Tumor cell spheroid-induced suppression of primary human cytotoxic T cells as a scalable in vitro model of exhaustion

Background: Cytotoxic T lymphocytes (CTL) are key effectors in the anti-tumor immune response. However, their function is commonly suppressed in tumors in the form of exhausted CTL. Understanding mechanisms of suppression and of therapeutics to overcome them is of substantial basic and translational importance yet hindered by limited access to large numbers of exhausted CTL in vitro.

Methods: Here we use three-dimensional tissue culture to generate primary human CTL with suppressed function. Using functional assays, a 21-antibody flow cytometry panel and determination of calcium signaling and CTL tumor cell couple maintenance, we show that these cells closely resemble exhausted CTL from tumors. For better understanding of in vitro human primary CTL as key tools in therapeutic development, before and after induction of suppression, we have determined the dependence of CTL function on methodology of generation, antigen dose and affinity across two T cell receptors and multiple tumor cell lines. As a further determination of their phenotype, we have investigated morphology and subcellular F-actin distributions of CTL as a key regulators of effector function.

Results: Primary human CTL formed cell couples with tumor target cells even in the absence of antigen. Yet, gradual stabilization of such cell couples was associated with increasing CTL effector function. Induction of suppression substantially destabilized CTL tumor cell couples.

Conclusion: This comprehensive characterization of the phenotype of in vitro primary human CTL, including a suppressed state, should facilitate their use in basic research, the development of CTL-targeting therapeutics and the determination of their mechanism of action.