The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering

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.

High-Affinity Ligands Can Trigger T Cell Receptor Signaling Without CD45 Segregation

“…By contrast, recent reports have shown that mechanical forces specifically applied to TCRs can initiate signaling in T cells ( 18 – 20 ), which may not require CD45 segregation for T cell activation. In addition, we recently reported that a high-affinity membrane-tethered anti-CD3 antibody effectively triggered TCR signaling even when elongated with very long tethers ( 21 ), which is not expected to effectively cause CD45 to physically segregate from engaged TCR complexes. The role of CD45 in initiation of TCR signaling is, therefore, uncertain.…”

“…We modulated the molecular topology of proteolipid surfaces by varying the lengths of the tethered anti-CD3 scFv. By addition of a short tether corresponding to one immunoglobulin (Ig)-like domain (~3.5 nm) ( 22 ) or a longer tether derived from the CD43 extracellular domain [19–45 nm depending on the measurement method ( 21 , 23 )], we could generate different dimensions of chimeric molecules. We examined a high-affinity anti-CD3 scFv (OKT3) that can activate T cells regardless of tether dimensions and a low-affinity anti-CD3 variant (OKT3 MA ) that can activate T cells when anchored to cells via a short but not long tether ( 21 ).…”

“…By addition of a short tether corresponding to one immunoglobulin (Ig)-like domain (~3.5 nm) ( 22 ) or a longer tether derived from the CD43 extracellular domain [19–45 nm depending on the measurement method ( 21 , 23 )], we could generate different dimensions of chimeric molecules. We examined a high-affinity anti-CD3 scFv (OKT3) that can activate T cells regardless of tether dimensions and a low-affinity anti-CD3 variant (OKT3 MA ) that can activate T cells when anchored to cells via a short but not long tether ( 21 ). We report here that an elongated high-affinity anti-CD3 scFv induced similar calcium mobilization, IL-2 secretion and cell proliferation in Jurkat T cells as those for short anti-CD3 scFv even though it induced significantly less segregation of CD45 from engaged TCRs at early times, suggesting that CD45 segregation from engaged TCRs is not mandatory for TCR triggering.…”

Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering

“…To date, all experiments testing axial positioning of TCR ligands have involved either the extension of TCR ligand height 12, 14 or the truncation of the CD45 extracellular domain 15, 16 . These flexible or “soft matter” approaches have non-linear effects on the inter-membrane spacing 12, 14, 17 and thus lateral spacing and affinity 18 . These caveats make it difficult to distinguish competing models 14, 15 .…”

“…These flexible or “soft matter” approaches have non-linear effects on the inter-membrane spacing 12, 14, 17 and thus lateral spacing and affinity 18 . These caveats make it difficult to distinguish competing models 14, 15 . If extension could be accomplished with a rigid, non-tilting structure, this would enable a more precise test of the KS model and a better general understanding of TCR triggering.…”