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: Itk and regulation of cytokine production: We have had a long interest in Itk, a tyrosine kinase involved in TCR signaling, mutations of which cause a human primary immunodeficiency associated with poor EBV clearance and immunopathology. Using Itk-deficient mice, we have previously studied defects associated with Itk-deficiency, including biochemical, transcriptional, developmental and functional defects, as well as alterations in cytokine production (Gomez-Rodriguez, Immunity 2009, JEM 2014; Nat Comm 2016). Defects are particularly profound in generation of IL-9-producing Th9 cells, a less-appreciated cell population that contributes to inflammatory responses in asthma, autoimmunity and cancer. In the last year (FY23), we focused on defects in Th9 differentiation, using newly-described conditional Itk-deficient mice to help provide evidence that regulation of IL-9 production by differentiated Th9 cells is less dependent on TCR restimulation, but are dependent on cytokine signals (Son et al Nat Immunol, 2023). We are continuing to use Itk-deficient mice to evaluate defects in Th9 differentiation and its rescue by IL-2, in order to explore how IL-2 rescues metabolic defects associated with weak TCR signaling, as well as investigate cross-talk between TCR and cytokine signaling. Our expertise in these areas have led to participation in invited reviews, as well as collaborative papers on phosphorylation-based signaling pathways (Castelo-Soccio L et al, Nat Rev Immunol 2023; Jiang Y et al, J. Autoimmunity 2023). We have also contributed to studies of humans with ITK mutations who develop TB, helping describe developmental defects uncovered with high-dimensional flow cytometry and parallel findings in Itk-deficient mice (Ogishi et al, JEM 2023). 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 described CD8+ cell defects in the 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), providing insight into the effects of activated PI3K on immune homeostasis and function, including CD4+ T and B cell-intrinsic and T cell-extrinsic phenotypes contributing to aberrant antibody production and autoimmunity, and a role for the commensal microbiome in the development of autoantibodies (Preite et al. Nature Immunol. 2018, Front Immunol 2019). We described CD8+ phenotypes affecting 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 long-lived effector cells (LLECs) that maintain anti-viral responses but with shorter duration than true memory cells. B. In the last year (FY23), we have been evaluating the role of PI3K and other signaling pathways during exhaustion in responses to chronic infection, revealing important roles for PI3K and for CD137 in balancing exhausted vs effector cells (Pichler et al. Immunity 2023 and in progress). We had previously uncovered signaling and transcriptional networks are required for long-term CD8 cell responses to chronic infection and showed that the transcription factor TCF1 both marks and is required for a population of stem- or progenitor-like CD8 cells that are 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; Yao et al Nature Immunol 2019, 2021; Pichler et al, Front Immunol 2022). We further found that PI3K plays an active role in suppressing expression of TCF1 (Cannons et al Cell Reports 2021, Pichler et al, in preparation). 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, diseases associated with expression of Type II immunity including TH2 cells. In the last year (FY23), we have shown that activated PI3Kd is a potent driver of IFN-g expression, preventing restriction of polarized cytokine production in vitro. Notably, house dust mite sensitization (a model of allergic asthma) of APDS mice led to markedly increased lung pathology that was distinct compared to WT, and associated with elevated IFNg and a paucity of Th2 cytokines. We have explored this immune-mediated pathology via scRNAseq technology and whether it may model Type I I-independent/steroid-resistant asthma, as well as the signaling, transcriptional and epigenetic mechanisms behind the altered patterns of cytokine production of activated PI3Kd T cells (Golec, in preparation). Our scRNA analyses have also contributed to studies of HDM-mediated pathology in collaboration with T. Nutman (NIAID) (Gazzinelli-Guimaraes et al, in press). 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 (Panchal et al, Frontiers Immunol., 2018) and SAP-deficient gene-targeted mice (Czar et al 2001), we have provided insight into the requirement for T:B cell interactions in the development and function of follicular T helper (Tfh) cells (Qi et al, Nature 2008; Cannons et al, Immunity 2010; Lu et al, Immunity 2011; Cannons et al Trends Immunol. 2013; Cannons et al. J. Immunol 2017; Panchal et al, Frontiers Immunol., 2018), which are the critical helper T cell population providing signals to B cells for germinal center formation and long-term humoral immunity. As an extension of this work, we have uncovered factors required for Tfh cell differentiation and function, including the transcription factor TCF1 (Wu et al, Cell Reports, 2015) and developed CRISPR mediated tools to inactivate multiple genes in mice and in primary T cells (Huang et al, Curr Protoc Immunol 2019; Huang et al, Nat Comm 2022) to identify and probe function of genes involved in Tfh cell differentiation. Our work provided 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. We have continued to expand our CRISPR-mediated mutagenesis tools to identify new regulators of lymphocyte adhesion and function that are regulated by PI3K (Johansen et al Front Immunol, 2021; Johansen et al Sci Signaling 2022)and 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 naive (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). In the last year, we have expanded our use of CRISPR libraries evaluation our phosphoinositide-focused, as well as whole genome to screen for effectors regulating cytotoxicity and cytokine production (Shibata, H, Golec, D et al, in progress).