Immunology Studies Research Articles

Musculoskeletal manifestations of systemic lupus erythmatosus.

Imaging studies suggest potential changes to the classification and assessment of inflammatory musculoskeletal lupus. This is important because of the burden of disease but the potential for new targeted therapies. Using our current classification and treatment, musculoskeletal symptoms continue to impact significantly on quality of life and work disability. Ultrasound and MRI studies suggested that new approaches to the diagnosis, classification, and evaluation of these symptoms are needed. Many patients with pain but no synovitis have ultrasound-proven joint and tendon inflammation but would not qualify for clinical trials or score highly on disease activity instruments. MRI studies show that erosions are more common than previously thought and may have a different pathogenesis than RA. Immunology studies suggest differences from other autoimmune synovitis, with a complex role for type I interferons. A wide range of biologic therapies appear more consistently effective for arthritis than some other manifestations. Changes to the selection of patients for therapy and stratification using musculoskeletal imaging may offer new approaches to clinical trials and the routine care of systemic lupus erythematosus patients with inflammatory musculoskeletal symptoms. Outcomes may thereby be improved using existing therapies. There are significant knowledge gaps that must be addressed to achieve these potential improved outcomes.

Interpreting ELISA analyses from wild animal samples: Some recurrent issues and solutions

Abstract Many studies in disease and ecological immunology rely on the use of assays that quantify the amount of specific antibodies (immunoglobulin) in samples. Enzyme‐linked immunosorbent assays (ELISAs) are increasingly used in ecology due to their availability for a broad array of antigens and the limited amount of sampling material they require. Two recurrent methodological issues are nevertheless faced by researchers: (1) the limited availability of immunological assays and reagents developed for non‐model species, and (2) the statistical determination of the cut‐off threshold used to distinguish individual samples that are likely to have or not to have antibodies against a specific antigen. Here, we outline two solutions to deal with these issues. First, we show that implementing two assays with differing detection methods can help validate the use of reagents, such as antibodies, in species different from their intended target. We illustrate this by comparing the quantification of specific vaccinal antibodies against Newcastle disease virus (NDV) using two ELISA approaches in four seabird species (Cory's shearwater, European shag, European storm petrel and Southern rockhopper penguin). Second, we provide a simple way to determine from the distribution of ELISA values whether the assayed samples are likely to be made of a single group of individuals (likely negative) or of two groups of individuals (negative and positive). We illustrate the use of this approach with two independent datasets: NDV antibody levels following vaccination and anti‐Borrelia antibody levels following natural exposure. The practical implementation of these methodological approaches could provide a way to efficiently apply ELISAs and other immune‐based assays to address questions in the growing fields of ecological immunology and disease ecology. A plain language summary is available for this article.

Survey of research on tumor immunology funded by National Natural Science Foundation of China

Tumor immunology is a very young field to study the relationship of tumor immunogenity, body immunity with tumor development and growth, immune response, antitumor immunity mechanism, tumor immunodiagnosis, and immunotherapy. It is a hot area in both life science and medicine. Tumor immunology is also an important area to which National Natural Science Foundation of China are heavily supporting. Here, we reviewed the 10-year history of grant application and supports on tumor immunology funded by National Natural Science Foundation of China (NSFC) from 2007 to 2016. Additionally, we showed the research progress and current situation of tumor immunology in China, and the scientific frontline and existing problems in this field were proposed as well. This paper summarized the situations of proposal application, and funded applications in the field of tumor immunology under the codes of Oncology by National Natural Science Foundation of China in 2007–2016. First, we analyzed the codes which under the category of tumor immunology. From the information we collected in the past decade, we observed robust increases in both application and funded application in regular projects, young investigator projects and area projects. The total volumes of applications are 3928 in the past decade and the annual growth rate ranged from 6.59% to 32.54%. The total funded projects are 711, with the same annual growth rate. We also analyzed the specific fields and the codes distributions of the funded projects in the China tumor immunology research, which including applications and funded applications of large projects (outstanding young investigator funds, excellent young investigator funds and key projects). Finally, we studied the sub-area which are most funded. We found most of the funded projects are focused on tumor microenvironment immunosuppression, tumor immune escape in relapse and metastasis, tumor immunotherapy, and immunotherapy targets identification and application. The results indicated the critical topic and research areas which the researchers might be interested in and the directions of national tumor immunology research. The findings also pointed out the current situation, trends and existing problems in tumor immunology study. All the information from this article will help the researchers to track the national tumor immunology research trend. It also helps the foundation administrators to make corresponding policy to facility the research projects in tumor immunology.

