Germinal center reactions in tertiary lymphoid structures associate with neoantigen burden, humoral immunity and long-term survivorship in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has traditionally been thought of as an immunologically quiescent tumor type presumably because of a relatively low tumor mutational burden (TMB) and poor responses to checkpoint blockade therapy. However, many PDAC tumors exhibit T cell inflamed phenotypes. The presence of tertiary lymphoid structures (TLS) has recently been shown to be predictive of checkpoint blockade response in melanomas and sarcomas, and are prognostic for survival in PDAC. In order to more comprehensively understand tumor immunity in PDAC patients with TLS, we performed RNA-seq, single and multiplex IHC, flow cytometry and predictive genomic analysis on treatment naïve, PDAC surgical specimens. Forty-six percent of tumors contained distinct T and B cell aggregates reflective of "early-stage TLS" (ES-TLS), which correlated with longer overall and progression-free survival. These tumors had greater CD8+ T cell infiltration but were not defined by previously published TLS gene-expression signatures. ES-TLS+ tumors were enriched for IgG1 class-switched memory B cells and memory CD4+ T cells, suggesting durable immunological memory persisted in these patients. We also observed the presence of active germinal centers (mature-TLS) in 31% of tumors with lymphocyte clusters, whose patients had long-term survival (median 56 months). M-TLS-positive tumors had equivalent overall T cell infiltration to ES-TLS, but were enriched for activated CD4+ memory cells, naive B cells and NK cells. Finally, using a TCGA-PDAC dataset, ES-TLS+ tumors harbored a decreased TMB, but M-TLS with germinal centers expressed significantly more MHCI-restricted neoantigens as determined by an in silico neoantigen prediction method. Interestingly, M-TLS+ tumors also had evidence of increased rates of B cell somatic hypermutation, suggesting that germinal centers form in the presence of high-quality tumor neoantigens leading to increased humoral immunity that confers improved survival for PDAC patients. AbbreviationsTLS: tertiary lymphoid structures; GC: germinal center(s); PDAC: pancreatic ductal adenocarcinoma; RNA-seq: RNA sequencing; BCRseq: B cell receptor sequencing; HEV: high endothelial venule; PNAd: peripheral node addressin; TMB: tumor mutational burden; TCGA: the cancer genome atlas; PAAD: pancreatic adenocarcinoma; FFPE: formalin fixed paraffin embedded; TIME: tumor immune microenvironment.

Transcriptional and immunohistological assessment of immune infiltration in pancreatic cancer.

Pancreatic adenocarcinoma is characterized by a complex tumor environment with a wide diversity of infiltrating stromal and immune cell types that impact the tumor response to conventional treatments. However, even in this poorly responsive tumor the extent of T cell infiltration as determined by quantitative immunohistology is a candidate prognostic factor for patient outcome. As such, even more comprehensive immunophenotyping of the tumor environment, such as immune cell type deconvolution via inference models based on gene expression profiling, holds significant promise. We hypothesized that RNA-Seq can provide a comprehensive alternative to quantitative immunohistology for immunophenotyping pancreatic cancer. We performed RNA-Seq on a prospective cohort of pancreatic tumor specimens and compared multiple approaches for gene expression-based immunophenotyping analysis compared to quantitative immunohistology. Our analyses demonstrated that while gene expression analyses provide additional information on the complexity of the tumor immune environment, they are limited in sensitivity by the low overall immune infiltrate in pancreatic cancer. As an alternative approach, we identified a set of genes that were enriched in highly T cell infiltrated pancreatic tumors, and demonstrate that these can identify patients with improved outcome in a reference population. These data demonstrate that the poor immune infiltrate in pancreatic cancer can present problems for analyses that use gene expression-based tools; however, there remains enormous potential in using these approaches to understand the relationships between diverse patterns of infiltrating cells and their impact on patient treatment outcomes.

Implementation and Validation of an Automated Flow Cytometry Analysis Pipeline for Human Immune Profiling.

Automated reagent preparation, sample processing, and data acquisition have increased the rate at which flow cytometry data can be generated. Furthermore, advances in technology and flow cytometry instrumentation continually increase the complexity and dimensionality of this data. Together, this leads to increased pressure on manual data analysis, which has inherent limitations including subjectivity of the analyst and the length of time needed for data processing. These issues can create bottlenecks in the data processing workflow and potentially compromise data quality. To address these issues, as well as the challenges associated with manual gating in a high-volume human immune profiling laboratory, we sought to implement an automated analysis pipeline. In this report, we discuss considerations for selecting an automated analysis method, the process of implementing an automated pipeline, and detail our successful incorporation of an automated gating strategy with flowDensity into our analysis workflow. This validated pipeline augments our laboratory's ability to provide rapid high-throughput immune profiling for patients participating in cancer immunotherapy clinical trials. © International Society for Advancement of Cytometry.

Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer.

The immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous - single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+ T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85-22% of CD8+ cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+ T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy.

Coordinated responses to individual tumor antigens by IgG antibody and CD8+ T cells following cancer vaccination.

BACKGROUND: One of today's greatest hurdles for cancer immunotherapy is the absence of information regarding which tumor antigens are already recognized by patients receiving immunotherapies, and whether those therapies then boost or generate an immune response against tumor proteins. For CD8+ T cells in particular, patient-specific immune recognition and responses at the level of individual tumor antigens are rarely characterized. Because of this, some immunologists have turned to serum antibodies as an alternative measure of antigen-specific anti-tumor immunity. In this work, we sought to simultaneously interrogate serum IgG and CD8+ T cell recognition of individual tumor antigens to determine whether antigen-specific serum IgG antibodies provide a window into the behavior of antigen-specific CD8+ T cell responses. Using antibody-based assays to evaluate immune response repertoires and focus T cell antigen exploration could afford substantial advantages for discovering and monitoring the anti-cancer immune responses of patients enrolled on clinical trials. METHODS: We vaccinated female BALB/c mice with a novel combination of an autophagosome-enriched vaccine derived from 4T1 mammary carcinoma along with poly-I:C adjuvant, then screened serum for IgG binding to arrays of 15mer peptides containing known mutation sites in 4T1. Simultaneously, we primed CD8+ T cell cultures from these same animals with 8-11mer peptides derived from these antigens. These primed T cells were then stimulated to measure recognition of the peptides or live 4T1 cells by IFNγ release. RESULTS: Vaccinated animals demonstrate increases in antigen-specific CD8+ T cell recognition of 4T1 tumor cells and peptides. For proteins confirmed in 4T1 cells and vaccine by mass spectrometry, there is a correlation between this increased CD8+ T cell IFNγ release and serum IgG binding to individual peptide antigens. CONCLUSIONS: These results suggest it is possible to observe some features of a patient's antigen-specific T cell repertoire via an antibody surrogate, which has implications for tumor antigen discovery and clinical monitoring of antigen-specific anti-tumor immunity.

Common gamma chain (γc) cytokines differentially potentiate TNFR family signaling in antigen-activated CD8(+) T cells.

BACKGROUND: Several members of the common gamma chain (gc) cytokine family are already approved (IL-2) or actively being developed as vaccine adjuvants and cancer immunotherapies. Studies have indicated that co-administration of gc cytokines may enhance the efficacy of immunotherapies that function via direct activation of co-stimulatory T cell receptors. To define the specific influence of gc cytokines on the co-stimulatory capacity of CD8(+) T cells and identify combinations with synergistic potential, we investigated the direct impact of gc cytokines on the differentiation and transcriptional profile of recently antigen-primed CD8(+) T cells. METHODS: Naïve CD8(+) T cells were activated with peptide-pulsed APCs. After 48 hours, CD8(+) T cells were harvested and re-cultured in media supplemented with IL-2, IL-4, IL-7, IL-15 or IL-21. After 24 hours, cells were analyzed by cytokine bead array, flow cytometry, and mRNA micro-array. Gene networks responsible for specific CD8(+) T cell functions were constructed through literature-meta review and publicly available annotation databases. Gene expression data from the experimental groups was imported into this network to visualize the impact of each gc cytokine on the functional polarization of recently-activated CD8(+) T cells. RESULTS: Among the gc cytokines, IL-2 induced the greatest increase in the expression of co-stimulatory receptors in recently-activated CD8(+) T cells. IL-2 increased significantly expression of 4-1BB, GITR, ICOS and OX40, at both the transcriptional and protein level. IL-2 also drove the greatest increase in cellular proliferation and the most robust shift towards a pro-survival phenotype, compared with the other gc cytokines. Both IL-4 and IL-21 enhanced expression of cytotoxic effector proteins, but drove distinct phenotypic polarizations, Th2/Tc2 and NK-like, respectively. CONCLUSIONS: Overall, these observations suggest that among gc cytokines, IL-2 may be uniquely capable of synergizing with therapeutic strategies that combine immunization with agonists of co-stimulatory T cell receptors. Previous studies have shown that the timing of IL-2 treatment relative to immunization plays a key role in defining the CD8(+) T cell response, and the findings from this study indicate that administration of exogenous IL-2 shortly after the initial antigen-priming event has concluded may augment the receptivity of these cells to subsequent TNFR co-stimulation.

