Cytotoxic T lymphocyte activation and recruitment mechanisms in the tumour microenvironment

Cytotoxic T lymphocytes (CTLs) employ advanced search strategies to find infected or malignant cells. In the tumour microenvironment (TME), following antigen recognition via the T cell receptor (TCR), CTLs stop to engage target cells, in which they induce cell death by delivering cytotoxic granules through a synapse. How the interaction between CTLs and tumour cells affects the migration characteristics of distant CTLs remained unexplored. Here, we found that after engaging tumour targets, CTLs rapidly start secreting the chemokines CCL3 and CCL4, which recruit distant CTLs via the CCR5 receptor. Responding CTLs switch from random search to a to highly directional motility towards the tumour mass. Newly arriving CTLs engage with tumour cells and add to the production of the chemotactic signal, thereby attracting further CTLs in a positive feedback loop. Additionally, the release of CCL3 and CCL4 from within tumour masses promotes the deep infiltration of CTLs, which otherwise amass at the periphery. Moreover, the TME is known to be immunosuppressive for CTLs due to the presence of transforming growth factor-β (TGF-β). We found that when exposed to TGF-β, CTLs continue to interact with cognate tumour cells but their killing capacity is gradually diminished over time. This lack of sustained cytotoxicity is due to an attenuation of Erk activity, a mediator of downstream TCR signalling. However, the upstream TCR signalling pathway remains unperturbed, allowing CTLs to stop and engage tumour cells. Furthermore, the inhibition of the TGF-β signalling pathway maintains a pool of memory T cell precursors, which can generate both memory and effector T cells. When TGF-β signalling is active, memory precursors proliferate rapidly and differentiate into effector T cells, losing their plasticity. Finally, we developed a ‘search and kill’ assay to quantify the migration behaviour and killing kinetics of CTLs within collagen matrices. Using this method, we discovered that CTLs from the Lifeact-EGFP reporter mouse increase in fluorescence intensity over time whilst eliminating cognate tumour cells. Enhanced GFP fluorescence is correlated with the production of IFN-γ and TNF-α, hallmarks of T cell activation. Therefore, we reveal that the Lifeact-EGFP reporter mouse is a valuable tool for live imaging to simultaneously study F-actin dynamics and monitor T cell activation in real time