Periodic Reporting for period 4 - TEMUBLYM (Teleost mucosal B1-like lymphocytes at the crossroad of tolerance and immunity)
Berichtszeitraum: 2021-10-01 bis 2022-09-30
B cells are one of the main players of immunity, responsible for the production of immunoglobulins. Despite this, many aspects of how B cells are regulated in teleost fish remain unclear. The main aim of this project was to pursue the phenotypic and functional characterization of fish B cells, focusing on mucosal B cells, under the assumption that fish B lymphocytes resemble mammalian B1 extrafollicular responses. For this, one of the main objectives was to obtain tools to differentiate naïve B cells from plasmablasts / plasma cells, to then study how these cells are regulated in mucosal surfaces. Additionally, we have undertaken extensive transcriptomic analysis of different B cell subsets from different sources at single cell level. Another important objective of the project was determining ways to breach peripheral tolerance. Along this line, a high number of different stimuli were tested, first in systemic tissues and afterwards in mucosal tissues. Finally, we also aimed to study the role of IgD in mucosal surfaces, having demonstrated the presence of cells exclusively expressing IgD at these sites. Our results greatly contribute to understand how fish B cells mount a protective mucosal immune response in the absence of T cell help from organized follicles. From a practical view, our work will be of great use for the future optimization of effective mucosal vaccination strategies, highly demanded by the aquaculture sector.
- Objective 1. Within this objective, we planned to obtain specific antibodies to differentiate between naïve B cells and plasmablasts / plasma cells. We have undertaken this in different ways. We have obtained a highly specific antibody against CD38A, and confirmed that IgM+CD38A+ corresponded to plasmablasts. We have also established a method to study plasmablasts based on IgD and MHC II surface expression and cell size. Finally, a flow cytometry method based on cell size and endoplasmic reticulum expansion has also been developed. We have also identified 4 homologs of Blimp1 in rainbow trout, confirming their implication in the B cell differentiation process and we have also undertaken a transcriptomic analysis of single B cells in different stages of maturation/differentiation to define additional markers for specific subsets. Along this line, the repertoire of light immunoglobulin chains has also been established at single cell level. We have also optimized an ELISPOT to quantify cells secreting specific antibodies. Through this technique, we were able to quantify the number of B cells secreting specific antibodies after the anal immunization of rainbow trout with different model antigens. Finally, we have performed a full transcriptomic analysis of stimulated and unstimulated B cells. In the past months of the project, we performed a transcriptomic analysis at single cell level of MHC II+ leukocyte populations in the intestinal mucosa.
- Objective 2. Within this objective, we had planned the production of rainbow trout knock-out (KO) for the TCR gene, thus lacking T cells to help us evaluate the precise contribution of T cells to B cell activation. After several unsuccessful attempts to produce KO trout, we performed the experiments in ZAP70-/- zebrafish. Another task within this objective was to attempt breaching peripheral tolerance in the intestine by different cytokines or TLR ligands. Before testing these stimuli in anal or oral stimulations, we studied their effects in rainbow trout splenic B cells. Thus, a broad characterization of the effects exerted by CpGs, Bacillus subtillis or different cytokines on splenic B cells was performed. The most promising molecules were also tested at intestinal level. In this objective we have undertaken some additional experiments not previously planned in the project. Thus, we have studied the response of B cells during proliferative kidney disease (PKD), Flavobacterium psychrophilum infection, red mark syndrome and to Yersinia ruckeri. Finally, we have also performed a phenotypic and functional characterization of B cells in the adipose tissue.
- Objective 3. We have purified secreted IgD from rainbow trout serum and we have investigated its effects on head kidney leukocytes. We have established that IgD-secreting cells constitute one of the main B cell subsets in some fish mucosa. We have analyzed the IgD repertoire in gills and gut compared to the spleen demonstrating that secreted IgD from gut and gills but not spleen show an V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. Additionally, we have recently undertaken a broad phenotypic and functional characterization of these IgD-secreting cells in skin and gills, clearly establishing that these cells have started a differentiation program to plasmablasts and establishing their response to different stimuli.
