Genomics approaches to investigate the evolution of germinal center responses

Specific Aims The germinal center (GC) is a site of rapid B cell proliferation and diversification that is induced in re- sponse to infection or immunization with T cell-dependent antigens. Within the GC, B cells undergo somatic hypermutation followed by affinity maturation to select high-affinity B cells. Development of GCs is dependent on B cell-T cell collaboration, with T cells providing CD40 ligand and cytokines that support the B cell response. These processes have been studied in detail in humans and in mouse models, given the crucial importance of GCs in anti-pathogen immune responses and in vaccine-specific responses. On the other hand, there is less understanding of the evolutionary processes that gave rise to the GC responses.GCs are found in mammals, including primitive mammals such as monotremes. However, they are absent from reptiles, amphibians and fish. Despite the lack of GCs in reptiles, many of the genes involved in development and function of GCs are present, including AICDA, BCL6, CD40, CD40LG, CXCR5, ICOS, ICOSLG, DOCK8, IFR4, IFR8, etc. Therefore, many of the genes involved in regulating GC responses predate the evolutionary origin of GCs and these genes may have been co-opted during evolution to serve new roles in GC biology. In the proposed study, advanced ge- nomics and bioinformatics techniques will be used to examine the spleen of the red-eared slider turtle Trachemys scripta with the goal of understanding the roles GC-related genes in this species and how the regulatory pro- grams driven by these pathways were co-opted to GC responses. scRNA-seq will be used to define immune cell subsets and types in the spleen of T. scripta, while spatial transcriptomics will be used to understand the spatial organization of these cells and their potential interactions. This information will help us to understand the regu- lation evolutionary transitions and biological adaptations. Comparisons to similar data in mice will be used to define how turtles differ from mice in terms of immune subsets and responses and to gain insight into how GC responses may have arisen from genetic programs controlling other pathways. While our studies focus on the GC response, the unbiased genomics approaches we are pursuing will yield valuable information on other as- pects of immunity, both adaptive and innate immune responses, that will be very valuable in understanding how the immune system developed in mammals.