Physiological Non-Responsiveness and Absence of Activation Induced Cell Death of T Cells Rapidly Re-Expressing Retrovirally Introduced TCRs after T Cell Activation.
Authors: van Loenen Marleen , Hagedoorn Renate , van Egmond Esther , Willemze Roelof , Falkenburg J.H. Frederik , Heemskerk Mirjam 
Published: April 2020

Abstract
TCR transfer to engineer tumor specific T cells may be an alternative strategy for adoptive immunotherapy. We previously have shown that TCR-transduced T cells are capable of recognizing targets both via the endogenous and the introduced TCR. Stimulation of the TCR induces internalization of TCRs leading to a refractory period with a high activation threshold. Since the introduced TCR is regulated by a viral promotor which is constantly activated, we investigated whether modulation of the introduced TCRs after antigen specific triggering occurred in a physiological manner compared to the endogenous TCR. CMV specific T cells were retrovirally transduced with the hematopoietic minor histocompatibility antigen HA-2 specific TCR. TCR transduced T cells were antigen specifically triggered via either the introduced HA-2 (HA-2 TCR) or the endogenous CMV specific TCR (CMV TCR). At various time points after stimulation cell surface expression of the TCRαß-complexes and the BV-chain of the CMV TCR and HA-2 TCR was studied with monoclonal antibodies. Tetramers specific for the CMV TCR or HA-2 TCR were used to distinguish the TCRs from chimeric TCRαß-complexes. Stimulation via the CMV TCR or the HA-2 TCR resulted in similar internalization of approximately 50% of the TCRs. Preferentially, but not solely, the triggered TCRs were internalized. In contrast to the kinetics of internalization, the kinetics of TCR re-expression after stimulation differed considerably between the endogenous CMV and the introduced HA-2 TCR. The introduced HA-2 TCR was already re-expressed at the cell surface 24h after stimulation, while 70% of the endogenous CMV TCR still was internalized 72h after stimulation. This rapid synthesis of HA-2 TCRs could lead to enhanced competition for cell surface expression. Indeed, when TCR cell surface expression of the HA-2 TCR restored, cell surface expression of the CMV TCR decreased even further. When T cells were analyzed 4h after stimulation for cytolytic reactivity, both T cells stimulated via the HA-2 and the CMV TCR were non-responsive, correlating with low TCR expression. Although the HA-2 TCR was re-expressed at the cell surface 48h after stimulation, still no cytolytic activity via either the HA-2 or the CMV TCR was found. At this timepoint the level of expression of adhesion molecules and the amount of intracellular granzyme B after an initial decline was comparable to non-stimulated T cells. However, cell surface expression of the CD8 coreceptor was still diminished, resulting in low T cell avidity. To analyze whether stimulation via rapidly re-expressed HA-2 TCRs would lead to antigen induced cell death (AICD), T cells were stimulated via the HA-2 TCR after an initial stimulation and analyzed at different timepoints. In agreement with their non-responsiveness in the functional study, no increased AICD after specific stimulation via the HA-2 TCR was observed. In conclusion, we observed physiological internalization of TCRs which were regulated by a retroviral promotor after antigen specific triggering, but the introduced TCR was more rapidly re-expressed at the cell surface. Despite different TCR make up early after stimulation, in these T cells physiological non-responsiveness and no increased AICD was found after stimulation of the re-expressed introduced TCR, illustrating that cell mechanisms other than TCR cell surface expression like CD8 downregulation are also involved in providing a protective refractory period.