- Objective 2. Within this objective, we had planned the production of rainbow trout knock-out (KO) for the TCR gene, thus lacking T cells to help us evaluate the precise contribution of T cells to B cell activation. After several unsuccessful attempts to produce KO trout, we performed the experiments in ZAP70-/- zebrafish. Another task within this objective was to attempt breaching peripheral tolerance in the intestine by different cytokines or TLR ligands. Before testing these stimuli in anal or oral stimulations, we studied their effects in rainbow trout splenic B cells. Thus, a broad characterization of the effects exerted by CpGs, Bacillus subtillis or different cytokines on splenic B cells was performed. The most promising molecules were also tested at intestinal level. In this objective we have undertaken some additional experiments not previously planned in the project. Thus, we have studied the response of B cells during proliferative kidney disease (PKD), Flavobacterium psychrophilum infection, red mark syndrome and to Yersinia ruckeri. Finally, we have also performed a phenotypic and functional characterization of B cells in the adipose tissue.
- Objective 3. We have purified secreted IgD from rainbow trout serum and we have investigated its effects on head kidney leukocytes. We have established that IgD-secreting cells constitute one of the main B cell subsets in some fish mucosa. We have analyzed the IgD repertoire in gills and gut compared to the spleen demonstrating that secreted IgD from gut and gills but not spleen show an V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. Additionally, we have recently undertaken a broad phenotypic and functional characterization of these IgD-secreting cells in skin and gills, clearly establishing that these cells have started a differentiation program to plasmablasts and establishing their response to different stimuli.
The results obtained in TEMUBLYM have significantly advanced our knowledge on how B cells are regulated in teleost fish, providing further evidence that fish B cells functionally and phenotypically resemble mammalian B1 cells. It has become evident that fish B cells are equipped with a wide range of innate receptors that allow them to directly sense pathogens, and that signaling through these receptors is an essential step to reach a full activation. Furthermore, we have demonstrated that fish B cells preferentially respond to T cell-independent stimuli both systemically and when the antigen is delivered through mucosal surfaces. Although fish B cells also respond to T cell dependent stimuli such as CD40L similarly to mammalian B2 cells, this CD40L and T cell-independent stimuli can synergize to achieve a higher degree of activation.
Throughout the project we have generated a great amount of information to contribute to a much deeper understanding of B cell functionality in fish. Within this objective, we have designed different strategies to distinguish naïve B cells and plasmablasts / plasma cells. We have also undertaken a transcriptomic analysis of single B cells in different stages of maturation/differentiation to define additional markers for specific subsets. Along this line, the repertoire of light immunoglobulin chains has also been established at single cell level. Remarkably, this study has revealed using single-cell transcriptomics, the transcription of distinct rearranged VLJLCL genes in single rainbow trout B cells, results that highlight the laxity of isotype exclusion in teleosts which strongly suggest that fish B cells can produce antibodies of different specificities.
Our results demonstrating that IgD-secreting plasmablasts play an important role in fish mucosal immunity have also been considered a major breakthrough in the field. Hence, we have established that IgD-secreting cells constitute one of the main B cell subsets in the rainbow trout intestine, gills and skin. We have performed a broad phenotypic and functional characterization of these cells, demonstrating that they have started a differentiation program to plasmablasts. In the intestine, we have established that this secreted IgD establishes a mutualistic relation with the gut microbiota.
Throughout the project we have generated a great amount of information to contribute to a much deeper understanding of B cell functionality in fish. Within this objective, we have designed different strategies to distinguish naïve B cells and plasmablasts / plasma cells. We have also undertaken a transcriptomic analysis of single B cells in different stages of maturation/differentiation to define additional markers for specific subsets. Along this line, the repertoire of light immunoglobulin chains has also been established at single cell level. Remarkably, this study has revealed using single-cell transcriptomics, the transcription of distinct rearranged VLJLCL genes in single rainbow trout B cells, results that highlight the laxity of isotype exclusion in teleosts which strongly suggest that fish B cells can produce antibodies of different specificities.
Our results demonstrating that IgD-secreting plasmablasts play an important role in fish mucosal immunity have also been considered a major breakthrough in the field. Hence, we have established that IgD-secreting cells constitute one of the main B cell subsets in the rainbow trout intestine, gills and skin. We have performed a broad phenotypic and functional characterization of these cells, demonstrating that they have started a differentiation program to plasmablasts. In the intestine, we have established that this secreted IgD establishes a mutualistic relation with the gut microbiota.