Recipient Organization
UNIV OF PENNSYLVANIA
(N/A)
PHILADELPHIA,PA 19104
Performing Department
5803 - Pathobiology
Non Technical Summary
Fish, like humans, have an immune system that protects them from pathogens. Like us, fish produce protective antibodies after infection, and these antibodies stop the pathogen from attacking the fish. In humans and other mammalian species, antibodies found in the blood are mainly induced in certain areas of the spleen that have a very well defined organization, and thus, such structures are also called organized lymphoid structures. However, it is believed that fish do not have such structures, and thus, it is unknown where antibody responses are induced in fish. Recent findings from our laboratory strongly suggest the presence of semi-organized lymphoid structures in the fish spleen, where antibody responses in these animals appear to be induced. This project will study these newly discovered semi-organized lymphoid structures and their role in the induction of fish antibody responses. Moreover, this project will study the mechanisms by which antibody responses are made stronger and more effective within these semi-organized lymphoid structures. Some of these mechanisms involve the selection and expansion of key B cell clones, the cells that produce antibodies that recognize pathogens, as well the mechanisms of hypermutation in antibodies, which improve the affinity of these antibodies for the pathogen.The results of this project will break the long-held dogma that fish do not have specialized lymphoid structures responsible for the induction of antibody responses. Importantly, the discovery of such structures in humans and other mammals was in the past a fundamental step to understanding how immune responses against pathogens are generated. This knowledge was in turn used to improve the efficacy of vaccines against human and animal pathogens. Thus, in addition to improving our knowledge on how fish fight pathogens through the induction of antibody responses, the results generated from this project will be critical in improving the efficacy of fish vaccines. In that regard, a growing number of farmed fish species require vaccination against different pathogens in order to survive in fish farms. However, many of the current fish vaccines are not very efficacious in inducing strong and protective antibody responses. Thus, the knowledge derived from this project will be instrumental for the future design of more efficacious fish vaccines. In the long term, the improvement of fish health through more efficacious vaccines will decrease fish losses due to disease in fish farms, thus, increasing the availability of fish protein to the general population at more affordable prices.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
Aquaculture is the fastest growing of all animal food sectors, in the global marketplace. While prevention and spread of infectious fish diseases are critical issues for this industry, the lack of basic knowledge on many fundamental aspects of the immune system of fish prevents the development of effective fish vaccines. It has been known for decades now that upon infection or immunization, teleost fish generate potent systemic IgM responses. More recently, it has also been shown that IgT responses are overwhelmingly induced in mucosal areas. However, the current paradigm states that teleost fish do not have organized lymphoid tissues (i.e., lymph nodes, germinal centers [GCs]). Thus, a central unresolved question has been where and how are antibody responses induced in these species in the absence of such organized lymphoid microstructures. It is likely that the lack of antibody reagents for fish immune cells has until recently hindered research efforts into answering this crucial question. In 2016, with funds from our previous award we reported for the first time the generation of monoclonal antibodies (mAbs) to trout CD4+ T cells, which represented the first mAbs generated against salmonid CD4+ T cells. These mAbs have been fundamental in this current granting period to start elucidating the interactions of B and T cells in trout lymphoid tissues.Our recent studies on the interactions of B and T cells in the spleen have led to the discovery that upon infection or immunization of fish, splenic melanomacrophage centers (MMC) contain semi-organized B and Tcell zones in close proximity, in which B cells are particularly seen proliferating a high rates. We have named these areas MMC-associated lymphoid microstructures (MMC-ALMs). Critically, our recent Illumina-sequencing-based studies of IgM repertoire show that upon infection or immunization, MMC-ALMs represent targeted areas in which Ag-specific IgM+ B cell clonal expansion occurs in teleosts. Based on these novel findings, the overarching hypothesis of this proposal is that MMC-ALMs represent semi-organized lymphoid tissue in which systemic IgM responses are induced in teleost fish. To address this hypothesis, we propose to phenotypically characterize the formation and induction of MMC-ALMs, and to address their roles and mechanisms underlying the processes of Ag-dependent B cell clonal expansion, somatic hypermutation and affinity maturation of systemic IgM responses.Our long-term goals are to understand the specific contributions of MMC-ALMs to systemic IgM responses of teleost fish. In the longer term, we plan to use the knowledge obtained from our proposed basic research investigations towards the development of novel vaccination strategies that promote improved systemic protective immune responses. Thus, the objectives of this proposal are:OBJECTIVE 1. Induction, development kinetics, and phenotypic characterization of MMC-associated lymphoid microstructures (MMC-ALMs). To date, little is known regarding the lymphoid structures and mechanisms involved in the induction of systemic IgM responses in teleost fish. Our preliminary data shows for the first time that upon immunogenic stimulation, trout develop semi-organized lymphoid microstructures associated to MMCs (MMCs-ALMs). In contrast to non-MMC areas, MMC-ALMs contain B and T cell zones in close proximity in which IgM+ B cells are seen proliferating a high rates. Moreover, Ag-specific IgM+ B cells are mainly detected within these MMC-ALMs. Based on these data, we hypothesize that upon immune stimulation, semi-organized lymphoid tissue in the form of MMC-ALMs are induced in the spleen of teleost fish. To address this hypothesis here we will expose fish or immunize them with either the parasite Ichthyophthirius multifiliis (Ich) or the antigen (Ag) DNP-KLH respectively. Spleen and serum from the stimulated and control fish will be collected at different time points to analyze Ich- and DNP-KLH- specific IgM titers, Ag-specific B cell numbers, Ag-specific CD4+ T cell proliferative responses, and the tissue distribution of proliferating and non-proliferating IgM+ B cells and CD4+ T cells in MMC-ALMs and non-MMC areas. In addition to obtaining novel knowledge on the phenotypic characteristics, development kinetics and maintenance over time of the induced MMC-ALMs, the resulting data will provide important clues for the development of correlates of immune activation and vaccine efficacy that may lead in the future to the rationale design of more efficacious vaccines in fish.OBJECTIVE 2. Occurrence of Ag-specific B cell clonal expansion and somatic hypermutation in MMC-ALMs upon pathogenic and antigenic stimulation. In mammals, Ag-specific B cell clonal expansion along with the processes of somatic hypermutation (SHM) and affinity maturation occur within organized lymphoid areas (i.e., lymph nodes). Our preliminary data shows that a high degree of IgM+ B cell proliferation are detected within MMC-ALMs upon pathogenic or antigenic stimulation. Moreover, we show Ig-repertoire data indicating that the top most frequent and largest B cell clonotypes are induced in MMC-ALMs. Combined, these data leads to hypothesize that the process of Ag-specific B cell clonal expansion mainly occurs in splenic MMC-ALMs. Moreover, based on the significant expression of AID detected in MMC-ALMs we hypothesize that the process of SHM occurs within such microstructures. To address these hypotheses, here we will perform an in-depth characterization of the kinetics and underlying mechanisms involved in the processes of B cell clonal expansion and SHM. Moreover, we will also evaluate whether affinity maturation of the IgM response follows after SHM. To assist us in the understanding of the cells and immune mechanism involved in these processes, we will perform transcriptomic studies of MMC-ALMs harvested by laser capture microdissection. Overall the results derived from the proposed studies will provide groundbreaking new data on the functional processes involved in the induction of IgM responses in MMC-ALMs.
Project Methods
Methods: For objective 1, fish will be infected with the Ich parasite or immunized with DNP-KLH, and reinfected or boosted once with Ich or the same Ag respectively a month later. Fish will be euthanized 1, 2, 4 weeks post-primary and 2, 4, 8 and 24 weeks post-secondary Ich exposures or boosting Ag immunizations. At each time point, serum and spleen will be harvested. Serum will be used in this aim to assess Ich- or DNP-KLH-specific IgM titers using ELISA methodology. We will use the spleen to assess Ag-specific CD4+ T cell proliferative responses, to evaluate the percentage of Ag-specific IgM+ B cells by immunohistochemistry (IHC) and immunofluorescence methodology, and to assess by IHC the localization of proliferating and non-proliferating IgM+ B cells and CD4+ T cells in MMC-ALMs and non-MMC areas. For objetive 2, the spleen and serum from Ich-infected fish, DNP-KLH-immunized fish and their respective controls will be harvested at different time points. MMC-ALMs and the non-MMC areas of the spleen will be harvested by laser capture microdissection (LCM). For the profiling repertoire and hypermutation analysis, IgHm (IgM heavy chain) cDNA from whole spleen tissue, along with that of individual MMC-ALMs and non-MMC areas will be amplified by 5'RACE. The obtained cDNA will be deep-sequenced using Illumina protocols. V(D)J rearrangements will be annotated, allowing Ig-repertoire analysis to assess clonotype diversity and sharing. The Ig sequences will be annotated at the gene level, based on our recent work which is an essential starting point also for the proposed somatic hypermutaion (SHM) studies. The assessment of hypermutation will be addressed by the study of the sequences previously obtained by 5'RACE and Illumina deep sequencing. In this aim, we will also perform transcriptome analysis of the different LCM-generated MMC-ALMs and non-MMCs, to identify key genes and gene signatures involved in the induction of Ag-specific IgM responses within MMC-ALMs.Efforts: As previously stated, The PD of this award teaches the subjects of immunology and microbiology for veterinarians. Some of the lectures taught involve fish immunology and fish pathogen subject areas, and in several instances the PD introduces results from his previous and current research projects in his formal classroom instruction.Evaluation: The evaluation of success of this project will be measured by the number and quality of publications in specialized scientific journals, as well as in the number of national and international conferences, and invited lectures where the work derived from this project is presented.