Investigating the Dependence of T Cell Receptor Triggering on CD45 Segregation

• Main Authors: Mohammad Ameen Al-Aghbar, 歐默漢
• Other Authors: Steve R Roffler
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/34992363163963417313

博士 === 國立陽明大學 === 分子醫學博士學位學程 === 105 === Current understanding of T cell receptor (TCR) triggering is based on the segregation of membrane tyrosine phosphatases having large ectodomains upon engagement between T cells and antigen presenting cells, thereby creating a kinases/phosphatases imbalance to phosphorylate immuno-receptor tyrosine-based activation motifs (ITAMs) in CD3 proteins associated with engaged TCRs. We recently observed that elongated OKT3 anti-CD3 single-chain antibodies (scFv) expressed on 3T3 fibroblasts can effectively trigger TCR signaling, inconsistent with a requirement for CD45 segregation from engaged TCRs. Here, we show that TCR triggering by elongated ligands depends on affinity rather than dimension, and CD45 segregation is not a mandatory step to initiate TCR triggering. We created elongated scFv with defined dimensions expressed on the surface of 3T3 APCs and or distributed over planar lipid bilayers to track CD45 movement by TIRF microscopy. We found that elongated scFv (OKT3-CD43) was unable to segregate CD45 from the ligated TCR microclusters, but inconsistent with the KS model, was able to induce T cell calcium flux, and Zap70 phosphorylation. However, within minutes of triggering, CD45 started to segregate partially from the TCR microclusters or pZap70, indicating that CD45 segregation follows rather than initiates TCR triggering. Using a mutated OKT3 scFv that showed low affinity to CD3ε (OKT3MA), we proved that TCR triggering is more dependent on ligand affinity rather than the ligand dimensions and CD45 physical segregation; short, low affinity scFv (OKT3MA-BGP) was able to trigger TCR similarly to wild type scFv (OKT3-BGP), while elongated low affinity OKT3MA scFv (OKT3MA-CD43) was unable to trigger TCR, nor segregate CD45. Longer follow up of T cells showed proliferation, IL-2 and IFN-γ secretion by the short ligands (high and low affinities) while only high affinity elongated ligand was able to produce productive T cell activation.