Dissecting mechanisms of CD8⁺ T cell-mediated control of metastatic melanoma

Abstract

Although CD8+ T cells can play an anti-tumoural role in cancer, they often fail to do so, in part due to the acquisition of an ‘exhausted’ or ‘dysfunctional’ state. Different T cell subsets have been identified in this context, including stem-like precursor exhausted cells (T_pex) that help sustain responses. More differentiated exhausted subsets downstream of T_pex include both dysfunctional terminally exhausted (T_tex) and more cytotoxic CX3CR1⁺ subsets. Indeed, immune checkpoint blockade therapy appears to induce proliferation of T_pex cells and enhance differentiation of the effector-like CX3CR1⁺ subset. Despite this emerging understanding, the precise features of the CD8⁺ T cell response that enable successful tumour control remain incompletely defined. This is particularly the case for lymph node metastases, which are often the first site of metastasis in patients. <br /> To address this, our group previously established a transplantable melanoma model whereby a proportion of mice develop metastases in the tumour-draining lymph node following curative-intent surgery of the orthotopic primary skin tumour. In this thesis, we further characterised this model and established situations of antigen-specific CD8⁺ T cell-driven control of lymph node metastases. We then interrogated the T cell response that was driving this control using cutting-edge techniques such as high-dimensional flow cytometry, single-cell RNA sequencing, and multiplex imaging. Firstly, we were able to maintain control of lymph node metastases over several weeks by transfer of in vitro-activated tumour-specific transgenic CD8⁺ T cells. In addition to ongoing control of metastases in some mice, others escaped therapy and rapidly grew out. We identified a less exhausted and more functionally activated T cell response in lesions under ongoing control relative to those that escaped therapy. These activated T cells preferentially localised to controlled lesions over the surrounding lymphoid tissue, and were associated with tumour cell cycle arrest, which may be contributing to the inhibition of metastasis outgrowth. Secondly, CD8⁺ T cell-driven spontaneous regression or eradication of lymph metastases was observed in Ifng^–/– mice, but not their wild-type counterparts. Relative to wild-type mice, tumour-specific T cells in metastases from Ifng^–/– mice showed greater differentiation, including an increase in the cytotoxic CX3CR1⁺ subset. Despite enhanced differentiation, T_pex cells were also maintained, which are known to be important for sustaining responses. In addition, T cells were more expanded in Ifng^–/– mice, which also likely contributed to successful tumour control. <br /> Overall, work in this thesis defined the spatial, phenotypic, and functional features of two different situations of CD8⁺ T cell-driven control of lymph node metastases. This provides novel insights into how successful CD8⁺ T cell responses to cancer are achieved and strong grounding for further work into unravelling the molecular mechanisms driving this. In turn, an improved understanding of these anti-tumoural responses could inform more effective and targeted clinical cancer immunotherapies in the future.