Real-Time Monitoring of T-Cell Function to Identify Tumor Evasion Mechanisms

Improving immunotherapy depends on a better understanding of the tumor microenvironment (TME). Recent research has shed light on tumor intrinsic factors that lead to exhaustion of immune cells, particularly T-cells. Identifying tumor intrinsic factors will help us develop effective immunotherapies. However, lack of real time T-cell monitoring has hindered identification of tumor intrinsic factors with immune suppressive roles. Calcium ions are used by T-cells to communicate and coordinate their activities. Thus, the intracellular levels of Ca2+ could be used to monitor T-cell function. We aim to develop a real-time T-cell monitoring platform based on calcium influx to evaluate the function of T-cells and identify tumor evasion mechanisms. The plasmid encoding a Ca2+ indicator (GCaMP6f) fused with another fluorescent protein (dTomato) was constructed using Gibson Assembly. After sequencing, constructed plasmid was analyzed using BLAST and SnapGene. Retrovirus expressing the constructed plasmid was generated by Plat-E cells transfection. The retrovirus was transduced into murine pmel T-cells. Flow cytometry analysis was used to determine the transduction efficiency. The changes of Ca2+ levels in response to TCR-independent stimulation (Ionomycin) and TCR-dependent stimulation (tumor) were determined to evaluate the performance of the real-time T-cell monitoring platform. We found that there was a 71% transduction efficiency of double positive GFP and dTomato expression in pmel T-cells. Dynamic changes of EGFP intensity in dTomato-positive T-cells after stimulation were observed in both experimental settings. Our data suggests that we successfully generated a virus vector which was used to express EGFP to monitor T-cell function.