Early regulation of T follicular helper cell differentiation via stroma-derived Notch signals

About This Grant

PROJECT SUMMARY The germinal center (GC) is a specialized microanatomical structure forming in secondary lymphoid organs in response to pathogens and immunogens. T follicular helper (Tfh) cells are a specialized subset of CD4+ T cells providing help to B cells within GCs. Tfh differentiation occurs in a stepwise fashion, first via polarization during initial priming by antigen-presenting cells and then via interactions with cognate B cells. The GC response provides a two-pronged system to generate durable protective immunity, as its main cellular outputs are antigen- specific memory B cells and long-lived plasma cells. While many key regulators of Tfh differentiation have been identified, many aspects of early Tfh differentiation remain to be elucidated. The Notch signaling pathway is highly conserved from mice to humans and has been demonstrated to play a role in effector T cell differentiation. This pathway is comprised of multiple ligand:receptor pairs which are differentially expressed across cell and tissue types. Of note, a subset of lymph node stromal cells termed fibroblastic reticular cells (FRCs) that are lineage traced by Ccl19-cre have been shown to be essential for both Tfh and effector CD8 differentiation. My own preliminary data indicates the same subset of cells provide necessary signals to antigen-specific, differentiating CD4 T cells within the first three days post immunization to drive the Tfh fate. In the absence of early Notch signals, differentiating T cells adopt a TH1-biased effector fate, evidenced by increased expression of the canonical TH1 transcription factor Tbet and increased production of TH1-associated cytokines such as IL-2, interferon gamma and tumor necrosis factor alpha. My preliminary data also suggests that the key Notch receptor, Notch1, and the key ligand, Delta like ligand 4, are upregulated 24 hours post immunization. Through a uLIPSTIC-based approach where cells in direct contact are irreversibly labeled, I have demonstrated that antigen-specific CD4 T cells interact with Ccl19-cre-expressing FRCs at the same 24 hour timepoint. However, little is known about the exact mechanisms behind receptor upregulation and the direct transcriptional targets following receptor:ligand interactions. Thus, I hypothesize that FRC niches control the early stages of Tfh differentiation and subsequent magnitude of GC responses via spatially regulated access to Dll4 Notch ligands. To explore this hypothesis, I will determine how the upregulation of Notch receptors is regulated post immunization. I will also define how differentiating Tfh cells interact with cellular partners, and how these interactions change in the absence of Notch signaling. Finally, I will determine how the Notch signaling pathway influences the transcription of known Tfh target genes through retroviral transduction and Cleavage Under Targets and Release using Nuclease (CUT&RUN) to identify the direct transcriptional targets of the Notch signaling pathway. Altogether, this proposal will identify direct Notch transcriptional targets in mature T cells for the first time and situate these events amongst the steps of Tfh differentiation, further elucidating the early mechanisms behind commitment to the Tfh fate.