Aggregation of IgM+, TCRβ+ and CD4+ Cells around melanomacrophage centers - evidence of an organized lymphoid structure in a teleost

Abstract The study of immunity in non-model organisms has the potential to reveal the evolved diversity of the immune system. However, such studies are hampered by the paucity of species-specific reagents. Advances in single-molecule RNA-ISH overcomes these setbacks and allows for the detection of potentially dozens of gene targets in a single tissue section. We examined the spatial expression of immune genes in the splenic tissue of a teleost, threespine stickleback, using the advanced multiplexed RNAScope Hiplex assay. Threespine stickleback, a small teleost found throughout the Northern Hemisphere, is an emerging immunologic model. It has been proposed that teleost humoral adaptive immunity initiates in the melanomacrophage center (MMC), a putative evolutionary precursor to germinal centers (GC), however this has not been directly tested. Teleosts lack GCs, yet still generate affinity matured antibodies in response to antigen challenge. We find that stickleback MMCs increase in size in response to antigen immunization but not to control injection, suggesting that MMC response is antigen driven, similar to the mammalian GC. Analysis of splenic tissue indicates that IgM mRNA expressing cells aggregate in discrete regions around stickleback MMCs. Similarly, CD4 and TCR-β expressing cells aggregate near IgM+ cells, comparable to the organization of B and T cells in mammalian GCs. These findings suggest that stickleback splenic tissues have an organized structure, focused around the MMCs, and support the hypothesis that MMCs could be the site of teleost adaptive immunity and the evolutionary precursors to GCs. This study also highlights the application of RNAScope in a non-model organism. Supported by NIH RO1AI146168