Abstract
The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation.
Highlights
The mean functional avidity is assessed by cellular assays such as the 51Chromium release cytotoxicity assay[28,29] or the IFN-γElispot assay[30], using titrated amounts of peptide
We tested whether the same goal can be achieved by the IFN-γElispot assay
In analogy to the cytotoxicity assay, we analyzed IFN-γproduction in the Elispot assay with titrated amounts of antigenic peptide to determine the EC50 values
Summary
The mean functional avidity is assessed by cellular assays such as the 51Chromium release cytotoxicity assay[28,29] or the IFN-γElispot assay[30], using titrated amounts of peptide. Current immunomonitoring techniques are insufficient with regard to the assessment of T cell avidity and TCR affinity. Elegant studies using a Streptamer-based assay[31] or the NTA-multimer technology[21,32], have allowed the direct and precise quantification of TCR: pMHC dissociation rates (koff) on living CD8 T cells, demonstrating that the koff rate correlated with the functional and protective capacity of antigen specific CD8 T cells. Two-dimensional (2D) measurements of TCR-pMHC interactions provide novel opportunities to characterize T cell affinity and antigen specificity[33]. The available techniques are primarily designed for monoclonal T cells, whereas the characterization of polyclonal T cell responses remains challenging. In the present study we attempted to evaluate the heterogeneity of T cell avidities, representing an additional parameter for the characterization of T cell responses. We demonstrate a new method to determine the heterogeneity of functional avidity, and characterize the avidity range of T cell clones and related natural polyclonal populations
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