Kinetic analysis of Fcγ receptor and T cell receptor interacting with respective ligands
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Low affinity Fcg receptor III (FcgRIII, CD16) triggers a variety of cellular events upon binding to the Fc portion of IgG. A real-time flow cytometry method was developed to measure the affinity and kinetics of such low affinity receptor/ligand interactions, which was shown as an easily operated yet powerful tool. Results revealed an unusual temperature dependence of reverse rate of CD16aTM dissociating from IgG. Except for a few studies using mammalian cell CD16s, most kinetics analyses use purified aglycosylated extracellular portion of the molecules, making it impossible to assess the importance of the receptor anchor and glycosylation on ligand binding. We used a micropipette adhesion frequency assay to demonstrate that the anchor length affects the forward rate and affinity of CD16s for IgG in a species specific manner, most likely through conformational changes. Receptor glycosylation dramatically reduced ligand binding by 100 folds. T cell receptor (TCR) is arguably the most important receptor in the adaptive human immune system. Together with coreceptor CD4 or CD8, TCR can discriminate different antigen peptides complexed with major histocompatibility complex (MHC) molecule (pMHC), which differ by as few as only one amino acid, and trigger different T cell responses. When T cell signaling was suppressed, TCR had similar affinity and kinetics for agonist and antagonist pMHC whose binding to CD8 was undetectable. TCR on activated T cell had a higher affinity for pMHCs, suggesting that TCRs organize themselves differently on activated T cells than on naïve T cells. In the absence of inhibitors for signaling, TCR binds agonist pMHC with several orders of magnitude higher affinity than antagonist pMHC. In addition, engagement of TCR by pMHC signals an upregulation of CD8 binding to pMHC, which is much stronger than the TCR-pMHC binding. The transition from weak TCR binding to the strong CD8 binding takes place around 0.75 second after TCR in contact with pMHC and can be reduced by several inhibitors of tyrosine and lipid phosphorylation, membrane rafts, and actin cytoskeleton. These results provide new insights to understanding T cell discrimination.
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