 2016, Dunning et al. Nat Immunol 2018). In our study we use the TFA to capture longitudinal immune signatures as dynamic "snapshots" of the patient's immune system after HCT. Hypotheses Fluctuations over-time in gene expression of allo HCT patients' immune system reflect the pathologic/disease control programs (GVHD/GVT) and may be used to identify diagnostic and predictive biomarkers. GVHD/GVT control immune programs depend on the "inner" interface between the donor immune-system and the recipient, and are influenced by external variables, as infections or drugs. These variables can affect the immune system-related gene expression and can be measured. Objectives To systematically measure gene expression signatures in immune perturbations post-allo HCT, in order to: Identify GVHD-related immune signatures consistent with clinical diagnosis of GVHD.Predict and stratify therapy-resistant GVHD and severe chronic GVHD, according to immune signatures.Identify links (causative and consequential) between GVHD, GVT, relapse, and other post-transplant immune perturbations (e.g. infections). Methods Enroll 250 allo HCT patients to populate a "GVHD cohort" and a "non-GVHD cohort" of 50 patients each, and 50 donors (healthy controls cohort) . Patients donate micro-quantities of blood (50 to 600 microliters), weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high throughput qPCR system, and normalized to the geometric mean Ct of 8 housekeeping genes. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Results Results of three series of patients' samples are shown to exemplify the potential of TFA as a method to study the mechanisms of GVHD and GVT. All three patients underwent myeloablative peripheral blood stem cell transplant from an HLA identical sibling donor. Two patients developed steroid responsive-acute GVHD (patient #1: GVHD stage I was diagnosed on day 38 post HCT, patient #4: GVHD stage III was diagnosed on day 21 post HCT). One patient (Patient #6) did not develop clinical GVHD, but routine skin biopsy on day 80 revealed apoptotic cells consistent with subclinical skin GVHD. Principal Component Analysis (PCA) of the three patients' series is shown in Figure 1, the dynamic transcriptomes according to TFA modules of patients #1 and #6 are shown in Figure 2, and representative TFA modular fingerprints are shown in Figure 3. Conclusion We anticipate that using the TFA approach will help to fill knowledge gaps instrumental to solve clinical dilemmas related to allo HCT complications, and to improve the clinical outcomes of allo HCT patients. Disclosures No relevant conflicts of interest to declare.

Neutrophil extracellular traps (NETs) are a recently identified, web-like, extracellular structure composed of decondensed nuclear DNA and associated antimicrobial granules. NETs are extruded into the extracellular environment via the reactive oxygen species (ROS)-dependent cell death pathway participating in inflammation and autoimmune diseases. Transketolase (TKT) is a thiamine pyrophosphate (vitamin B1)-dependent enzyme that links the pentose phosphate pathway with the glycolytic pathway by feeding excess sugar phosphates into the main carbohydrate metabolic pathways to generate biosynthetic reducing capacity in the form of NADPH as a substrate for ROS generation. In this work, TKT was selected as a lead candidate from 24 NET-associated proteins obtained by literature screening and knowledge gap assessment. Consequently, we determined whether TKT influenced NET formation in vitro. We firstly established that the release of ROS-dependent NETs was significantly decreased after purified human PMNs were pretreated with oxythiamine, a TKT inhibitor, and in a concentration dependent manner. As a cofactor for TKT reaction, we evaluated the release of NET formation either in vitamin B1 treatment or in combined use of oxythiamine and vitamin B1, and found that those treatments also exerted a significant suppressive effect on the amount of NET-DNA and ROS production. The regulation of TKT by oxythiamine and/or vitamin B1 may therefore be associated with response to the modulation of NET formation by preventing generation of excessive NETs in inflammatory diseases.

