Fantastic T cells and how to track them: Methods for tracking adoptively transferred T cells and enhancing efficacy of cellular therapies

Cancer remains a devastating and deadly disease despite vast research efforts into developing curative therapies. However, with the emergence of cancer immunotherapy, the treatment of cancer has revolutionized, bringing hope to cancer patients. One approach to cancer immunotherapy is harnessing the anti-tumor potential of T cells, by adoptive cell therapy (ACT).

ACTs with chimeric antigen receptor (CAR) T cells have shown great efficacy in hematological cancers but unfortunately, clinical success of ACTs has been disappointing in solid tumors. This is thought to be due to various reasons, such as loss of target antigen on tumor cells, abnormal tumor vasculature that hinders tumor infiltration of T cells, and an immunosuppressive tumor microenvironment (TME).

Here we aimed at improving the efficacy of ACTs in solid tumors by developing methods for better evaluation of the behavior of adoptively transferred T cells in vivo. Firstly, we showed that tethering IL-12 to T cells before ACT, resulted in prolonged antitumor response in two preclinical murine cancer models.

IL-12 tethered T cells induced activation and expansion of naïve tumor-specific T cells, specific for other tumor-antigens than the targeted antigen by the transferred cells. We demonstrated that this effect was mediated through activation of cross-presenting dendritic cells (DCs), however blocking natural killer (NK) cells reduced the DC activation, indicating the evolvement of NK cells as well.

Secondly, we sought to develop methods for tracking adoptively transferred T cells in vivo. We compared the loading of 64Cu-radiolabeled cationic micelles and liposomes onto T cells and investigated if the loading affected T cell biodistribution in vivo using PET/CT imaging. Micelles were successfully loaded onto T cells and allowed for in vivo tracking of T cells for up to 40 hours without influencing the natural trafficking of the loaded T cells.

However, in vivo tracking of T cells with liposomes might be better suited for theranostic purposes for drug-delivery systems. Lastly, we developed a method for analyzing tumor infiltration of adoptively transferred T cells by ex vivo high-resolution 3D imaging. We optimized T cell staining with Cell Trace Far Red (CTFR) and showed that CTFR-stained T cells could be visualized in optically cleared tissues with light sheet microscopy.

Furthermore, we demonstrated that we could visualize and analyze three parameters at once: CTFR-stained T cells, tumor vasculature and ICAM-1 integrin expression on vascular endothelium.