Dynamics of T-cell driven immune responses to radiotherapy in a murine model of melanoma

Background: Radiotherapy has been shown to lead to anti-tumour immune responses and there is increasing interest in developing rational treatment strategies to bolster this effect. One area of interest is the use of combination radiotherapy-drug treatments.

Favourable results have been reported from addition of drugs that target the DNA damage response (DDR) as well as immunotherapy.

Regulatory T cells (Tregs), characterised by expression of the forkhead box P3 (Foxp3) transcription factor, are believed to drive tumour immune escape mechanisms both within and outside the tumor microenvironment and have been associated with poor prognosis in several cancers.

The proportion of Tregs reportedly rises post-irradiation as they are relatively radio-resistant and are preferentially selected.

It is unclear if this time-dependent rise of Tregs is clinically significant and, if so, what the optimal timing of drugs to boost anti-tumour immunity would be to circumvent this.

Aims: We aim to determine how radiotherapy impacts on Treg activation in real-time and if this can inform the optimal timing of addition of combination systemic therapies. Methods: An established murine model of melanoma will be used and focal irradiation of tumour will be conducted.

Initial experiments will compare the therapeutic effect of 3 different dose-fractionation regimens (2 Gy x 5 fractions; 4 Gy x 4 fractions and 8 Gy x 3 fractions) to determine the optimal dose-fractionation to be used for ongoing experiments.

Foxp3-Tocky system will be used to analyse irradiated murine models of melanoma at 3 different time points (2, 6 and 10 days post-irradiation) with T-cell flow cytometry and quantitative timer data analysis using the Tocky system to differentiate Tregs numbers between the new, persistent and arrested phases.

Immunohistochemistry staining for intratumoural Tregs (CD25+ Foxp3+) will also be performed at different time points post-irradiation.

How the results will be used: The results of this research will be used to inform the optimal temporal relationship of combination therapies such as immunotherapy and DDR-targeted drugs against with radiotherapy in the early phase clinical trial setting.