Abstract
The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens.
Highlights
The kinetic parameters that govern T cell activation, co-stimulation and functionality remain a point of discussion in the immunological field
We interrogated the 2D affinity and kinetics of human CD8 interacting with peptide-bound major histocompatibility complexes (pMHC) by testing these HCV-specific CTLs interacting with PPI peptide in complex with pMHC (Figure 2A)
Despite occupying a large range of 2D T cell receptor (TCR) affinity, the adhesion frequency curves of CD8 interacting with pMHC showed a consistent trend of approaching saturation with similar receptor and ligand site densities (Figure 2B)
Summary
The kinetic parameters that govern T cell activation, co-stimulation and functionality remain a point of discussion in the immunological field. SPR measures the number of immobilized ligands [peptide-bound major histocompatibility complexes (pMHC)] which bind to solubilized receptors [T cell receptor (TCR)] over time or vice versa to determine the rate constants that describe their binding and disassociation Studies using this method have converged upon the 3D off-rate as the most accurate predictor of T cell cytolytic capacity [1,2,3]. Using a micropipette adhesion assay, Huang et al independently showed that 2D off-rate was faster than its 3D counterpart and a larger dynamic range of affinity were present in 2D compared to that of 3D, which was predominantly due to a wide range of on-rates and a small range of off-rates They found that 2D affinity and kinetic parameters correlated better with T cell proliferative response to peptide stimulation compared to their 3D counterparts [4]
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