The role of CD8+ T cells in the regression of squamous cell carcinoma

Abstract

Cutaneous squamous cell carcinoma (SCC) is one of the most common cancers, with a high risk of mortality if left untreated. The rate of SCC is highly related to UV exposure and immune status. The “gold standard” treatment for SCC is surgery; however, this does not prevent tumour recurrence, and patients with one SCC have a high incidence (~60%) of secondary SCCs in 10 years. An alternative way to treat SCCs and induce long-term protective immunity is through immunotherapy. This strategy is specifically suitable because SCCs exhibit high mutational load and are considered to be highly immunogenic. The main aim of this study is to elucidate critical factors for the immune-mediated rejection of SCC. UV-induced SCCs often fail to grow when transplanted into normal syngeneic mice but can establish in immunodeficient or immunosuppressed mice. Taking advantage of this intrinsic feature, an SCC regression model has been developed in the Wells Lab: transplanted SCC establish and grow in mice immunosuppressed with tacrolimus, and upon withdrawal of tacrolimus, tumours undergo immune-mediated rejection. Through the depletion of individual immune cell subsets at the time of tacrolimus withdrawal, I demonstrated the critical role for CD8+ T cells, but not CD4+ T cells, gamma-delta T cells, nor NK cells, in driving the elimination of established SCC tumours. This phenomenon was also critically dependent on IFN-γ, although IFN-γ did not show a direct cytotoxic effect on SCC cells. IFN-γ-neutralisation abrogated SCC regression presumably by reducing CD8+ T cell infiltration and secretion of IFN-γ-inducible CXCL9, CXCL10 and CCL5 within the tumour microenvironment. A strong positive correlation was revealed between the expression of CXCL10 and CD8+ T cell abundance in tumours. Indeed, blockade of the CXCL10 receptor CXCR3 at the time of tacrolimus withdrawal prevented CD8+ T cell infiltration, which blocked SCC regression. To identify the key sources and targets of IFN-γ for SCC surveillance, I created several SCC chimeric mouse models which consisted of FVBxC57BL/6J F1 recipients and C57BL/6J bone marrow donors. This model allowed me to take advantage of the ample variety of C57BL/6J KO strains in conjunction with the FVB SCC cell line. The data uncovered using these chimeric mouse models revealed an important role of immune subsets as producers of IFN-γ, but not as recipients of IFN-γ signals as the lack of IFN-γ-receptor on these cells did not impact tumour control. Together, these findings suggest a key role for IFN-γ in driving the expression of chemokines within the tumour environment that enable the destruction of established SCC by CD8+ T cells. To investigate the mechanisms whereby CD8+ T cells eliminate SCC tumours, I created chimera mouse models reconstituted with bone marrow cells from Perforin-/-, TRAIL-/-, mFasL-/- and sFasL-/- strains and assessed the capacity of SCC tumours to establish compared to recipients that received wild-type cells. Only the absence of perforin production from hematopoietic cells permitted SCC tumours to establish and led to lower overall survival. The adoptive transfer of CD8+ T cells into tumour-bearing Perforin-/- chimeras showed signs of tumour rejection and growth arrest in 2 out of 5 animals while all tumours in the control group kept growing (n=6). Together, these findings suggest an important role of perforin as a key effector molecule in CD8+ T cell-associated SCC tumour control. In addition to the SCC tumour models, I generated a UV-induced tumour mouse model using the BPV236 cell line. The BPV236 tumours in C57BL/6J background were histologically defined as spindle cell carcinoma. Similar to other UV-induced tumours, BPV236 did not have the ability to grow in immunocompetent mice. A series of immune subset-depleting experiments showed that BPV236 tumours could establish and grow on C57BL/6J mice in the absence of CD8+ T cells. To investigate the dominant T cell-associated anti-tumour mechanisms controlling BPV236 tumour growth, BPV236 cells were injected into mice deficient in expression of IFN-γ, CXCR3, perforin, TRAIL, sFasL, or mFasL. My data suggested that IFN-γ and mFasL were critical in the control of BPV236 tumour establishment and growth, while CXCR3, perforin, TRAIL or sFasL were less important. In summary, this project provides evidence of the importance of CD8+ T cells and the cytokine IFN-γ in the control of UV-induced tumours, both SCC and spindle cell carcinoma. Notably, the dominant effector pathway(s) for CD8+ T cell mediated tumour control changed depending on the UV-induced tumour type. This is an important finding which supports the deep characterisation of different malignancy types to inform suitable selective treatments. These findings also indicate a need for treatments aimed at activating or reviving CD8+ T cells as a whole, rather than those that focus on individual killing mechanisms, considering that an accurate diagnosis of UV-induced skin tumours is not currently achievable without histopathology.