Summary We present an approach integrating structural and computational biology with immunological tests to identify epitopes in the OppA antigen from the Gram-negative pathogen Burkholderia pseudomallei , the etiological agent of melioidosis. The crystal structure of OppA Bp , reported here at 2.1 A resolution, was the basis for a computational analysis that identified three potential epitopes. In parallel, antigen proteolysis and immunocapturing allowed us to identify three additional peptides. All six potential epitopes were synthesized as free peptides and tested for their immunoreactivity against sera from healthy seronegative, healthy seropositive, and recovered melioidosis patients. Three synthetic peptides allowed the different patient groups to be distinguished, underlining the potential of this approach. Extension of the computational analysis, including energy-based decomposition methods, allowed rationalizing results of the predictive analyses and the immunocapture epitope mapping. Our results illustrate a structure-based epitope discovery process, whose application may expand our perspectives in the diagnostic and vaccine design fields.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Background: Members of the ADAM (a disintegrin and metalloprotease domain) family have emerged as critical regulators of cell-cell signaling during development and homeostasis. ADAM9 is consistently overexpressed in various human cancers, and has been shown to play an important role in tumorigenesis. However, little is known about the involvement of ADAM9 during immune-mediated processes. Results: Mining of an extensive compendium of transcriptomic datasets identified important gaps in knowledge regarding the possible role of ADAM9 in immunological homeostasis and inflammation: 1) The abundance of ADAM9 transcripts in the blood was increased in patients with acute infection but, 2) changed very little after in vitro exposure to a wide range of pathogen-associated molecular patterns (PAMPs). 3) Furthermore it was found to increase significantly in subjects as a result of tissue injury or tissue remodeling, in absence of infectious processes. Conclusions: Our findings indicate that ADAM9 may constitute a valuable biomarker for the assessment of tissue damage, especially in clinical situations where other inflammatory markers are confounded by infectious processes.

The major risk factor for melioidosis, an infectious disease caused by B. pseudomallei, is diabetes mellitus. More than half of diabetic melioidosis patients in Thailand were prescribed glibenclamide. Recent evidence demonstrates that glibenclamide reduces pro-inflammatory cytokine production by polymorphonuclear neutrophils (PMNs) of diabetic individuals in response to this bacterial infection. However, the mechanisms by which glibenclamide affects cytokine production are unknown. We found that PMNs from glibenclamide-treated diabetic individuals infected with live B. pseudomallei in vitro showed lower free glutathione (GSH) levels compared with those of healthy individuals. Glibenclamide decreased GSH levels and glutathione peroxidase (GPx) of PMNs after exposed to live B. pseudomallei. Moreover, glibenclamide reduced cytokine production and migration capacity of infected PMNs, whereas GSH could restore these functions. Taken together, our data show a link between the effect of glibenclamide on GSH and PMN functions in response to B. pseudomallei that may contribute to the susceptibility of diabetic individuals to B. pseudomallei infection.

Septicemia is the most severe form of melioidosis caused by the Gram-negative bacterium, Burkholderia pseudomallei. Here, we show that levels of IL-27p28 transcript and protein were both significantly elevated in patients with sepsis, particularly melioidosis and in patients with unfavorable disease outcome. Moreover, human monocytes/macrophages and neutrophils were the major source of IL-27 during infection. The addition of exogenous IL-27 in vitro resulted in significantly increased bacterial survival, reduced B. pseudomallei-induced oxidative burst, and enhanced IL-1β and TNF-α production by purified neutrophils from healthy subjects. Finally, blockade of endogenous IL-27 in neutrophils using soluble IL-27 receptor antagonist prior to infection led to significantly reduced survival of bacteria and decreased IL-1β, but not TNF-α production. These results indicate a potential role for IL-27 in the suppression of anti-bacterial defense mechanisms that might contribute to disease severity in sepsis. The targeting of this cytokine may be beneficial in the management of human sepsis.

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a life-threatening disease of humans. Within host cells, superoxide is an important mediator of pathogen killing. In this study, we have identified the B. pseudomallei K96243 sodC gene, shown that it has superoxide dismutase activity, and constructed an allelic deletion mutant of this gene. Compared with the wild-type, the mutant was more sensitive to killing by extracellular superoxide, but not to superoxide generated intracellularly. The sodC mutant showed a markedly decreased survival in J774A.1 mouse macrophages, and reduced numbers of bacteria were recovered from human polymorphonuclear neutrophils (PMNs) when compared with the wild-type. The numbers of wild-type or mutant bacteria recovered from human diabetic neutrophils were significantly lower than from normal human neutrophils. The sodC mutant was attenuated in BALB/c mice. Our results indicate that SodC plays a key role in the virulence of B. pseudomallei, but that diabetics are not more susceptible to infection because of a reduced ability of PMNs to kill by superoxide.