Complete genome sequence of the marine fish pathogen Vibrio anguillarum harboring the pJM1 virulence plasmid and genomic comparison with other virulent strains of V. anguillarum and V. ordalii.

We dissected the complete genome sequence of the O1 serotype strain Vibrio anguillarum 775(pJM1) and determined the draft genomic sequences of plasmidless strains of serotype O1 (strain 96F) and O2ß (strain RV22) and V. ordalii. All strains harbor two chromosomes, but 775 also harbors the virulence plasmid pJM1, which carries the anguibactin-producing and cognate transport genes, one of the main virulence factors of V. anguillarum. Genomic analysis identified eight genomic islands in chromosome 1 of V. anguillarum 775(pJM1) and two in chromosome 2. Some of them carried potential virulence genes for the biosynthesis of O antigens, hemolysins, and exonucleases as well as others for sugar transport and metabolism. The majority of genes for essential cell functions and pathogenicity are located on chromosome 1. In contrast, chromosome 2 contains a larger fraction (59%) of hypothetical genes than does chromosome 1 (42%). Chromosome 2 also harbors a superintegron, as well as host "addiction" genes that are typically found on plasmids. Unique distinctive properties include homologues of type III secretion system genes in 96F, homologues of V. cholerae zot and ace toxin genes in RV22, and the biofilm formation syp genes in V. ordalii. Mobile genetic elements, some of them possibly originated in the pJM1 plasmid, were very abundant in 775, resulting in the silencing of specific genes, with only few insertions in the 96F and RV22 chromosomes.

The genus Listonella MacDonell and Colwell 1986 is a later heterotypic synonym of the genus Vibrio Pacini 1854 (Approved Lists 1980)--a taxonomic opinion.

We analysed the taxonomic position of the genus Listonella based on phylogenetic, genomic and phenotypic data. The species of the genus Listonella were nested within the genus Vibrio according to the 16S rRNA gene sequence-based phylogenetic tree. The closest neighbour of Vibrio (Listonella) anguillarum strains LMG 4437(T) and ATCC 68554 (=strain 775) was Vibrio ordalii LMG 13544(T), with more than 99.5% 16S rRNA gene sequence similarity. Furthermore, Vibrio (Listonella) pelagius is highly related to Vibrio splendidus. According to average amino acid identity (AAI), multilocus sequence analysis (MLSA) and Karlin genome signature, the closest neighbour of L. anguillarum ATCC 68554 is V. ordalii LMG 13544(T), with 95% AAI, 98% MLSA and 5 in Karlin. V. anguillarum ATCC 68554 and Vibrio cholerae N16961 had 77% similarity in AAI, 85% in MLSA and 14 in the Karlin signature. Phenotypic analyses of previously published data for V. (L.) anguillarum and V. (L.) pelagius revealed that the genus Listonella is extremely similar to the genus Vibrio. V. ordalii and L. anguillarum strains yielded up to 67% DNA-DNA hybridization. There are only a few phenotypic features that might be used to discriminate these two species: L. anguillarum is positive for the Voges-Proskauer reaction, citrate utilization, starch hydrolysis, lipase activity and acid production from glycerol, sorbitol and trehalose, whereas V. ordalii is negative for these traits. We suggest that the genus Listonella is a later heterotypic synonym of the genus Vibrio and propose to use the names Vibrio anguillarum and Vibrio pelagius in place of Listonella anguillarum and Listonella pelagia, respectively.

Comparison of lactate, base excess, bicarbonate, and pH as predictors of mortality after severe trauma in rhesus macaques (Macaca mulatta).

Social group housing of rhesus macaques at biomedical facilities is advocated to improve the psychologic wellbeing of these intelligent and social animals. An unintended outcome of social housing in this species is increased intraspecific aggression resulting in cases of severe multiple trauma and posttraumatic shock. The metabolic correlates of oxygen debt are likely important quantifiers of the severity of posttraumatic shock and may serve as useful guides in the treatment of these cases. The purpose of this retrospective study was to evaluate venous blood lactate, base excess, bicarbonate, and pH as predictors of mortality. These 4 variables were assessed in 84 monkeys with severe traumatic injury and shock. Data were available from blood samples collected prior to resuscitation therapy and the day after resuscitation therapy. The pre- and postresuscitation therapy levels of the variables then were tested for association with 6-d survival. When measured prior to resuscitation therapy, all variables were strongly correlated with each other and had a statistically significant association with survival. No single variable had both strong specificity and high sensitivity when measured prior to resuscitation therapy. Survival analysis showed that as the number of categorical indicators of acidosis increased, 6-d survival decreased. Analysis of the 4 variables after resuscitation therapy indicated that lactate was the only variable significantly associated with survival in our study.

