Physiological TCR Modulation after Antigen Specific Triggering of Introduced TCRs under Control of a Retroviral Promotor.
Authors: van Loenen Marleen M. , Hagedoorn Renate S. , van Egmond Esther H.M. , Willemze Roel , Falkenburg J.H.Frederik , Heemskerk Mirjam H.M. 
Published: April 2020

Abstract
TCR transfer to engineer tumor specific T cells may be an alternative strategy for adoptive immunotherapy. We have previously shown that TCR-transduced T cells are capable of recognizing targets both via their endogenous TCR and the introduced TCR. Since the introduced TCR is regulated by a different promotor, we investigated whether triggering and modulation of the introduced TCR occur in a physiological manner compared to the endogenous TCR. Because introduction of a TCRα-chain and TCRβ-chain will lead to formation of chimeric TCR-complexes with the endogenous TCRα- and β-chain, we used models in which both specificities of the TCRs were known and tetramers were available to distinguish each TCR. CMV PP65 specific T cells were transduced with a retroviral construct encoding the hematopoietic minor histocompatibility antigen HA-2 specific TCR (HA-2-TCR). TCR-transduced T cells were antigen specifically triggered using EBV-LCLs that presented endogenously processed PP65- or HA-2-antigen. At various time points after stimulation cell surface expression of the TCRαβ-complexes and the BV-chain of the endogenous TCR was studied with monoclonal antibodies (mAbs). No mAb directed against the BV-chain of the introduced TCR was available. Tetramers specific for the endogenous or introduced TCR were used to distinguish the TCRs from chimeric TCRαβ-complexes. We observed that after stimulation of either the endogenous or the introduced TCR the total amount of TCRs decreased to 40% and 70% of unstimulated cells, respectively. When the endogenous TCR was triggered, HA2- and PP65-tetramer stainings as well as the endogenous BV chain were diminished after 1 day. Stimulation of the introduced TCR decreased the introduced TCR expression after 1 day, and the endogenous TCR expression measured by tetramers and BV specific mAb was marginally decreased. Two days after stimulation of the introduced TCR and 3 days after stimulation of the endogenous TCR, the total amount of TCRαβ-complexes was restored. Staining with specific BV-mAb and tetramers demonstrated that the endogenous TCR expression, both after triggering of the endogenous as well as the introduced TCR, was still decreased at day 3. These data indicate that TCRαβ-complexes on the surface at days 2 and 3 mainly consisted of the introduced HA-2-TCR. TCR expression of non-transduced T cells was still decreased at day 3 after specific stimulation. Functional analysis of the T cells in a chromium release assay after 1 day of TCR triggering demonstrated that the T cells exerted reduced cytolytic activity that correlated with the downmodulation of TCR expression of either the introduced and endogenous TCR. The lytic activity of the T cells was restored at day 2 or 3 and correlated with the tetramer stainings. In conclusion, we observed physiological downmodulation of TCRs which were regulated by a retroviral promotor after antigen specific triggering. However, the introduced TCR was more quickly re-expressed at the cell surface, probably due to the increased retroviral promotor activity. The downmodulation upon specific triggering of both introduced and endogenous TCRs implies that cell mechanisms other than promotor activity are also involved in regulation of TCR cell surface expression.