Molecular Basis for γδ T Lineage Specification

Research Inst Of Fox Chase Can Ctr, Philadelphia PA

Investigators

Linked publications & trials

Abstract

Project 3 has been removed, but the overall scope of Projects 1, 2, and 4 have not been altered. Instead, the nature of the support provided by the leaders of Project 3 has been reduced and converted to paid consultation. Project 1: T lymphocytes comprise two major lineages,  and , which play critical, partially-distinct roles in host defense.  and  T cells arise from a common progenitor in the thymus; however, the molecular processes responsible for specification of these lineages during development in the thymus remain incompletely understood and this represents a major gap in knowledge. We seek to address this knowledge gap. In doing so, we have provided compelling evidence that specification of the  and  T cell fates is controlled by differences in T cell receptor (TCR) signal strength. These signaling differences regulate fate by proportional induction of Id3, which causes graded repression of the function of E box DNA binding proteins (E proteins; E2A and HEB). Nevertheless, the targets through which these E proteins regulate  T lineage commitment and effector fate specification remain unclear, and this presents a major gap in knowledge. To address this knowledge gap, we (Projects 1/2/4) employed a comprehensive, genome-wide approach, which revealed a number of important insights into the mechanism by which strong TCR signals remodel E protein binding to specify fate. First, strong TCR signals have selective effects on E proteins, markedly repressing E2A binding to the genome, while preserving HEB binding. The sparing of HEB binding is important because HEB function is required for development of interleukin-17 (IL-17) producing  T cells (Proj1/2/4). Second, we have identified Tcf7/TCF1 as both a novel E protein target and a critical cofactor that cooperates with E proteins in controlling  lineage commitment. Consequently, in the current proposal we seek to address two major questions relating to how E proteins control TCF1 expression and the targets through which they cooperate to control lineage fate. We will do so according to two aims: Aim1 seeks to interrogate the mechanism by which 3 critical E protein bound elements (EPE) near the Tcf7 locus regulate Tcf7/TCF1 expression and whether individual E protein family members (E2A, HEBCan, and HEBAlt) play distinct roles. In Aim 2, we will focus on the E/TCF targets through which the  lineage fate is facilitated. We have determined that many of the E/TCF co-bound targets regulate cell metabolism and  lineage commitment is coupled with changes in lipid composition. We will employ a series of gain and loss of function strategies to explore the importance of these targets in regulating  lineage commitment and peripheral effector fate. In addition, we will profile at a genome-wide scale how the reduction in TCF1 expression caused by strong TCR signals cooperates with E protein remodeling to specify the  lineage fate. We will do so using a novel method termed STARR-Seq (with Project 2 and Core B) that interrogates the regulatory element repertoire at a genome-wide scale in single cells. Consequently, in the current proposal, we integrate the expertise of all Project leaders (1, 2, and 4) and the Genomics Core to elucidate the mechanistic basis by which changes in E protein binding controls  lineage commitment and adoption of the IL-17 effector fate and determine how the molecular remodeling in mouse model systems aligns with that during development of human  T cells (Proj2). Project 2: The critical role played by γδ T cells in host defense and immunopathologies has been well-established and the unique features of γδ T cells are being harnessed for emerging cellular therapies. While there has been much effort in characterizing mouse γδ T cell development, very little is known about the regulatory mechanisms that control human γδ T cell selection and functional diversification into type 1 (IFN-γ+TBET+) and type 3 (IL-17+RORγT+) effector fates. Despite views that human and mouse γδ T cells are poorly conserved based on distinct TCRγ and TCRδ loci, our preliminary analysis of public genomics data suggests an alignment in the role for TCR signal strength and downstream pathways during mouse and human γδ fate specification. To investigate this possibility, we are capitalizing on the strengths and synergy between the Ciofani and Adams labs. This collaboration creates an unprecedented opportunity to interrogate the links between TCR ligand engagement, TCR signal strength, downstream regulatory networks, and developmental outcomes during primary human γδ T cell differentiation at single-cell resolution and at genome-wide scale. Using human γδTCR/ligand models established in the Adams Lab and using the developmental and genomics expertise of the Ciofani group, we will directly