- 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.