Draft sequencing and assembly of the genome of the world\u2019s largest fish, the whale shark: Rhincodon typus Smith 1828

BackgroundThe whale shark (Rhincodon typus) has by far the largest body size of any elasmobranch (shark or ray) species. Therefore, it is also the largest extant species of the paraphyletic assemblage commonly referred to as fishes. As both a phenotypic extreme and a member of the group Chondrichthyes – the sister group to the remaining gnathostomes, which includes all tetrapods and therefore also humans – its genome is of substantial comparative interest. Whale sharks are also listed as an endangered species on the International Union for Conservation of Nature’s Red List of threatened species and are of growing popularity as both a target of ecotourism and as a charismatic conservation ambassador for the pelagic ecosystem. A genome map for this species would aid in defining effective conservation units and understanding global population structure.ResultsWe characterised the nuclear genome of the whale shark using next generation sequencing (454, Illumina) and de novo assembly and annotation methods, based on material collected from the Georgia Aquarium. The data set consisted of 878,654,233 reads, which yielded a draft assembly of 1,213,200 contigs and 997,976 scaffolds. The estimated genome size was 3.44Gb. As expected, the proteome of the whale shark was most closely related to the only other complete genome of a cartilaginous fish, the holocephalan elephant shark. The whale shark contained a novel Toll-like-receptor (TLR) protein with sequence similarity to both the TLR4 and TLR13 proteins of mammals and TLR21 of teleosts. The data are publicly available on GenBank, FigShare, and from the NCBI Short Read Archive under accession number SRP044374.ConclusionsThis represents the first shotgun elasmobranch genome and will aid studies of molecular systematics, biogeography, genetic differentiation, and conservation genetics in this and other shark species, as well as providing comparative data for studies of evolutionary biology and immunology across the jawed vertebrate lineages.

The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS.

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroides lacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.

The future of immune checkpoint cancer therapy after PD-1 and CTLA-4.

The adaptive immune system plays an important role in eradicating malignant cells. Co-stimulatory and co-inhibitory signals to T cells though immune checkpoint receptors are involved in tumorigenesis and metastasis. Exploitation of immune checkpoint inhibitors, PD-1 and CTLA-4, with monoclonal antibodies has created impressive clinical responses. Many other immune checkpoint co-inhibitors and co-stimulators exist, including the B7 superfamily and tumor necrosis factor receptors superfamily. Here, we will examine co-inhibitors and co-stimulators beyond PD-1 and CTLA-4 that are being investigated in active clinical trials. We will review the immunology and preclinical studies that support investigation of these targets. Finally, we will briefly discuss the potential for immunotherapy to be combined with other treatment modalities.

Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy

Abstract The humanized BLT (bone marrow-liver-thymus) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain HLA (human leukocyte antigen) haplotypes or monogene deficiencies. In order to circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ−/−) recipient mice, we were able to expand one primary BLT mouse into a colony of 4–5 proBLT (propagated BLT) mice in 6–8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to that of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2 restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable pre-clinical research tool.

Abstract A85: Investigating endogenous anti-tumor T cell responses in native tumor microenvironments

