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 the intersection of T cell receptor (TCR) and IL-2 signaling: 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 (Schaeffer et al Science 1999, JEM 2000, Nature Immunol 2001; Broussard et al, Immunity 2006; Horai et al, Immunity 2007; 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. Using conditional Itk-deficient mice we helped provide evidence that IL-9 production by differentiated Th9 cells is more dependent on cytokine signals than TCR restimulation, in contrast to other T helper lineages (Son et al Nat Immunol, 2023). We have continued to use Itk-deficient mice to evaluate defects in Th9 differentiation and its rescue by IL-2. Our data suggests that Itk and TCR signaling play critical roles in metabolic reprogramming upon T cell activation, which we show is amplified and maintained by IL-2. We have used Itk-deficient cells and Itk-inhibited mouse and human cells to explore how IL-2 rescues metabolic defects associated with weak TCR signaling, and to investigate cross-talk between TCR and cytokine signaling (Kaul et al, in preparation). Additional studies include collaborative work on TCR signaling and the role of Itk and the adaptor molecule SLP-76 in the activation of the NFkB transcription factor (with L. Berg and D. Yablonski). Our expertise in these areas have also led to participation in invited reviews, methods papers, as well as collaborative papers on phosphorylation-based signaling pathways and human immunodeficiencies (Kaul and Schwartzberg, Trends Pharm Sci 2024; Kaul et al JOVE 2025, in press; Band et al, submitted, BioRvix 2025). 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 Cell Reports 2021) 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). We have continued to evaluate the role of PI3K in CD8 T cell dysfunction that arises in response to chronic infection, revealing important roles for PI3K in balancing exhausted vs effector cells (Pichler et al. submitted). 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, and for responses to checkpoint blockade (Wu et al, Sci Immunol 2016; 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 during acute infection (Cannons et al Cell Reports 2021). We have now found that activated PI3Kd leads to a reduction of FoxO1-dependent TCF-1+ stem-like progenitor CD8+ T cells that are required for sustaining antigen-specific T cells in response to chronic viral infection. Nonetheless, mice expressing activated PI3Kd maintained CD8+ T cell responses that were skewed instead towards effector-like cells in a FoxO1-independent manner, associated with an amplified IL-21-STAT3 response axis and improved viral control. Activated PI3Kd limited TOX expression, prevented epigenetic changes associated with T cell exhaustion, and promoted effector differentiation and function from both progenitor stem-like cells and cells with an exhausted phenotype. Together, this work uncovers a key role for PI3Kd activation in shaping the balance and plasticity between effector function and exhaustion while uncoupling T cell persistence from progenitor cells during chronic infection. 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 (Pichler et al, submitted). B. 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. Nonetheless, other data from our group and others have found evidence for increased Type I immunity with increased IFNg-producing cells in patients and mice expressing activated PI3Kd (Preite et al Nat Immunol 2018 and Immunol Rev 2019). Using mice expressing activated PI3Kd, a model for APDS, we found aberrant expression of proinflammatory Th1-signature genes under Th2-inducing conditions, both in vivo and in vitro (Golec et al, in revision). Specifically, we have found that activated PI3Kd is a potent driver of IFN-g expression, preventing restriction of Th2 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 to evaluate signaling, transcriptional and epigenetic mechanisms behind the altered patterns of cytokine production of activated PI3Kd T cells. We further found that this dysregulation was driven by a robust PI3K-IL-2-Foxo1 signaling loop, fueling Foxo1-inactivation, loss of Th2-lineage restriction and extensive epigenetic reprogramming. Surprisingly, ablation of Fasl, a Foxo1-repressed gene, restored normal Th2 differentiation and TCR signaling. BioID and imaging analyses revealed Fas interactions with TCR-signaling components, which were supported by Fas-mediated potentiation of TCR signaling that could occur in the absence of FADD, an adaptor that links Fas to apoptotic signaling. Our results highlight a novel Fas-FasL signaling pathway as a critical intermediate in phenotypes driven by activated-PI3K, thereby linking two key pathways of immune dysregulation (Golec, in revision). Our scRNA analyses have also contributed to studies of HDM-mediated pathology in collaboration with T. Nutman (NIAID) (Gazzinelli-Guimaraes et al, JACI Glob 2024). In all of these studies, we complement our use of mouse models with studies of patient cells, as well as studies using CRISPR-mediated mutagenesis to dissect signaling pathways. We have contributed to other laboratories’ work by helping with CRISPR technology (Perry et al, JOVE 2025; Xhu et al, BioRvix 2024, under revision; Band et al, BioRvix 2025, submitted), in addition to our own CRISPR studies of signaling and T cell differentiation (Johansen et al Sci Signaling 2022 and Trends Immunol 2024; Huang et al, Nat Comm 2022 and Curr Protoc Immunol 2019). In addition, I am mentoring 2 additional fellows from the laboratory of Dr. Y. Belkaid, who has left NIH. Their studies of T cell immunity in the mammary gland and skin complement and are complemented by the work and techniques developed in my lab.