The role of TCR, ligand, and signal strength in human γδ T cell development

Research Inst Of Fox Chase Can Ctr, Philadelphia PA

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Abstract

PROJECT SUMMARY/ABSTRACT - 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, 3, 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 (the latter in collaboration with Project 3). 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 (with Project 3) 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.