Abstract Genetically-engineered mouse models (GEMMs) exist for most types of cancer in which autochthonous tumors develop into advanced disease in the presence of a fully functional immune system. These GEMMs would be ideal for investigating questions in cancer immunology, including those concerning the commonalities and differences between immune responses that target different cancers, and how the tumor microenvironments (TMEs) associated with different cancer types impact the functions of tumor-infiltrating immune cells. However, unlike human tumors, tumors in GEMMs have few somatic mutations and thus require exogenous neoantigens (neoAgs) to drive interactions with anti-tumor T cells. There are only a handful of mechanisms to program tumors to express neoAgs, and thus, GEMMs for most tumor types remain inaccessible for cancer immunology studies. Here, we describe the “iNversion INducible Joined neoAg” (NINJA) mouse, which can be used to program GEMM tumors with a common neoAg, and thus stimulate a common set of tumor-specific CD8 T cells. NINJA will be useful for addressing many key unanswered questions cancer immunology, including comparisons of immune responses across several cancer types and comparisons of anti-tumor T cell responses targeting de novo expressed neoAgs at different stages in tumor development. NINJA mice will also be invaluable as a preclinical platform for testing immunotherapeutics, and for investigating why certain immunotherapeutic regimens are more effective when treating some cancers and not others. NINJA is unique, because neoAgs are encoded within the genome, yet NINJA avoids central and peripheral tolerance, a common problem with other systems. This is achieved because the DNA sequences encoding the full-length neoAgs are discontinuous in the OFF state and, thus, neoAgs are silent. Induction of neoAgs is highly regulated, requiring a permanent inversion of a DNA element in NINJA, which allows production of the full-length NINJA neoAgs and GFP (a marker of neoAg expression). This inversion is regulated by Flippase (FlpO), which itself is highly regulated, requiring cells are exposed to Cre recombinase, and then doxycycline and tamoxifen. Therefore, NINJA is compatible with most Cre-inducible GEMMs of cancer. Activating NINJA with adenoviral infection results in a strong, NINJA-specific CD8 T cell response while, in contrast, activation in the setting of lung tumors leads to T cell dysfunction. Thus, NINJA is a powerful, highly versatile system for investigating T cell function in many settings, including cancer, immune tolerance, and autoimmunity. Citation Format: Nikhil Joshi, Tyler Jacks. Investigating endogenous anti-tumor T cell responses in native tumor microenvironments. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A85.

An Integrated Workflow To Assess Technical and Biological Variability of Cell Population Frequencies in Human Peripheral Blood by Flow Cytometry.

In the context of large-scale human system immunology studies, controlling for technical and biological variability is crucial to ensure that experimental data support research conclusions. In this study, we report on a universal workflow to evaluate both technical and biological variation in multiparameter flow cytometry, applied to the development of a 10-color panel to identify all major cell populations and T cell subsets in cryopreserved PBMC. Replicate runs from a control donation and comparison of different gating strategies assessed the technical variability associated with each cell population and permitted the calculation of a quality control score. Applying our panel to a large collection of PBMC samples, we found that most cell populations showed low intraindividual variability over time. In contrast, certain subpopulations such as CD56 T cells and Temra CD4 T cells were associated with high interindividual variability. Age but not gender had a significant effect on the frequency of several populations, with a drastic decrease in naive T cells observed in older donors. Ethnicity also influenced a significant proportion of immune cell population frequencies, emphasizing the need to account for these covariates in immune profiling studies. We also exemplify the usefulness of our workflow by identifying a novel cell-subset signature of latent tuberculosis infection. Thus, our study provides a universal workflow to establish and evaluate any flow cytometry panel in systems immunology studies.

Exploring the Association of Surface Plasmon Resonance with Recombinant MHC:Ig Hybrid Protein as a Tool for Detecting T Lymphocytes in Mice Infected with Leishmania (Leishmania) amazonensis.

A surface plasmon resonance- (SPR-) based recognition method applying H-2 Ld:Ig/peptides complexes for ex vivo monitoring cellular immune responses during murine infection with Leishmania (Leishmania) amazonensis is described. Lymphocytes from lesion-draining popliteal lymph nodes were captured on a carboxylated sensor chip surface previously functionalized with H-2 Ld:Ig (DimerX) protein bound to synthetic peptides derived from the COOH-terminal region of cysteine proteinase B of L. (L.) amazonensis. In computational analysis, these peptides presented values of kinetic constants favorable to form complexes with H-2 Ld at neutral pH, with a Gibbs free energy ΔG° < 0. The assayed DimerX:peptide complexes presented the property of attaching to distinct T lymphocytes subsets, obtained from experimentally infected BALB/c mice, in each week of infection, thus indicating a temporal variation in specific T lymphocytes populations, each directed to a different COOH-terminal region-derived peptide. The experimental design proposed herein is an innovative approach for cellular immunology studies of a neglected disease, providing a useful tool for the analysis of specific T lymphocytes subsets.

Modelling and Simulation of the Dynamics of the Antigen-Specific T Cell Response Using Variable Structure Control Theory.

