Comparative investigation of the role of tumor-infiltrating and peripheral blood γδ T cells in melanoma rejection under checkpoint therapy
Immune checkpoint blockade (ICB) in metastatic melanoma (MM) was the first successful immunotherapy in solid cancers. Meanwhile, ICB utilizing antagonistic antibodies to the checkpoint molecules PD-1±CTLA-4 on the surface of effector T cells are standard of care for a steadily increasing list of advanced solid cancers. However, despite the groundbreaking success of ICB over the past decade, still only 50% of MM patients respond durably, but many nonetheless suffer side effects. Thus, there is the urgent need to improve today´s ICB strategies.
γδ T cells are “unconventional” T cells with MHC-unrestricted cytotoxicity, which are a direct target of ICBs. The studies proposed here aim at a better understanding of the contribution of γδ T cells to cancer immunosurveillance under ICB and of their (impaired) role in MM rejection. This may lead to the identification of potential means of modulating these cells to reinvigorate/boost their anti-cancer functionality. In our previous FOR2799 project, we showed that i) peripheral blood (PB) Vδ1 T cell frequencies are a predictive biomarker for successful ICB, ii) patients with a poor outcome exhibit an accumulation of terminally differentiated, potentially exhausted PB Vδ1 T cells that might be dominated by a few expanded clones, iii) γδ T cell functionality is impaired in MM before and during ICB, relative to healthy donors. We also have preliminary evidence for the trafficking of γδ T cell clones between tumor and PB.
Here, we propose an in-depth analysis of ICB-associated, tumor-infiltrating γδ T cells to investigate their phenotype and function utilizing high-dimensional single cell proteomic and transcriptomic analyses approaches, in correlation with respective clinical data. This analysis will compare the impact of ICB on PB Vδ1 T cell signatures in MM patients with the most extreme terminally differentiated phenotype (poor prognosis under ICB) with patients exhibiting a more effector cell-dominated profile with a better prognosis. Both PB and tumor samples will be studied in parallel, including the TCR repertoire, functional properties and high dimensional phenotyping. These studies will also include spatial multiplexed in-situ proteomic analyses to gain insight into the orientation of γδ T cells in the tumor microenvironment. Finally, γδ T cell cytotoxicity and potential translational strategies to boost it will be studied in our recently established in vitro melanoma model testing novel agents, for example a new bispecific antibody.
From this project, we expect to generate new insights into the contribution of γδ T cells to MM rejection that will allow us to formulate new hypotheses for future exploitation of these unconventional T cells in strategies both including and independent of ICB.
Investigation of the role of γδ T-cells in melanoma rejection under checkpoint therapy
A crucial contribution of T-cells to cancer immunosurveillance and therapy has been intensively studied and documented over the years. Groundbreaking success was recently achieved through the introduction of checkpoint blockade which releases T-cells from the physiological negative feedback mechanisms exploited by tumors to escape immune attack. Blockade of inhibitory receptors such as CTLA-4 and PD-1 expressed on activated T-cells has seen survival of some stage IV melanoma patients dramatically increased. However, it remains the case that not all patients respond, but all may suffer side effects. Further improvements of today’s treatment strategies is still urgently required.In the present proposal, we focus on the numerically minor subset of γδ T-cells, which has a major role in defense against microbial and non-microbial challenges but has been under-investigated in the context of checkpoint blockade in cancer. Results from in vitro and in vivo experiments suggest that these unique T-cells sense infected and/or malignant cells, contribute efficiently to the regulation of immunity and thus to the elimination of these cells. Little is known about the structures which are recognized by γδ T-cells in an MHC-independent manner. Peripheral blood γδ T-cells consist usually of a majority carrying the Vδ2-TCR and a minority of cells expressing Vδ1- and other Vδ-TCRs. The Vδ2 T-cell subset is associated with the expression of semi-invariant public TCRs, which recognize phospho-antigens (pAgs) or pAg-triggered cell surface molecules such as butyrophilin. In contrast, Vδ1 T-cells are focused on a private repertoire with a few high frequency clonotypes.The role of both Vδ1 and Vδ2 T-cells in cancer immunosurveillance is controversial. We and others have reported both, positive and negative associations of γδ T-cell frequencies with melanoma patient survival. Here, we propose a comprehensive analysis of γδ T-cells in stage IV melanoma patients prior to and under PD-1 blockade to investigate their role in a) melanoma rejection and b) the potential direct and indirect immunmodulatory effects of checkpoint blockade (γδ T-cells express checkpoint molecules just as do T-cells). Deep-phenotyping (using CyTOF), investigation of (poly)functionality, proliferative capabilities, and γδ TCR-repertoire analysis of peripheral blood γδ T-cells will be contrasted with investigations of the presence or absence of γδ T-cell subsets infiltrating the tumor, allowing a comprehensive interpretation of resulting data combined with clinical meta-data available for all patients that have been recruited into our biobank. Additionally, we aim to investigate tumor-specific γδ T-cell clones and their amplification by “γδ T-cell boosters” in vitro.The environment of FOR2799 will provide optimal conditions for a sustainable exchange and effective research strategy targeting exploitation of γδ T-cells in future cancer treatment regimes.