Retroviral Interference with Execution Pathways in Target Cells To Unravel Mechanisms of Cytotoxic T Cell-Mediated Cell Death.
Authors: de Vries J.F. , von dem Borne P.A. , Heemskerk M.H.M. , Willemze R. , Falkenburg J.H.F. , Barge R.M.Y. 
Published: April 2020

Abstract
Cytotoxic T lymphocytes (CTLs) mediate target cell death by different effector mechanisms. We investigated whether a correlation exists between the kinetics of CTL-induced killing of the target cell and the different apoptotic pathways executed by the CTL. Different CTL clones were isolated using from a patient with CML after receiving donor lymphocyte infusions from an HLA-identical donor. These CTL clones recognized minor antigens expressed on the EBV-LCL cells from the patient. Since these clones were not all equally effective in killing the same target cells, we hypothesized that these T cells may induce different apoptotic pathways. In order to study the different execution pathways in the target cell, we generated retroviral constructs encoding the anti-apoptotic genes FADD-DN and FLIP, both inhibiting death receptor-mediated killing, and the granzyme B (GrB)-inhibitor PI-9. Using a retroviral delivery system, these constructs were transduced to the EBV-LCL cells. The cells highly overexpressing the gene of interest were sorted based on coexpression of a reporter gene, which was confirmed by Western Blot analysis. Both the wildtype (EBV-WT) and the transduced EBV cells (EBV-FADD-DN, EBV-FLIP and EBV-PI-9) were used as targets and the different CTL clones as effectors (E:T ratio = 1:1) in a quantitative CFSE-based cytotoxicity assay using Flow-Count Fluorospheres. To analyze the mechanism of immediate cell death caused by rapidly killing CTL clones, EBV-WT cells were exposed for 2 hours (h) to these clones, resulting in 30% lysis. This lysis could neither be blocked by FADD-DN or FLIP, nor by PI-9. Furthermore, no inhibition was obtained using the general caspase-inhibitor z-VAD-FMK, indicating that the killing was caspase-independent. In agreement with this observation, specific triggering of the Fas receptor pathway by an agonistic Fas antibody (100 ng/ml) did not induce any apoptosis within 2 h. To study CTL-induced cell death after a longer period, we analyzed CTL clones with different rates of killing and measured cytotoxicity after 5 and 24 h. Rapidly killing clones induced 60–70% cell death of the EBV-WT within 5 h, whereas slowly killing CTL clones did not show cytotoxicity after 5 h, but induced 50–60% cell death of EBV-WT after 24 h of incubation. Inhibition of CTL-mediated cell death by overexpression of the different anti-apoptotic proteins was comparable for all types of clones, showing 10–35% inhibition by either FADD-DN or FLIP and 20–55% by PI-9, indicating that both pathways are involved. As control, Fas antibody-induced apoptosis was almost completely inhibited in EBV-FADD-DN and EBV-FLIP, demonstrating highly effective block of the Fas-receptor pathway by these constructs. These results indicate that despite different kinetics of killing, all T cell clones used both the death receptor pathway and GrB release to kill its targets. In conclusion: 1. Interference with the Fas receptor or the GrB effector pathway cannot prevent CTL-mediated target cell death within the first 2 h of exposure, indicating that this lysis is probably directly perforin-mediated. 2. Both slowly and rapidly killing CTL clones use various effector mechanisms to kill their target cells, including both the death receptor and GrB pathways. In case of slower mediated cell death, not the execution pathway induced by the CTL, but affinity between TCR and MHC/peptide complex and other effector/target interactions more likely determine the kinetics of CTL-mediated cell death.