2D Kinetic Analysis of TCR and CD8 Coreceptor for LCMV GP33 Epitopes

Abstract

The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the in vitro analyses of CD8 T cell responses to this epitope make use of an altered peptide ligand (APL) in which the cysteine from the original 9-mer peptide (KAVYNFATC) is substituted by a methionine at position 41 (KAVYNFATM). In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.

CD8 co-receptor enhances T cell activation without any effect on initial attachment

“…This is consistent with a pioneer study made with atomic force microscopy, revealing that CD8 was not increasing adhesion efficiency for an activating peptide in a mouse system (Puech 2011). This may however seem at variance with recent reports that CD8 might increase the lifetime of TCR/pMHC interactions (Kolawole 2018, Hong 2018. However, while it seems well demonstrated that T cell activation may enhance the stability of the interaction between TCR born by CD8+ cells and cognate pMHCs (O'Rourke 1990, Hong 2018, Kolawole 2018, it is not clear whether this phenomenon can occur during the first seconds following initial TCR engagement.…”

“…This may however seem at variance with recent reports that CD8 might increase the lifetime of TCR/pMHC interactions (Kolawole 2018, Hong 2018. However, while it seems well demonstrated that T cell activation may enhance the stability of the interaction between TCR born by CD8+ cells and cognate pMHCs (O'Rourke 1990, Hong 2018, Kolawole 2018, it is not clear whether this phenomenon can occur during the first seconds following initial TCR engagement. Also, the effect of CD8 on TCR-pMHC interaction was reported to depend on tested TCR (Zhong 2013).…”

“…In aforementioned reports on correlation between T cell activation and lifetime of TCR-pMHC bonds, Liu et al used a mutated MHC to prevent CD8 binding (Liu 2014) and Robert et al (Robert 2012 used a cell-free system including only TCR and pMHC. However, other experiments suggested that CD8 might significantly increase the resistance of TCR-pMHC interaction by forming a trimolecular complex (Jiang 2011, Kolawole 2018, Hong 2018, but this occurred after a lag of 1 second or more (Jiang 2011, Hong 2018 ) and required tyrosine phosphorylation events. Since phosphorylation is a well known key step of T lymphocyte activation (Smith-Garvin 2009), and this may involve the p56lck Src kinase associated with CD8, it is not clear whether CD8 might enhance lymphocyte activation by potentiating signaling events with a consecutive binding enhancement, or it might enhance signaling events independently of the binding process.…”

TCR–pMHC kinetics under force in a cell-free system show no intrinsic catch bond, but a minimal encounter duration before binding

“…There is no doubt that catch bonds can be detected with the flow chamber (9, 39), including by our group (40). Forming catch bonds at 10 to 15 pN has been proposed to increase ligand discrimination by strongly increasing differences in bond lifetimes between catch bond forming agonists peptides and slip bond-forming irrelevant or antagonist peptides (19)(20)(21)(22). Using the laminar flow chamber, nonspecific TCR-pMHC interactions could not be detected here, and so presumably had lifetimes shorter than the detection threshold of 180 ms.…”

“…For these reasons, efforts have been made to measure TCR-pMHC 2D dissociation kinetics and the effect of mechanical force thereon. Independent studies using the biomembrane force probe (BFP) (19)(20)(21)(22) or optical tweezers (23) on live cells, or using optical tweezers in cell-free experimental setup (23,24) reported that activating TCR-pMHC interactions exhibit a decrease in off-rate when exposed to mechanical force in the 10-to 15-pN range, i.e., catch bonds. It was therefore suggested that T cells might probe the TCR-pMHC bond by exerting a pull on the order of 10 pN, with activating peptides displaying a nonintuitive increase bond lifetime that made it 10-fold higher than the lifetime of nonactivating peptides (19).…”