Experimental and mathematical studies in immunology have revealed that the dynamics of the programmed T cell response to vigorous infection can be conveniently modelled using a sigmoidal or a discontinuous immune response function. This paper hypothesizes strong synergies between this existing work and the dynamical behaviour of engineering systems with a variable structure control (VSC) law. These findings motivate the interpretation of the immune system as a variable structure control system. It is shown that dynamical properties as well as conditions to analytically assess the transition from health to disease can be developed for the specific T cell response from the theory of variable structure control. In particular, it is shown that the robustness properties of the specific T cell response as observed in experiments can be explained analytically using a VSC perspective. Further, the predictive capacity of the VSC framework to determine the T cell help required to overcome chronic Lymphocytic Choriomeningitis Virus (LCMV) infection is demonstrated. The findings demonstrate that studying the immune system using variable structure control theory provides a new framework for evaluating immunological dynamics and experimental observations. A modelling and simulation tool results with predictive capacity to determine how to modify the immune response to achieve healthy outcomes which may have application in drug development and vaccine design.

Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34+ hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ-/-) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

An In Vitro Model for the Study of the Macrophage Response Upon Trichophyton rubrum Challenge.

Diversity in the macrophage models currently employed in immunology studies may lead to opposed results and interpretations. In this study, we aimed to analyze the suitability of J774 macrophage-like cells as a model for the interaction between the dermatophyte Trichophyton rubrum and macrophages. J774 cells were competent in fungal phagocytosis, but succumbed to hyphal growth. Nevertheless, they could also secrete IL-1β in response to the dermatophyte. On the opposite direction, inflammatory, thioglycollate-induced peritoneal macrophages did not succumb to fungal growth and showed no significant IL-1β production. The proteomic profiling of these cells uncovered vimentin and plastin-2 as proteins whose abundance was altered by the fungal interaction. Our study indicates that this cell line could be an interesting tool in the investigation of T. rubrum infection biology.

Standardization and quality control for high‐dimensional mass cytometry studies of human samples

Mass cytometry (CyTOF), a mass spectrometry-based single cell phenotyping technology, allows utilization of over 35 antibodies in a single sample and is a promising tool for translational human immunology studies. Although several analysis tools are available to interpret the complex data sets generated, a robust method for standardization and quality control within and across studies is needed. Here we report an efficient and easily adaptable method to monitor quality of individual samples in human immunology studies and to facilitate reproducible data analysis. Samples to be assessed are spiked with a defined amount of reference peripheral blood mononuclear cells from a healthy donor, derived from a single large blood draw. The presence of known standardized numbers and phenotypic profiles of these reference cells greatly facilitates sample analysis by allowing for: 1) quality control for consistent staining of each antibody in the panel, 2) identification of potential batch effects, and 3) implementation of a robust gating strategy. We demonstrate the utility of this method using peripheral blood and bronchoalveolar lavage samples from HIV+ patients by characterizing their CD8+ T-cell phenotypes and cytokine expression, respectively. Our results indicate that this method allows quality control of experimental conditions and results in highly reproducible population frequencies through a robust gating strategy. © 2016 International Society for Advancement of Cytometry.

The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

Élie Metchnikoff (1845-1916): celebrating 100 years of cellular immunology and beyond.

The year 2016 marks 100 years since the death of Élie Metchnikoff (1845-1916), the Russian zoologist who pioneered the study of cellular immunology and who is widely credited with the discovery of phagocytosis, for which he was jointly awarded the Nobel Prize in Physiology or Medicine in 1908. However, his long scientific career spanned many disciplines and has had far-reaching effects on modern immunology beyond the study of phagocytosis. In this Viewpoint article, five leading immunologists from the fields of phagocytosis, macrophage biology, leukocyte migration, the microbiota and intravital imaging tell Nature Reviews Immunology how Metchnikoff's work has influenced past, present and future research in their respective fields.

