Abstract
T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.
Highlights
T cells play a central role in the adaptive immune response by killing viral-infected or cancer cells and by regulating other immune cells
We previously reported that membrane-anchored anti-CD3 scFv with short tethers effectively activated T cells, whereas elongation of the tether resulted in progressive loss of T cell activation [14, 17], consistent with the Kinetic segregation (KS) model of T cell activation
The anti-CD3 scFv in our previous studies were constructed from the 145-2C11 (2C11) antibody, which is a hamster anti-mouse CD3ε antibody with a reported relatively low affinity (Kd ~ 7 × 10−8 M) [25, 26]
Summary
T cells play a central role in the adaptive immune response by killing viral-infected or cancer cells and by regulating other immune cells. T cell activation is initiated by binding of a cognate T cell receptor (TCR) to short stimulatory peptides in the context of major histocompatibility complex (pMHC) [1, 2]. The TCR α/β chains have short cytoplasmic tails, but all proximal signaling events are transduced through the associated CD3 molecules [3]. The individual CD3 molecule possesses one to three intracellular motifs for tyrosine phosphorylation, known as immune-receptor tyrosine-based activation motifs (ITAMs) [3, 4]. ZAP70 phosphorylates the adapter proteins, linker for T cell activation (LAT), and SH2 domain-containing leukocyte protein of 76 kDa (SLP76), which leads to activation of downstream signaling and expression of effector functions [5, 6]
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