Genetic and Biochemical Approaches to Tyrosine Kinase and Lymphocyte Signaling

在过去的一年里，我们的工作涵盖了几个主要领域，建立在我们以前的工作基础上：

In the last year, our work has covered several major areas that build on our previous work: I. Phosphoinositide 3 Kinase (PI3K) delta: As part of a collaborative study, we previously helped characterize activating mutations affecting PI3Kdelta, a hematopoietic-specific catalytic subunit in patients with sino-pulmonary infections, mucosal lymphoid nodules, decreased circulating lymphocytes, lymphoproliferation, and EBV viremia. Our work initially focused on characterization of CD8+ cell defects in these patients, which showed elevated activation of downstream PI3K targets, including increased pAKT, and mTOR downstream targets (Lucas et al, Nature Immunol. 2014; Cannons et al, Front Immunol 2018). To further understand these defects, we generated a mouse model that recapitulates multiple features of the disease, and have used these mice to provide new insight into the requirements for PI3K in immune homeostasis and function. We previously published work dissecting T and B cell-intrinsic and T cell-extrinsic components to these phenotypes, including autoimmunity, and have revealed a major role for the commensal microbiome in the development of autoantibodies (Preite et al. Nature Immunol. 2018, Front Immunol 2019). Our more recent work has revealed a major role for PI3K in driving effector T cell function, including Fas-mediated cell death, at the expense of T cell-mediated immunity (Cannons et al, Front Immunol. 2018 Cannons et al, submitted). We are now starting to probe the role of PI3Kd in tissue-specicific immunity, including hypersensitivity. III. Regulation of Tfh cells and humoral immune responses: Another major focus of our work was SAP, mutations of which cause the genetic disorder X-linked proliferative syndrome (XLP1),characterized by fatal EBV-infection, lymphomas, and antibody defects (Cannons et al. J. Immunol 2017; Panchal et al, Frontiers Immunol., 2018). Using gene-targeted mice we generated (Czar et al 2001), we previously showed that SAP-/- T cells failed to provide essential signals for B cells to generate germinal centers and long-term antibody responses, the hallmarks of successful vaccination (Crotty et al. Nature 2004; Cannons et al. 2006). Our work has provided insight into the requirement for T:B cell interactions in the development and function of Tfh cells (Qi et al, Nature 2008; Cannons et al, Immunity 2010; Lu et al, Immunity 2011), which are the critical helper T cell population providing signals to B cells for germinal center formation and long-term humoral immunity, required for an organism to respond appropriately to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). Stemming from this work, we have continued to explore genes important for Tfh cell differentiation and function, including the transcription factor TCF1, a component of the Wnt signaling pathway, that is selectively expressed and required for Tfh cells in response to viral infection (Wu et al, Cell Reports, 2015). To further uncover genes important for Tfh differentiation, we have developed new CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al, PLOS One, 2016 ; Huang et al, Curr Protoc Immunol 2019, Roy et al, In Press). We are using these tools to identify and probe function of genes involved in Tfh cell differentiation, uncovering a role for the VHL-HIF1a axis (Huang et al, in revision). In a related set of studies, we have identified Rasa3 as a new regulator pf lymphocyte adhesion (which is required for Tfh cell interactions with other cells in vivo) (Johansen et al, In progress). We have also collaborated with other groups to use these tools to better understand requirements for T cell adhesion and migration via regulation of integrin activation (Roy et al, In Press). IV. In work over the last few years, we have found that similar signaling and transcriptional networks are required for long-term CD8 cell responses to chronic infection, and that the transcription factor TCF1 both marks and is required for a population of stem-like CD8 cells with remarkable transcriptional and metabolic overlap with Tfh cells (Wu et al, Sci Immunol 2016). These stem- or progenitor-like cells are now recognized as critical for maintaining responses to chronic infection and cancer and are the cells that are responsible for responses to checkpoint blockade therapies. In the last year, we have used single-cell RNAseq methods to learn more about this population during chronic infection and their differences from memory cells during acute infection. We have now identified another transcription factor, TOX, that is critical for the maintenance of T cells during chronic infections as well as cancer (Yao et al Nature Immunol 2019). Work from our lab, as well as 5 other groups published concurrently in Nature, Immunity, PNAS and Hepatology, revealed that TOX promotes expression of factors associated with exhaustion, but which are critical to allow T cells to persist during chronic infection. This work has important implications for understanding and potentially manipulating T cell responses to chronic infections such as HIV and HCV, as well as cancer (Blank et al, Nature Rev Immunol 2019). Finally, integrating our work on chronic infection and longterm immunity with other studies in the lab, we have found that PI3K plays an important role in the regulation of TCF1. IV. We have continued to collaborate with others on generation of new mouse models of human genetic diseases, including L. Notarangelo and L. Biesecker (Lindhurst et al, Hum Mol Genet 2019) and on immunological and signaling characterization of mouse models (Kanellopoulou et al, JEM 2019; Collins et al, Cell, 2019). V. In collaboration with Drs. Kalpana Manthiram and Dan Kastner, we have been helping characterize a periodic fever associated with tonsillitis and aphthous ulcers, PFAPA. Dr. Manthiram has shown genetic associations with a number of loci including IL-12Rb, STAT4 and associated with other inflammatory disorders such as Behcets Disease (Manthiram et al PNAS 2020). Additional work has shown altered regulation of Tfh and GC B cells. As that Dr. Manthiram has just joined our laboratory, we will expand this line of work over the next year.