Background
Chronic stimulation of CD8 T cells, either as a result of chronic infection or exposure to antigens within the tumor microenvironment, can lead to a state of exhaustion whereby T cells no longer elicit a productive effector response. Re-mobilization of exhausted tumor-specific CD8 T cells (TEX) presents a possible therapeutic strategy to generate more robust anti-tumor T cell responses.
The success of biologics targeting receptors and ligands associated with exhausted T cells such as PD-1/PD-L1 and CTLA-4 has spurred an acceleration of research to unravel the molecular processes that drive or maintain the exhausted state with the goal of developing treatments that target these pathways. In addition, the recent finding that T cell exhaustion causes epigenetic changes through the process of epigenetic scarring make histone deacetylase (HDAC) inhibitors attractive candidates for combination therapies. Effective therapeutic development necessitates reliable human in vitro models that closely mimic disease conditions. To achieve this, we adapted an in vitro TEX model via application of flow cytometry and time lapse imaging techniques to kinetically assess TEX cell phenotype and effector function.
Methods
T cell exhaustion was induced by repeated stimulation over an 8-day period, during which exhaustion markers TOX, PD-1, CTLA-4, LAG-, TIGIT and CD39 were assessed by flow cytometry at regular intervals. In addition, cytokine release was assessed by TR-FRET as well as proliferation by tritiated thymidine. On day 8, a cytotoxicity assay was run to confirm exhaustion of the T cells and an MLR was performed with exhausted vs effector T cells to assess proliferative capacity by tritiated thymidine.
Results
Persistent and repeated rounds of polyclonal human CD8 T cell stimulation recapitulated distinctive characteristics of exhausted T cells including high expression of checkpoint receptors and upregulation of transcription factors known to suppress immune activation. Over 8 days and 4 rounds of stimulation TEX cells lost effector capabilities, producing lower levels of pro-inflammatory cytokines and becoming refractory to re-stimulation through the TCR. In functional cytotoxicity assays TEX cells exhibited substantial deficits in target killing compared to donor matched Teff cell counterparts. Exhaustion could be reversed with the addition of IL-2, however, anti-PD1 treatment alone had no effect.
Conclusions
Overall, this in vitro TEX assay models key aspects of CD8 T cell exhaustion and offers a versatile and accessible platform to test novel therapeutics targeting exhaustion pathways either individually or in combination with checkpoint inhibitors to test if they fully restore effector function.
Ethics Approval
The study was performed under ethical approval from the NHS health research Authority, London – Queen Square Research Ethics Committee (UK REC reference: 17/LO/0221). All participants gave informed consent before taking part.