test the effect of TCR signal strength on human γδ T cell selection and regulatory programming, parallel to the proposed work of Projects 1 and 4 in mouse models. We focus on CD1d and HLA-A2, ligands that have been characterized biochemically, structurally, and functionally by the Adams lab. Both the Adams and Ciofani labs have access to healthy pediatric human thymic samples through Biobanking programs at their respective institutions. Here, we propose an integrative approach to define the role of TCR, ligand and signal strength during human γδ T cell development. In Aim 1, we will couple scRNA-seq with detection of antigen-specificity via engineered barcoded tetrameric reagents (CITE-seq) to directly test the influence of TCR engagement of known γδ T cell ligands in repertoire honing, and the developmental and effector status of γδ thymocytes in vivo. The goal of Aim 2 is to directly test the role of TCR signaling strength on γδ T cell selection and effector fate using a reductionist approach in vitro. We will employ an array of well-characterized γδTCR-ligand pairs of various affinities and signal outputs in the context of the OP9-DL4 T cell differentiation system to assess how modulation of TCR signal strength influences human γδ versus αβ lineage specification. Lastly, in Aim 3, we will identify the regulatory networks that translate differences in TCR signal strength into γδ T cell fate. For this, we propose a novel multimodal single cell mRNA gene expression and high-throughput reporter assay (scSTARRseq) that will allow us to simultaneously map cellular states and profile the activity of thousands of cis elements to capture the regulatory associations between TCR signal strength and γδ versus αβ developmental trajectories. Altogether, this proposal will establish a comprehensive understanding the regulatory mechanisms governing human γδ T cell development. Project 4 T lymphocytes consist of two major lineages, αβ and γδ, that arise from common progenitors. The separation of these two lineages is instructed by TCR signaling, with weak and strong signals favoring development of the  and cell lineages, respectively. The signaling axis through which differences in signal strength are executed involves sequential activation of tyrosine and ERK MAPK kinases, that ultimately terminate with induction of the critical antagonist of E protein function, Id3. TCR-signals of differing strength elicit proportional induction of Id3, which antagonizes E2A and HEB occupancy of genomic sites, in a graded fashion. How strong versus weak TCR signals instruct  versus  T cell fate through graded reduction in E protein function remains a critical gap in knowledge to be addressed by this program. Here we hypothesize that gradients of TCR signal strength instruct differences in Id3 gene expression dynamics and propose that these differences underpin the mechanism that instructs differentiation into either the  or  lineage fate. To test this possibility, we engineered an approach that for the first time, enabled monitoring of mRNA synthesis in live immune cells that were derived from primary lymphoid organs. Using this approach, we were able to track mRNA synthesis in live immune cells that were derived from the bone marrow. We found that pre-TCR and -TCR signals activated distinct Id3 transcriptional bursting signatures at the nuclear envelope. Here we seek to determine how weak versus strong TCR-induced Id3 transcriptional bursting signatures are associated with the  versus  lineage fate decision and how TCR-induced Id3 gene expression dynamics is initiated at the nuclear envelope. Specifically, we would convert TCR-induced fluctuations in Id3 mRNA abundance from analog to digital signals. We would describe ligand-TCR interactions in terms of digital codes that define the spatiotemporal control of Id3 gene expression dynamics. We would examine where the Id3 locus is localized and whether and how the nuclear positioning of the Id3 locus is regulated by antigen receptor signaling. This project is heavily integrated with all other projects. In collaboration with Project 1, we would determine whether and how in response to pre-TCR and TCR signals ERK signal duration modulates transcriptional bursting frequencies, ON- and OFF-times and bursting amplitudes. In collaboration with Project 1, we would examine how feed-back circuitry involving Id3, E2A and HEB, instructs Id3 gene expression dynamics in response to pre-TCR and -TCR signals. In collaboration with Project 2, we would monitor Id3 gene expression dynamics in KN6- T cells in response to exposure of tetramers that interact with a gradient of affinities for the KN6-TCR.This aim will be done in close collaboration with Project 1. Ultimately, it is our goal, in collaboration with our PO1 partners, to establish a new paradigm that defines TCR signals elicited in response to self- or non-self-antigens by their digital Id3 bursting codes.