Development, stability, and antigen specificity of T follicular regulatory phenotype cells - PROJECT SUMMARY/ABSTRACT Many human autoimmune diseases are associated with the expansion of self-reactive B cells and subsequent autoantibodies targeting self-constituents. Even in healthy individuals, an appreciable fraction of self-reactive B cells exists, implying the existence of extrinsic layers that restrain overt humoral autoimmunity. Foxp3+ T follicular regulatory (Tfr) cells—defined in part by their localization within the B cell follicle and the germinal center (GC)—are one such layer. Selective removal of Tfr cells in mice at the peak of an immune response results in a substantial increase of autoreactive antibodies, while low Tfr cell frequencies are negatively associated with human autoimmune diseases, further suggesting a key role in the regulation of B cell tolerance. Most previous work has studied Tfr cells at the peak of a GC reaction, meaning that their contributions at steady state and following cessation of a GC are unknown. Without knowledge of the entire life cycle of Tfr cells— including their initial development, phenotype differentiation, and maintenance outside of a GC—we will remain ignorant to additional roles that these cells likely play in maintaining immunity. Early adoptive transfer studies have suggested that the majority of Tfr cells derive from existing regulatory T cell (Treg) precursors. Alternatively, some lines of evidence controversially hint that a small subset of Tfr cells may also differentiate from Foxp3neg T helper cells of foreign antigen specificity, though such populations have not been consistently identifiable. Based on these data, we hypothesize that most Tfr-phenotype cells elicited during a GC reaction represent stable pre- existing thymus-derived Treg cells reactive to self-antigens. As proposed below, we will define the development, differentiation, and stability of Tfr cells. We will also assess the extent to which Tfr cells are reactive to foreign versus self-antigens. To accomplish these aims, we will generate T cell receptor retrogenic mice expressing monoclonal populations of Tfr biased clones of interest, allowing us to study naturally occurring Tfr cells at the clonal level. We will track the entire in vivo life cycle of individual Tfr biased clones, both at steady state and after cessation of a GC response, providing key insights into the biology of these cells. Finally, we will investigate the antigen specificity of Tfr cells by assessing their in vitro reactivity to dendritic cells or B cells loaded with self or foreign antigens. The findings obtained from this project will provide us with a clear model of the complete developmental trajectory and potential foreign reactivity of Tfr cells. This model will enable us to further study these cells in previously unappreciated contexts, thereby improving our understanding of how Tfr cells may succeed—or fail—to prevent autoimmunity.
