The Functional Activity of Genetically Engineered T Cell Receptor Transferred T Cells Is Highly Dependent on Pairing Properties of the Transferred TCR α and β Chains.
Authors: Heemskerk Mirjam H.M. , Hoogeboom Manja , Hagedoorn Renate , Kester Michel G.D. , Willemze Roel , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
The genetic engineering of T lymphocytes is an attractive strategy to specifically redirect T cell immunity towards viral infections and malignancies. Transfer of virus- or tumor-specific TCRs has demonstrated to endow T cells with redirected antigen specificity. We demonstrated redirected anti-leukemic reactivity of CMV specific T cells using gene transfer of minor histocompatibility antigen HA-2 specific TCRs. The HA-2-TCR-modified T cells exerted high cytolytic activity against HA-2 expressing target cells, including leukemic cells, and not against target cells negative for the HA-2 mHag. After cloning of the TCR-transferred T cells, we demonstrated that the HA-2-TCR cell surface expression, measured by HA-2-tetramer staining, was variable on the transduced T cell clones, and that the cytolytic capacity of the T cells correlated with the level of HA-2-TCR expression.
Since we could demonstrate that this variation in HA-2-TCR expression was not due to differences in transgene expression, we investigated whether the endogenous TCRs influenced the expression of the introduced TCR. CMV-A2 specific T cells were isolated from peripheral blood and transduced with the HA-2-TCR. In control transduced CMV specific T cells we observed 5 different high affinity CMV specific TCRs. CMV specific T cells transduced with the HA-2-TCR that expressed predominantly the HA-2-TCR, expressed only one of these types of CMV-TCR, and in CMV specific T cells with low HA-2-TCR expression two different types of CMV-TCRs were found. These data indicated that the level of expression of the introduced TCR is strongly influenced by the endogenous TCR. To investigate whether this was due to differences in promotor activity of the endogenous and retrovirally introduced TCR, the three CMV-TCRs were characterized and transferred into unselected peripheral T cells. T cells transferred with the weak competitior CMV-TCR that was strongly downregulated in CMV specific T cells by introduction of the HA-2-TCR, showed low CMV specific cytotoxicity and no tetramer staining. In contrast, T cells transferred with the strong competitor CMV-TCR that was modestly downregulated in CMV specific T cells by introduction of the HA-2-TCR, revealed strong CMV specific cytotoxic activity and tetramer staining. These data demonstrate that the introduced and endogenous TCRs compete for cell surface expression, and that this competition is dependent on characteristics of the different TCRs and independent of whether the TCR is retrovirally introduced or naturally expressed. To investigate whether the cell surface expression of the different TCRs was determined by preferential pairing properties of the individual TCR chains, TCR α and β deficient Jurkat 76 cells were transduced with the three CMV-specific TCRs or with chimeric TCRs consisting of the TCR α chain of one TCR with the TCR β chain of another TCR. TCRαβ membrane expression revealed that TCRs with a strong competitor phenotype expressed higher levels of TCRαβ than the TCR that was a weak competitor. TCRαβ expression of Jurkat cells transduced with chimeric TCRs indicated that the expression level of the different TCRs was determined by the pairing properties of the individual TCR α and β chains and not by differences in protein expression. In conclusion these data demonstrated that introduced and endogenous TCRs compete for cell surface expression in favor of the TCR that has the highest intrinsic pairing properties.