Genetic and Biochemical Approaches to Tyrosine Kinase and Lymphocyte Signaling

National Institute Of Allergy And Infectious Diseases

Investigators

Linked publications & trials

Abstract

In the last year, our work has covered several major areas that build on our previous work: I. Phosphoinositide 3 Kinase (PI3K) delta-mediated regulation of adaptive immunity: A. As part of a collaborative study, we previously helped characterize immunodeficient patients expressing activating mutations affecting PI3Kdelta in a disease now known as Activated PI3K delta Syndrome APDS, characterized by immunodeficiency and immune dysregulation. We initially described CD8+ cell defects in these patients (Lucas et al, Nature Immunol. 2014; Cannons et al, Front Immunol 2018) and generated a mouse model that recapitulates multiple features of the disease (Preite et al Nat Immunol 2018). Studies of these mice have provided insight into the effects of activated PI3K on immune homeostasis and function, including CD4+ T and B cell-intrinsic and T cell-extrinsic phenotypes that contribute to aberrant antibody production and autoimmunity, and uncovered a role for the commensal microbiome in the development of autoantibodies (Preite et al. Nature Immunol. 2018, Front Immunol 2019). In the last year we extended our studies revealing a major role for PI3K in driving CD8+ effector T cell function at the expense of central memory through accentuation of mTOR, Myc and IL-2 signaling and repression of TCF1 expression, leading to altered transcription and epigenetic circuits (Cannons et al, Cell Reports, 2021). Our work revealed a role for PI3K activity in driving the generation of recently described long-lived effector cells (LLECs) which maintain anit-viral responses but with shorter duration than true memory cells. B. T cell regulation during exhaustion: Over the last few years, we have uncovered signaling and transcriptional networks are required for long-term CD8 cell responses to chronic infection, showing that the transcription factor TCF1 both marks and is required for a population of stem- or progenitor-like CD8 cells that are now recognized as critical for maintaining responses during exhaustion induced by chronic infection and cancer (Wu et al, Sci Immunol 2016); these cells are responsible for responses to checkpoint blockade therapies (Blank et al, Nature Rev Immunol 2019; Pichler et al, Front Immunol 2022). Using single-cell RNAseq to learn more about this population, we have delineated requirements for their development and maintenance during chronic infection and differences of these cells from memory cells during acute infection, including 1) the identification of the transcription factor, TOX, that promotes expression of factors that are critical to allow T cells to persist during exhaustion (Yao et al Nature Immunol 2019). In recent work, we have found that PI3K plays an active role in suppressing expression of TCF1, which is important both for CD8 cell memory in acute infection and development of this progenitor-like population in chronic infection (Cannons et al Cell Reports 2021) and have uncovered a role for Bach2, another PI3K-repressed transcription factor is required for the development/maintenance of this progenitor/stem cell-like CD8 population (Yao et al. Nat Immunol 2021). In the last year, we have started to evaluate the role of PI3K and its effectors in responses to chronic infection, revealing important roles for PI3K in balancing exhaustion vs effector cells. This work has important implications for understanding and potentially manipulating T cell responses in chronic infections such as HIV and HCV, as well as in T cell-mediated treatments for cancer. C. Patients with APDS also have increased evidence of hypersensitivity, including asthma and eosinophilic esophagitis. We have now started to probe the role of PI3Kd in tissue-specific immunity, including hypersensitivity. We find that activated PI3Kd drives expression of multiple effector cytokines in vitro, but most notably IFN-g, preventing restriction of polarized cytokine production in vitro. Notably, house dust mite sensitization (a model of allergic asthma) of activated PI3Kd mice led to markedly increased lung pathology compared to WT, but this was associated with elevated IFNg induction and a relative paucity of Th2 cytokines. We are exploring the consequences of this immune-mediated pathology via scRNAseq technology and whether it may model Type I I-independent/steroid-resistant asthma, as well as mechanisms behind the altered patterns of cytokine production of activated PI3Kd T cells. II Regulation of Tfh cells and humoral immune responses: Through our work on 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) and SAP-deficient gene-targeted mice we generated (Czar et al 2001), we have previously shown 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 collaborative work 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. Thus, our work provided insight into the signals required for developing appropriate longterm humoral responses to distinct infectious organisms and vaccines (Cannons et al Trends Immunol. 2013). As an extension of this work, we have focused on uncovering factors required for Tfh cell differentiation and function, including the transcription factor TCF1, which is required for Tfh cells in response to viral infection (Wu et al, Cell Reports, 2015). Our expertise in Tfh cell biology has further contributed to studies of other groups as well (Zhao et al Mol. Cell 2022). To extend these studies, we have developed 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) and have used these tools to identify and probe function of genes involved in Tfh cell differentiation (Huang et al, Nat Comm 2022). Our work has helped uncover a role for the VHL-HIF1a axis in regulating both Tfh cell differentiation and expansion (Huang et al, Nat Comm 2022). This work both provides evidence for positive and negative feedback loops in Tfh cell differentiation and demonstrates the power of targeted CRISPR-mediated screens for T cell function and differentiation in vivo. III. CRISPR-mediated mutagenesis screens for immune cell function. We have expanded our CRISPR-mediated mutagenesis tools to identify new regulators of lymphocyte adhesion, which is key for lymphocyte interactions, migration and function and which is regulated in part by PI3K (Johansen et al Front Immunol, 2021). Focusing on PI3K effectors, we have uncovered a role for Rasa3, GTPase activating protein that inhibits the GTPase Rap1, as a critical negative regulator of naive T cell adhesion and trafficking that keeps nave (and likely other specific T cell populations) in a less-adhesive state, allowing them to precisely regulate their trafficking upon activation in order to generate appropriate immune responses (Johansen et al, Sci Signaling 2022).