Gene networks and the neuroendocrine regulation of puberty.

A sustained increase in pulsatile release of gonadotrophin releasing hormone (GnRH) from the hypothalamus is an essential, final event that defines the initiation of mammalian puberty. This increase depends on coordinated changes in transsynaptic and glial-neuronal communication, consisting of activating neuronal and glial excitatory inputs to the GnRH neuronal network and the loss of transsynaptic inhibitory tone. It is now clear that the prevalent excitatory systems stimulating GnRH secretion involve a neuronal component consisting of excitatory amino acids (glutamate) and at least one peptide (kisspeptin), and a glial component that uses growth factors and small molecules for cell-cell signaling. GABAergic and opiatergic neurons provide transsynaptic inhibitory control to the system, but GABA neurons also exert direct excitatory effects on GnRH neurons. The molecular mechanisms that provide encompassing coordination to this cellular network are not known, but they appear to involve a host of functionally related genes hierarchically arranged. We envision that, as observed in other gene networks, the highest level of control in this network is provided by transcriptional regulators that, by directing expression of key subordinate genes, impose an integrative level of coordination to the neuronal and glial subsets involved in initiating the pubertal process. The use of high-throughput and gene manipulation approaches coupled to systems biology strategies should provide not only the experimental bases supporting this concept, but also unveil the existence of crucial components of network control not yet identified.

MonkeySNP: a web portal for non-human primate single nucleotide polymorphisms.

UNLABELLED: MonkeySNP is a web-based resource created by the Genetic Resource and Informatics Program at the Oregon National Primate Research Center to facilitate access to non-human primate (NHP) single nucleotide polymorphisms (SNP) data. MonkeySNP is a mirror of the NCBI dbSNP database and contains additional NHP subpopulation genotype data and visual genotype displays to support SNP review and selection. AVAILABILITY: http://monkeysnp.ohsu.edu/snp/ SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Expression of a tumor-related gene network increases in the mammalian hypothalamus at the time of female puberty.

Much has been learned in recent years about the central mechanisms controlling the initiation of mammalian puberty. It is now clear that this process requires the interactive participation of several genes. Using a combination of high throughput, molecular, and bioinformatics strategies, in combination with a system biology approach, we singled out from the hypothalamus of nonhuman primates and rats a group of related genes whose expression increases at the time of female puberty. Although these genes [henceforth termed tumor-related genes (TRGs)] have diverse cellular functions, they share the common feature of having been earlier identified as involved in tumor suppression/tumor formation. A prominent member of this group is KiSS1, a gene recently shown to be essential for the occurrence of puberty. Cis-regulatory analysis revealed the presence of a hierarchically arranged gene set containing five major hubs (CDP/CUTL1, MAF, p53, YY1, and USF2) controlling the network at the transcriptional level. In turn, these hubs are heavily connected to non-TRGs involved in the transcriptional regulation of the pubertal process. TRGs may be expressed in the mammalian hypothalamus as components of a regulatory gene network that facilitates and integrates cellular and cell-cell communication programs required for the acquisition of female reproductive competence.

Single nucleotide polymorphisms (SNPs) distinguish Indian-origin and Chinese-origin rhesus macaques (Macaca mulatta).

BACKGROUND: Rhesus macaques serve a critical role in the study of human biomedical research. While both Indian and Chinese rhesus macaques are commonly used, genetic differences between these two subspecies affect aspects of their behavior and physiology, including response to simian immunodeficiency virus (SIV) infection. Single nucleotide polymorphisms (SNPs) can play an important role in both establishing ancestry and in identifying genes involved in complex diseases. We sequenced the 3' end of rhesus macaque genes in an effort to identify gene-based SNPs that could distinguish between Indian and Chinese rhesus macaques and aid in association analysis. RESULTS: We surveyed the 3' end of 94 genes in 20 rhesus macaque animals. The study included 10 animals each of Indian and Chinese ancestry. We identified a total of 661 SNPs, 457 of which appeared exclusively in one or the other population. Seventy-nine additional animals were genotyped at 44 of the population-exclusive SNPs. Of those, 38 SNPs were confirmed as being population-specific. CONCLUSION: This study demonstrates that the 3' end of genes is rich in sequence polymorphisms and is suitable for the efficient discovery of gene-linked SNPs. In addition, the results show that the genomic sequences of Indian and Chinese rhesus macaque are remarkably divergent, and include numerous population-specific SNPs. These ancestral SNPs could be used for the rapid scanning of rhesus macaques, both to establish animal ancestry and to identify gene alleles that may contribute to the phenotypic differences observed in these populations.

