Extended Data Fig. 4: Vaccination activates T cells in the tumour microenvironment.

To test the effect of treatment number on IP immune responses, IP cells were collected and analysed for activation and functional status using flow cytometry on Day 19 following tumour challenge in mice vaccinated 1x, 2x, 3x, 4x or those receiving no treatment (PBS; no Tx) on days 4, 6,11 and 17. a) Proportion of IP CD4 and CD8 cells with effector memory phenotypes (CD44⁺ CD62L^low). b) Representative dot plot for effector memory CD4⁺ IP cells (CD44 vs CD62L. c) Representative plot for IFNγ expression by IP CD4⁺ cells from with no vaccination vs 2x vaccination. d) Proportion of IP CD4⁺ cells expressing the regulatory markers CTLA4 and FoxP3. e) Proportion of CD4⁺ and CD8⁺ IP cells expressing PD1 and Tim3. n = 3 biological replicates; unpaired, two-tailed, parametric t-tests, SD error bars, *p < 0.05, **p < 0.01, ***p < 0.001.