A kinetic study of the T cell recognition mechanism

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Huang, Jun
Zhu, Cheng
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The mechanism of T cell recognition is the central but unsolved puzzle of adaptive immunology. The difficulties come from the multichain structure of TCR/CD3, the binate binding structure of the pMHC molecule, the diversity of the peptides presented on the APC, the critical role of coreceptor CD4/8, the communication between TCR and coreceptor CD4/8, the complex environment of interactions taking place and the binding and signaling coupled process of recognition. Most studies were using the 3D kinetic measurements or biological functional assays to address the mechanism of the T cell recognition. However, those assays are usually either lacking of physiology relevance or missing of the initial recognition signals. Here a 2D micropipette adhesion assay with high temporal resolution (-second) was used to address the in situ kinetics of molecular interaction at the membrane of live T cells. The aim of this project is to advance our understanding to the T cell recognition mechanism. The micropipette adhesion assay was firstly used to address a simple case, the resting state pMHC-CD8 interaction. In the absence of TCR-pMHC interaction, the pMHC-CD8 interaction has a very low affinity that depends on the MHC alleles and the lipid rafts of the T cell membrane where CD8 resides, but not on the peptide complexed to the MHC and whether the CD8 is an a a homodimer or an αβ heterodimer. For cognate pMHC, following the initial observation in the F5 T cell system, the binding also displays a two-step curve in the OTI T cell system. The first-step binding occurs before one second and has a very fast on-rate and off-rate (>2s ⁻¹), and the secondstep binding follows immediately but reaches a much higher level of binding. It was identified that the first-step binding is mediated by the TCR-pMHC interaction, and the second-step binding is triggered by the TCR-pMHC interaction but mediated by CD8- pMHC binding. The two-step binding is the unique property of cognate pMHC, and it can be abolished by disrupting the lipid rafts, inhibiting the Src family protein tyrosine kinases (PTK) or protein tyrosine phosphatase (PTP). The finding of two-step binding identifies a CD8-dependent signaling amplification pathway. The data also indicated the active communication between TCR and CD8 in the antigen recognition. The crosstalk between TCR and CD8 was further dissected using two anti-CD8 antibodies 53.6.7 and CT-CD8a. 53-6.7 can significantly enhance the binding of pMHC to the T cell. Although the enhancement is directly mediated by MHC-CD8 interaction, the enhancing role of this antibody is TCR dependent. Blocking the TCR-pMHC interaction on OTI T cell or expressing CD8 alone on a hybridoma abolished the enhancement. The enhancement is also dependent on the integrity of lipid rafts and the normal function of PTP. In contrast, the antibody CT-CD8 can inhibit the binding of pMHC to the T cells and interfere with the TCR-pMHC interaction. The enhancing or inhibitory role of these two anti-CD8 antibodies is reversely correlated with the affinities of TCR-pMHC interactions. Only 53-6.7, but not CT-CD8 antibody, can phosphorylate and activate Lck. The data demonstrated a dual way crosstalk between TCR and CD8, and indicated the importance of cooperation of TCR and CD8 in antigen recognition. In the physiology condition, the TCR must accurately and efficiently recognize the cognate peptide from thousands of surrounding endogenous peptides. There is an argument regarding whether the endogenous peptides plays a role in helping the TCR recognition. Our results demonstrated that the nonstimulatory peptides can significantly enhance the T cell recognition sensitivity. In the presence of nonstimulatory peptide, the TCR can efficiently detect a single antigenic pMHC. The enhancement of recognition is due to the CD8 binding to the nonstimulatory pMHC. Blocking the CD8 binding can paralyze the enhancement. In contrast, it was found that the presence of antagonist can inhibit the binding of agonist pMHC to the T cells, and the inhibition occurs in the initial recognition step. Based on the data, an "amplification and competition" model was proposed to explain the molecular mechanism of the enhancement and inhibition function of the nonstimulatory and antagonist peptides in the T cell recognition, respectively.
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