Comprehensive profiling of lung tumor infiltrating lymphocytes using high throughput TCR sequencing

Tumor infiltrating lymphocytes (TILs) are T and B cells that have the ability to migrate into the tumor microenvironment (TME), recognize malignant cells, and promote their clearance. Recently, we have seen therapeutic success for adoptive cell therapy (ACT) with ex-vivo expanded TILs in a range of solid cancers. The therapeutic success of these targeted therapies relies on persistence of tumor-specific TILs in circulation in patients after ACT administration. Some of the main development challenges for large-scale production of these therapies are the absence of fast and contamination-free TIL expansion protocols as well as the need for technologies that are able to assess the functional state and clonotype makeup of the TIL product prior to infusion. Here we developed methods that overcome these challenges and demonstrate their utility by generating a lung tumor TILs multimodal single cell dataset. Specifically, we showcase that the CliniMACS Prodigy® Platform is able to quickly and reliably expand tumor infiltrating T cells in less than 2 weeks. At the end of expansion we take a subset of these expanded cells and perform single cell immune profiling sequencing to characterize their transcriptional state and dissect their T cell receptor (TCR) diversity. Using the whole transcriptome data, we are able to identify all expected T cell subsets and characterize their cellular states. Furthermore, we show we are able to detect paired TCR CDR3α/β sequences in more than 92% of the cells profiled. Using this information, we estimate the frequency of each CDR3 clonotype in the sample and identify hyperexpanded clonotypes that might be tumor reactive and worth further investigation. Overall, we have developed methods for the rapid expansion and deep characterization of TILs. We performed a proof of concept experiment that demonstrated the utility of our methods and identified expanded clonotypes that can be used to inform the design of future immunotherapies. We hope our methods will enable immunologists to create safer, faster and patient-specific treatments that achieve long-term tumor remission.