Background
CD8+ T cells are frequently unable to adequately control tumour cells, resulting in tumour progression. Peripheral tolerance limits initial T cell activation in the lymphoid organs and consequent tissue infiltration while exhaustion restrains established effector cell functions. Despite extensive preclinical evidence that tumours can exploit peripheral tolerance to limit T cell activation in lymphoid organs, most cancer immunotherapy studies have focused on how therapies impinge upon exhausted CD8+ T cells within the tumour. In contrast, the capacity of CD8+ T cells in the draining lymph node to predict immunotherapy responsiveness and overall patient outcome remains largely unexplored, mainly due to unavailability of fresh lymph nodes for research purposes.
Methods
Our multidisciplinary team designed a protocol, which allows us to collect blood and fresh tissue of the primary tumour, normal tissue, metastatic tumour, normal tissue (metastatic site) as well as two draining lymph nodes and one non-draining lymph node from patients with stage IV colorectal cancer presenting with synchronous resectable disease. Samples were processed according to the novel FixNCut protocol, cryopreserved and later enriched for CD45+ and CD8+ cells by FACS sorting, hashtagged with anti-human Total Seq A antibodies and LMOs before performing scRNAseq and TCR sequencing according to the 10Xpipeline.
Results:
We demonstrated that the FixNCut protocol allows fixation, cryopreservation and FACS enrichment of cells before scRNAseq and TCR sequencing. Separation of hashtag signals with anti-human Total Seq A antibodies was superior to MULTI-seq LMOs. First analysis of our data identified a diversity of CD8+ T cell clusters that are shared across patients, including some lymph node restricted clusters. Further analysis is being conducted to determine if these clusters have markers of CD8+ T cell tolerance.
Conclusion:
We generated a novel dataset which will allow us to quantify the prevalence of CD8+ T cell tolerance in colorectal cancer.