Single-cell transcriptome uncovers heterogeneity and immunity of leukocyte subpopulations after vaccination with inactivated Edwardsiella tarda in flounder (Paralichthys olivaceus)

From an evolutionary point of view, the immune system of poikilothermic vertebrates is special and mysterious, and its immunity depends on the conserved components and functional complexity of immune cells. Unraveling the diversity and functional differences of immune cells can contribute to efficient and scientific vaccinations. Here we established a model of formalin-killed Edwardsiella tarda vaccination within flounder (Paralichthys olivaceus), and investigated the classification, kinetic and immune response of leukocytes by single-cell RNA sequencing. The results revealed that the leukocytes of flounder were divided into 12 clusters, including important immune cells such as granulocytes, monocytes/macrophages, T/B lymphocytes, DCs and NK-like cells. The sub-classification was performed for granulocytes, monocytes/macrophages and T/B lymphocytes, in which B cells could be classified into naive B cells, activated B cells and antibody-secreting cells according to their activation status. The percentages of granulocytes, myeloid progenitor cells and hematopoietic stem cells increased after inactivated vaccine immunization, and the percentages of activated B cells and antibody-secreting cells also increased significantly in the leukocyte populations. In addition, a positive correlation was found between the activation of B cells, secretion of specific antibodies and the protection efficacy of the inactivated vaccine. Our results have critical implications for the identification of immune cells and understanding of the adaptive immune response in teleost fish.