Peripheral Shaping of γδ TCR Repertoires
Starting from the question of how peripheral signals shape the T cell receptor (TCR) repertoire of γδ-T cells, this subproject of FOR 2799 aims to answer two important questions in this field, namely: what are the cognate ligands of γδ TCR and what is the role of γδ T cells during an immune response?
Apart from a large population of innate human γδ T cells, all of which carry a similar, low variance Vγ9Vδ2 TCR and all of which bind to the same butyrophilin molecules, other human γδ T cells typically use a highly diverse TCR repertoire of Vδ1 or Vδ3 chains that pair with six variable Vγ chains. The pool of unique TCRs among these more adaptive clones is very large and rarely shared between individuals. It has been hypothesized that their highly diverse TCRs are similar to B-cell receptors and bind randomly to surface antigens. However, in recent years, a number of unique Vδ1- or Vδ3-TCRs have been shown to bind to the major histocompatibility complex (MHC) or MHC-related proteins.
In the first funding period of FOR 2799, we investigated the antigen recognized by a set of CMV-responsive human γδ TCRs. We identified a Vγ3Vδ1 TCR (designated TCR04) that was specifically reactive against a B-cell lymphoma cell line. Staining of lymphoma cells with soluble versions of TCR04 and subsequent genome-wide CRISPR/Cas9 knock-out screening led to the identification of HLA-DR (MHC II) as the cognate antigen for TCR04.
In the upcoming funding period, we will now further determine the underlying molecular determinants of HLA-DR recognition by TCR04 and similar related γδ-TCR. In addition, we will use new technologies to track the progression of adaptive γδ T cell responses to viral infections by simultaneous single cell RNA sequencing and single cell TCR sequencing. Finally, we plan to identify additional relevant antigens that activate adaptive γδ T cells.
This proposal is part of the DFG research group FOR 2799 "Receiving and Translating Signals via the γδ T Cell Receptor" and is designed to collaborate with all other projects in the network.
Peripheral shaping of γδ TCR repertoires
In this project, we will further investigate how γδ TCR repertoires are shaped and which cognate ligands can trigger γδ TCR signaling. We hypothesize that the peripheral γδ TCR repertoire is shaped through recognition of specific antigen via the γδ TCR and that antigen-specific triggering of the γδ TCR induces intracellular signaling events leading to proliferation and survival of the respective γδ T cells. The principal method to test our hypotheses is already established in our group, namely the analysis of human γδ TCR repertoires by next generation sequencing of rearranged TRG and TRD gene pools. We will use it to explore how exposure to microbial, viral, or neoplastic stimuli will modulate the peripheral γδ TCR repertoire in cord blood, pediatric, and adult cohorts. Furthermore, we will establish new methods to perform efficient high throughput analysis of paired TRG and TRD TCR chains. We will share and advance this know-how with the entire DFG Research Unit. In return, new collaborations will enable us to identify how individual γδ TCR recombinations respond to neoplasm and microbial exposure and warrant access to γδ T cells from clinically relevant cancer patient cohorts. In addition, we will combine γδ TCR repertoire analyses and protein biochemistry to identify cognate ligands of responsive γδ TCR clones. We expect that cognate antigens of γδ TCRs may either be directly of microbial or viral origin or danger-indicating self-structures that were induced by tumor formation as well as by microbial or viral infection. Defining how γδ TCRs recognize microbial antigen and/or danger-associated self-ligands will pave the way for the therapeutic manipulation of immune surveillance by γδ T cells.This project proposal is part of the DFG Research Unit FOR 2799 „Receiving and Translating Signals via the γδ T Cell Receptor“ and designed to collaborate with all other projects within the research group.