# Ligand-dependent preTCR function

> **NIH NIH P01** · DANA-FARBER CANCER INST · 2024 · $131,094

## Abstract

ABSTRACT
  T cells mediate adaptive immune recognition essential for self vs. non-self discrimination via clonally
distributed T cell receptors (TCRs) generated in the thymus through  and  gene rearrangements. Sculpting of
the T cell repertoire through both positive and negative selection occurs at the CD4+CD8+ double positive (DP)
stage of thymic development where  TCRs first appear. Prior to the DP stage, individual  chains associate
with the invariant pre-T (pT), forming preTCRs on the surface of early thymocytes. pT consists of a C-like
Ig domain lacking a V domain. Signaling through preTCR was considered a ligand-independent, autonomous
process. Our recent data show otherwise. Using NMR spectroscopy, interactions between two distinct preTCR
 chains and peptide/MHC (pMHC) ligands are observed involving canonical V CDR loops and a hydrophobic
patch accessible on the molecular surface of the unpaired preTCR V domain. These observations have been
recapitulated and extended using biomembrane force probe (BFP) and optical tweezers (OT) analyses of
preTCRs on intact early thymocytes. Mutation of preTCR V CDR and patch residues impacts early thymocyte
proliferation and developmental progression to the DP stage in both fetal thymic organ culture (FTOC) and OP9-
DL4 stromal cell-dependent differentiation systems. PreTCR-pMHC interaction triggers thymocyte calcium flux,
revealing ligand-dependent signaling. Here we pursue three aims to define preTCR mechanobiology. In Aim 1,
we will use high throughput next generation sequencing (NGS) of thymocyte subpopulations to determine 
repertoire changes as a consequence of preTCR-ligand interaction in vitro using single chain pMHC expressing
stroma and thymocyte progenitors as well as by performing in vivo analysis in B6 MHC sufficient and deficient
mice. We shall identify  chains selected for loss or survival during thymic development and perform RNAseq
on B6 Rag2-/- thymocytes transduced with each type, determining the relationship of preTCR ligation,
biomechanics and transcriptome at population and single cell level. In Aim 2, structural features and
mechanobiology of the preTCRs will be interrogated with single molecule (SM) OT experiments to assess the
impact of diverse preTCRs on structural transitions, bond lifetimes, hopping between compact and extended
states and their relationship to thymic development. In addition, structural studies by NMR in collaboration with
Project 3, Core B, Core C and X-ray crystallography shall define individual molecular population states of ligated
and unligated preTCR conformers. In Aim 3, we will determine if TCRs, distinct from TCRs and preTCRs in
lacking the elongated C chain FG loop element, manifest bond prolongation under force. Both human and
mouse TCRs with defined CD1c or CD1d ligand specificities as well as G8  that interacts with T22 will be
compared using SM, single molecule single cell (SMSC) and single cell (SC) OT analyses with chim...

## Key facts

- **NIH application ID:** 10877947
- **Project number:** 5P01AI143565-05
- **Recipient organization:** DANA-FARBER CANCER INST
- **Principal Investigator:** ELLIS L REINHERZ
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $131,094
- **Award type:** 5
- **Project period:** 2020-07-29 → 2025-09-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10877947

## Citation

> US National Institutes of Health, RePORTER application 10877947, Ligand-dependent preTCR function (5P01AI143565-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10877947. Licensed CC0.

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