Identification and isolation of high avidity tumor-specific CD8 T cells by experimentally assessing pMHC-TCR binding parameters with soluble pMHC complexes
- 323 Downloads
KeywordsSingle Cell Level High Avidity Tumoricidal Activity Cell Antigen Receptor Subsequent Isolation
Adoptive transfer of high avidity tumor-specific CD8 T cells is a promising treatment for late stage cancers. Because most tumor antigens are self-antigens, tumor-specific T cells typically express low affinity T cell antigen receptors (TCR) due to thymic negative selection, which accounts mostly for their poor tumoricidal activity. To identify rare, most tumor-reactive T cells we developed a robust and accurate screening assay. The functional avidity of T cell responses is related to pMHC-TCR binding parameters, with an assumed optimal range of pMHC-TCR affinity for the most potent interaction. We recently described the development of NTAmers, reversible pMHC multimers built on switchable Ni2+-NTA-His-tag complexes, that allow accurate measurement of monomeric pMHC-TCR dissociation kinetics on living tumor-specific CD8 T cells by flow cytometry. We found a good correlation between off-rates and cellular responses on cloned CD8 T cells from cancer patients. Moreover we developed pMHC dimers allowing measurement of pMHC-TCR association and dissociation kinetics on cancer-specific T cells. Combined measurement of pMHC-TCR on and off rates allowed accurate identification and isolation of functional high avidity tumor-specific CD8 T cells. In vivo experiments confirmed that high avidity CD8 T cells isolated from a cancer patient conferred superior tumor control. This novel technology has further allowed us to describe the functional involvement of the CD8 co-receptor in this interaction. Using CD8-binding deficient pMHC complexes, we observed that CD8 increases on CD8 T cells the off-rate by 3-4 fold but has no discernable effect on the on-rate.
In conclusion, NTAmers and pMHC dimers enable precise measurement of pMHC-TCR binding parameters under physiological conditions and subsequent isolation of live functionally active tumor-specific CD8 T cells. Translation of these techniques to the single cell level for high-throughput screening and isolation of rare and highly functional tumor-specific CD8 T cells directly from cancer patients is in progress.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.