# Ligands for Human Synovial Gamma/Delta T Cells

> **NIH NIH R21** · UNIVERSITY OF VERMONT & ST AGRIC COLLEGE · 2021 · $164,263

## Abstract

Project Summary
gd T cells remain an enigma of the immune system. Much of this results from an ignorance of the ligands for gd T cells.
gd T cells often localize at epithelial barriers, sites of inflammation with infection, autoimmunity, or tumors, and appear
to promote tissue homeostasis at these sites. Consistect with this, a recent seminal study reported that intratumor gd T
cells are the most favorable prognostic immune population across 39 cancer types in humans. However, compared to
ab T cells, almost nothing is known regarding the specificity of gd T cells or the structure of the TCR-gd complexed with
its ligands. From our studies in human Lyme and rheumatoid arthritis we have developed a panel of synovial Vd1 gd T
cell clones. From three of these we cloned the TCR-gd and produced a soluble TCR-gd (sTCR-gd) that was tetramerized
for flow cytometry. This reagent revealed a broad spectrum of staining of a panel of 15 tumor cell lines, as well as
activated monocytes and T lymphocytes. The staining patterns were combined with RNAseq data on the same tumor
lines and bioinformatics performed to narrow the list of candidate ligands. We also determined that the ligand(s) is a
protein that contains needed glycosaminoglycans (GAGs). This was confirmed by both enzymatic digestion of GAGs and
CRISPR/Cas9 deletion of a sulfate transporter the promotes GAG formation. We have also produced initial crystals of the
sTCR-gd for eventual structure analysis, and lentivirally transfected the TCR-gd into TCR-negative Jurkat T cells. The
Specific Aims are thus:
Specific Aim 1. Identify synovial TCR-gd candidate ligands using RNAseq and bioinformatics. We will expand the
number of tumor lines examined to the full panel of 24 cell lines for which RNAseq data is available. Similar analyses will
be performed on activated monocytes and T cells. This will increase the power of the bioinformatics analysis.
Specific Aim 2. Confirm TCR-gd candidate ligands and pathways of synthesis using CRISPR/Cas9. We will perform
CRISPR/Cas9 of targeted candidate ligands from Aim 1. In parallel, we will perform an independent genome-wide
CRISPR/Cas9 screen to also identify molecules and pathways involved with ligand synthesis
This project combines the expertise of three co-PIs: Dr. Ralph Budd in human synovial gd T cell biology, Dr. Simon Davis
in novel bioinformatics approaches to identifying cell surface ligands for orphan receptors, and Dr. Gavin Wright in
genome-wide CRISPR/Cas9 analyses. It also uses novel reagents, being the only laboratory that has human syonovial gd T
cell clones, and unique soluble TCR-gd from three clones. Furthermore, all the required technqiues are in place, as are
the collaborators. The findings would provide an enormous advance for human gd T cell biology, and enable future
studies on the regulation of TCR-gd ligands as well as the structure of a synovial TCR-gd in complex with its ligand.

## Key facts

- **NIH application ID:** 10142365
- **Project number:** 5R21AI151407-02
- **Recipient organization:** UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
- **Principal Investigator:** Ralph C Budd
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $164,263
- **Award type:** 5
- **Project period:** 2020-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10142365

## Citation

> US National Institutes of Health, RePORTER application 10142365, Ligands for Human Synovial Gamma/Delta T Cells (5R21AI151407-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10142365. Licensed CC0.

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