Abstract A12: The immune composition of a spontaneous mouse breast cancer

Abstract The MMTV-PyMT mouse strain is a widely used model for the study of breast cancer. In this strain, the mouse mammary tumor virus (MMTV) promoter drives expression of the polyoma middle T (PyMT) oncogene in breast epithelium resulting in widespread transformation of the tissue and formation of progressive tumors that eventually metastasize to the lungs and lymph nodes, closely recapitulating the human disease. It is know that MMTV-PyMT tumors are infiltrated by large numbers of immune cells and the role of some immune populations in cancer progression and metastasis has been described. However a detailed characterization of the myeloid and lymphoid populations infiltrating MMTV-PyMT during tumor progression is yet to be performed. In this study we used comprehensive flow cytometry panels to enumerate and quantify B cells, αβ T cells (CD4 and CD8), γδ T cells, NK T cells, NK cells, neutrophils, monocytes, dendritic cells and macrophages within MMTV-PyMT tumors in different stages of progression. Given that hypoxia is a prominent aspect of breast cancer we extended this characterization to strains with targeted deletion of hypoxia-inducible factor (HIF) 1α and HIF2α in myeloid cells. We found lymphoid cells to be the predominant immune populations within PyMT tumors particularly B cells and αβ T cells (CD4+ and CD8+) but their frequencies did not markedly change with progression or tumor mass. Surprisingly the majority of CD8 T cells expressed the NK cell marker NK1.1 (&amp;gt;80%). In addition we found dendritic cells (CD11chigh, MHC IIhigh) to be the prevalent myeloid population and that the frequency of this population had a strong positive correlation with tumor mass. Myeloid deletion of HIF1a, but not HIF2a, resulted in increased tumor mass and dendritic cell infiltration while other immune population frequencies remained unchanged. Whether dendritic cells directly affect tumor growth and to what extent HIF1α is involved in this process remains to be elucidated. We expect that this immune characterization of PyMT tumors will serve as a reference for future breast cancer immunology studies and possibly assist in the development of new immunotherapies. Citation Format: Pedro Velica, Helene Rundqvist, Milos Gojkovic, Randall S. Johnson. The immune composition of a spontaneous mouse breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A12.

The use and abuse of immune indices in nutritional immunology studies

SUMMARY The immune system is complex, with redundant and overlapping functions. Identifying the correct immune parameters to study the immune status of an animal is challenging. Nutritional immunologists often measure different isotypes of antibodies, hematological parameters, parasite loads, functional assays of macrophages and T cells, pro- and anti-inflammatory cytokines, phosphorylation status of proteins involved in signaling pathways, and several other parameters to identify the immune status of birds. Papers in which authors misinterpret immune parameters are common. This article discusses the drawbacks of some of the common interpretations of several immunological assays available for nutritional immunologists.

EN

Bats are reported as reservoir host for several viruses, which cause significant illness in human and animals. Some of the bat transmitted zoonotic viral diseases such as Ebola, Hendra, Nipah and rabies may cause severe human casualties. They also harbor several other viruses such as MERS and SARS corona viruses, which may cause disease in human through direct spillover to human or through an intermediate host or vectors. Being reservoir hosts bats do not get affected by these viruses. This probably may happen due to the specificity of bat immune system, which reacts differently with viral pathogens in comparison to their other mammalian counterparts. Although bats are important reservoir hosts for several zoonotic viruses, very little information is available regarding host/virus relationships as only few experimental studies have been done on bat colonies, lack of expertise for study of bat immunology and antiviral responses and difficulty in conducting field work. However, with the advancement in epidemiology and molecular biology, these problems can be addressed, which will provide the insight into interactions of bats and zoonotic viruses. It may also clarify regarding virus persistence in nature and various associated risk factors which might facilitate viral transmission to animals and humans. Koushlesh Ranjan 1,* , Minakshi Prasad 2 and Gaya Prasad 3

Characterization of immune-related genes in the yellow catfish Pelteobagrus fulvidraco in response to LPS challenge.

Fish are considered an excellent model for studies in comparative immunology as they are a representative population of lower vertebrates linked to invertebrate evolution. To gain a better understanding of the immune response in fish, we constructed a subtractive cDNA library from the head kidney of lipopolysaccharide-stimulated yellow catfish (Pelteobagrus fulvidraco) using suppression subtractive hybridization (SSH). A total of 300 putative EST clones were identified which contained 95 genes, including 27 immune-related genes, 7 cytoskeleton-related genes, 3 genes involved in the cell cycle and apoptosis, 9 respiration and energy metabolism-related genes, 7 genes related to transport, 24 metabolism-related genes, 10 genes involved in stress responses, seven genes involved in regulation of transcription and translation and 59 unknown genes. Using real-time quantitative reverse transcription PCR, a subset of randomly selected genes involved in the immune response to lipopolysaccharide challenge were investigated to verify the reliability of the SSH data which identified 16 up-regulated genes. The genes identified in this study provide novel insight into the immune response in fish.