Hypoxia-associated L-2-hydroxyglutarate in regulation of germinal center responses

About This Grant

PROJECT SUMMARY/ABSTRACT Germinal centers (GCs) are sites where B cells engage in T cell-dependent selection and ultimately differentiate to antibody-secreting plasma cells or long-lived memory B cells, a process that generates high- quality antibodies and helps to provide protection against pathogens and maintain immunological memory. Previous studies have shown that GCs are hypoxic compared to surrounding tissues and further suggest that this hypoxic environment is crucial for GC B cell responses. However, the mechanisms by which germinal center hypoxia governs GC B cell selection and differentiation are not fully understood. Hypoxia induces generation of the metabolite L-2-hydroxyglutarate (L-2HG), an inhibitor of α-ketoglutarate (αKG) dependent dioxygenases, including ten-eleven translocation (TET) and Jumonji-C domain containing lysine (K) demethylase (KDM) enzymes. TET and KDM enzymes mediate DNA and histone demethylation, respectively, and are known to be critical regulators of B cell fate. In mice with B cell specific deletion of L-2HG dehydrogenase (L2HGDH), the enzyme that metabolizes L-2HG to αKG, I observed an expansion of GC B cells and skewing towards memory fates. However, it is unknown how L-2HG-directed epigenetic changes affect DNA and histone methylation at key B cell regulatory genes, and how these changes may contribute to GC B cell fate decisions. Based on these preliminary results, I hypothesize that L-2HG produced under germinal center hypoxia influences B cell fate decisions through epigenetic reprogramming. In Aim 1, I will determine the role of L-2HG in regulation of GC B cell differentiation and memory commitment. I will use genetic models of B cell specific L2hgdh deficiency and overexpression to study the effects of altered L-2HG levels on GC B cell differentiation and memory. I will also use an immunization-infection model of influenza A to examine the functional effects of enhanced L-2HG levels on immunological memory. In Aim 2, I will elucidate the mechanisms of L-2HG-mediated epigenetic reprogramming in B cells. Using CUT&Tag and whole genome bisulfide sequencing (WGBS), I will assess the effects of increased L-2HG levels on histone and DNA methylation and assess transcriptional changes with single-cell RNA sequencing. I will further test the effects of DNA methylation inhibition to rescue the transcriptional, epigenetic, and differentiation changes induced by L-2HG treatment in B cells. Together, these studies will provide clear genetic evidence for an important role of L-2HG in memory B cell lineage commitment and define the precise mechanisms by which L-2HG influences transcriptional and epigenetic states in B cells.