ABSTRACT
Tumors generate an immune-suppressive environment that prevents effective killing of tumor cells by CD8+ cytotoxic T cells (CTL). It remains largely unclear upon which cell type and at which stage of the anti-tumor response mediators of suppression act. We have combined an in vivo tumor model with a matching in vitro reconstruction of the tumor microenvironment based on tumor spheroids to identify suppressors of anti-tumor immunity that directly act on interaction between CTL and tumor cells and to determine mechanisms of action. An adenosine 2A receptor antagonist, as enhanced by blockade of TIM3, slowed tumor growth in vivo. Engagement of the adenosine 2A receptor and TIM3 reduced tumor cell killing in spheroids, impaired CTL cytoskeletal polarization ex vivo and in vitro and inhibited CTL infiltration into tumors and spheroids. With this role in CTL killing, blocking A2AR and TIM3 may complement therapies that enhance T cell priming, e.g. anti-PD-1 and anti-CTLA-4.
Subject(s)
Cell Death , Cytoskeleton/physiology , Cytosol/physiology , Hepatitis A Virus Cellular Receptor 2/genetics , Receptor, Adenosine A2A/genetics , Adenosine A2 Receptor Agonists/pharmacology , Animals , Cell Line, Tumor , Female , Hepatitis A Virus Cellular Receptor 2/metabolism , Male , Mice , Mice, Inbred BALB C , Receptor, Adenosine A2A/metabolismABSTRACT
The killing of tumor cells by CD8+ T cells is suppressed by the tumor microenvironment, and increased expression of inhibitory receptors, including programmed cell death protein-1 (PD-1), is associated with tumor-mediated suppression of T cells. To find cellular defects triggered by tumor exposure and associated PD-1 signaling, we established an ex vivo imaging approach to investigate the response of antigen-specific, activated effector CD8+ tumor-infiltrating lymphocytes (TILs) after interaction with target tumor cells. Although TIL-tumor cell couples readily formed, couple stability deteriorated within minutes. This was associated with impaired F-actin clearing from the center of the cellular interface, reduced Ca2+ signaling, increased TIL locomotion, and impaired tumor cell killing. The interaction of CD8+ T lymphocytes with tumor cell spheroids in vitro induced a similar phenotype, supporting a critical role of direct T cell-tumor cell contact. Diminished engagement of PD-1 within the tumor, but not acute ex vivo blockade, partially restored cell couple maintenance and killing. PD-1 thus contributes to the suppression of TIL function by inducing a state of impaired subcellular organization.