The identification of novel ovarian proteases through the use of genomic and bioinformatic methodologies.

Proteolytic activities are essential for follicular growth, ovulation, as well as for luteal formation and regression. Using suppression subtractive hybridization (SSH), a novel mouse ovary-selective gene (termed protease serine 35, Prss35) was identified. Analysis of the mouse genome database using the Prss35 sequence led to the identification of a homologous protease (protease serine 23, Prss23). PRSS35 possesses general features that are characteristic of serine (Ser) proteases, but is unique in that the canonical Ser that defines this enzyme family is replaced by a threonine (Thr). In contrast, PRSS23 possesses the standard catalytic Ser typical for this family of proteases. As determined by real-time polymerase chain reaction (PCR), the Prss35 mRNA levels increased around the time of ovulation and remained elevated in the developing corpus luteum. Steroid ablation/replacement studies demonstrated progesterone-dependent regulation of Prss35 gene expression prior to follicle rupture. Prss35 gene expression was localized to the theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory and ovulatory follicles, as well as to the developing corpus luteum. In contrast, Prss23 mRNA levels decreased transiently after ovulation induction and again in the postovulatory period. Prss23 gene expression was noted primarily in the granulosa cells of the secondary/early antral follicles. PRSS35 and PRSS23 orthologs in the rat, human, rhesus macaque, chimpanzee, cattle, dog, and chicken were identified and found to be highly homologous to one another (75-99% homology). Collectively, these results suggest that the PRSS35 and PRSS23 genes have been conserved as critical ovarian proteases throughout the course of vertebrate evolution.

Applying task analysis to describe and facilitate bioinformatics tasks.

OBJECTIVE: To document bioinformatics tasks currently per-formed by researchers in genomics and proteomics in an effort to recognize unmet informatics needs and challenges, identify system features that would enhance the performance of those tasks, and inform the development of new bioinformatics tools. DESIGN: A cross-sectional study of bioinformatics tasks performed by OHSU investigators involved in genomics and proteomics research was conducted using task analysis techniques. RESULTS: Four major categories emerged from 22 bioinformatics tasks reported by 6 research laboratories. These were: 1) gene analysis, 2) protein analysis, 3) biostatistical analysis, and 4) literature searching. Analysis of the data also raised the following challenging issues: 1) lack of procedural documentation, 2) use of home-grown strategies to accomplish goals, 3) individual needs and preferences, and 4) lack of awareness of existing bioinformatics tools. CONCLUSION: Task analysis was effective at documenting bioinformatics tasks performed by researchers in the fields of genomics and proteomics, at identifying potentially desirable system features and useful bioinformatics tools, and at providing a better understanding of some of the unmet needs and challenges faced by these researchers.

BioQuery: an object framework for building queries to biomedical databases.

SUMMARY: BioQuery is an application that helps scientists automate database searches. Users can build and store queries to public biomedical databases, and receive periodic updates on the results of those queries when new data is available. The application is implemented on a portable object framework that can provide database-searching capability to other applications. This framework is easily extensible, allowing users to develop plug-ins that provide access to new databases. BioQuery thus provides end-users with a complete database searching interface and updating service, and gives developers a toolkit to provide database-searching capability to their applications. AVAILABILITY: Free to all users: http://www.bioquery.org.

Delivering bioinformatics training: bridging the gaps between computer science and biomedicine.

Biomedical researchers have always sought innovative methodologies to elucidate the underlying biology in their experimental models. As the pace of research has increased with new technologies that 'scale-up' these experiments, researchers have developed acute needs for the information technologies which assist them in managing and processing their experiments and results into useful data analyses that support scientific discovery. The application of information technology to support this discovery process is often called bioinformatics. We have observed a 'gap' in the training of those individuals who traditionally aid in the delivery of information technology at the level of the end-user (e.g. a systems analyst working with a biomedical researcher) which can negatively impact the successful application of technological solutions to biomedical research problems. In this paper we describe the roots and branches of bioinformatics to illustrate a range of applications and technologies that it encompasses. We then propose a taxonomy of bioinformatics as a framework for the identification of skills employed in the field. The taxonomy can be used to assess a set of skills required by a student to traverse this hierarchy from one area to another. We then describe a curriculum that attempts to deliver the identified skills to a broad audience of participants, and describe our experiences with the curriculum to show how it can help bridge the 'gap'.