Neutrophils play a key role in the control of Burkholderia pseudomallei, the pathogen that causes melioidosis. Here, we show that survival of intracellular B. pseudomallei was significantly increased in the presence of 3-methyladenine or lysosomal cathepsin inhibitors. The LC3-flux was increased in B. pseudomallei-infected neutrophils. Concordant with this result, confocal microscopy analyses using anti-LC3 antibodies revealed that B. pseudomallei-containing phagosomes partially overlapped with LC3-positive signal at 3 and 6 h postinfection. Electron microscopic analyses of B. pseudomallei-infected neutrophils at 3 h revealed B. pseudomallei-containing phagosomes that occasionally fused with phagophores or autophagosomes. Following infection with a B. pseudomallei mutant lacking the Burkholderia secretion apparatus Bsa Type III secretion system, neither this characteristic structure nor bacterial escape into the cytosol were observed. These findings indicate that human neutrophils are able to recruit autophagic machinery adjacent to B. pseudomallei-containing phagosomes in a Type III secretion system-dependent manner.

This work was supported by National Institutes of Health–National Institute of Allergy and Infectious Diseases Large Scale T Cell Epitope Discovery Program Contract HHSN27220090046C (to R.B. and D.A.), the Welton Foundation (to R.B.), the National Institute for Health Research Biomedical Research funding scheme (to R.B. and D.A.), and a Wellcome Trust Intermediate Clinical Fellowship award (WT100174AIA; to S.D.). There is an urgent need for a better understanding of adaptive immunity to Burkholderia pseudomallei, the causative agent of melioidosis that is frequently associated with sepsis or death in patients in Southeast Asia and Northern Australia. The imperative to identify vaccine targets is driven both by the public health agenda in these regions and biological threat concerns. In several intracellular bacterial pathogens, alkyl hydroperoxidase reductases are upregulated as part of the response to host oxidative stress, and they can stimulate strong adaptive immunity. We show that alkyl hydroperoxidase reductase (AhpC) of B. pseudomallei is strongly immunogenic for T cells of 'humanized' HLA transgenic mice and seropositive human donors. Some T cell epitopes, such as p6, are able to bind diverse HLA class II heterodimers and stimulate strong T cell immunity in mice and humans. Importantly, patients with acute melioidosis who survive infection show stronger T cell responses to AhpC relative to those who do not. Although the sequence of AhpC is virtually invariant among global B. pseudomallei clinical isolates, a Cambodian isolate varies only in C-terminal truncation of the p6 T cell epitope, raising the possibility of selection by host immunity. This variant peptide is virtually unable to stimulate T cell immunity. For an infection in which there has been debate about centrality of T cell immunity in defense, these observations support a role for T cell immunity to AhpC in disease protection. Publisher PDF

Burkholderia pseudomallei is a Gram-negative bacterium responsible for melioidosis, a serious and often fatal infectious disease that is poorly controlled by existing treatments. Due to its inherent resistance to the major antibiotic classes and its facultative intracellular pathogenicity, an effective vaccine would be extremely desirable, along with appropriate prevention and therapeutic management. One of the main subunit vaccine candidates is flagellin of Burkholderia pseudomallei (FliCBp). Here, we present the high resolution crystal structure of FliCBp and report the synthesis and characterization of three peptides predicted to be both B and T cell FliCBp epitopes, by both structure-based in silico methods, and sequence-based epitope prediction tools. All three epitopes were shown to be immunoreactive against human IgG antibodies and to elicit cytokine production from human peripheral blood mononuclear cells. Furthermore, two of the peptides (F51-69 and F270-288) were found to be dominant immunoreactive epitopes, and their antibodies enhanced the bactericidal activities of purified human neutrophils. The epitopes derived from this study may represent potential melioidosis vaccine components., Author Summary Melioidosis is an infectious disease caused by Burkolderia pseudomallei that poses a major public health problem in Southeast Asia and northern Australia. This bacterium is difficult to treat due to its intrinsic resistance to antibiotics, poor diagnosis, and the lack of a licensed vaccine. Vaccine safety is a prime concern, therefore recombinant protein subunit and/or peptide vaccine components, may represent safer alternatives. In this context, we targeted one of the main subunit vaccine candidates tested to date, flagellin from B. pseudomallei (FliCBp) that comprises the flagellar filament that mediates bacterial motility. Based on the knowledge that activation of both cell-mediated and antibody-mediated responses must be addressed in a melioidosis vaccine, we identified B and T cell immunoreactive peptides from FliCBp, using both sequence-based and structure-based computational prediction programs, for further in vitro immunological testing. Our data confirm the accuracy of sequence-based epitope prediction tools, and two structure-based methods applied to the FliCBp crystal structure (here-described), in predicting both T- and B-cell epitopes. Moreover, we identified two epitope peptides with significant joint T-cell and B-cell activities for further development as melioidosis vaccine components.