Extended Data Fig. 4: T cell phenotype and apoptosis in liver metastasis.

a Flow cytometry histograms showing phenotype of intrahepatic CD45⁺CD8⁺KSP-tetramer⁺ T cell (green) and total CD8 T-cell pool (blue) in mice with subcutaneous MC38 tumours (S.C., bottom) and subcutaneous and liver MC38 tumours (S.C. + liver, top). b Flow cytometry quantification of cleaved caspase-3 of OT-I cells in mice that bearing subcutaneous MC38-OVA tumour and sham (PBS, n = 11), MC38-Luc (n = 10) or MC38-OVA (n = 10) liver tumour. Unactivated CD45.1⁺CD45.2⁺OT-I cells were adoptive transferred and analysed 12 days after adoptive transfer. Data from two independent experiments were pooled. One-way ANOVA with Tukey’s correction, mean ± SD. c Frequency of KSP-tetramer⁺CD8⁺ cells expressing cleaved caspase-3 in liver of subcutaneous MC38 tumour-bearing mice with (n = 11) and without (n = 6) liver tumours. Unpaired two-tailed Student’s t-test, mean ± SD, data from two independent experiments were pooled. d Cell number of cleaved caspase-3 expressing OT-I cells from indicated location. OT-I cells were activated in vitro and labeled with CFSE, then intravenously transferred. Cells were analysed 4 days after transfer. One-way ANOVA with Dunnett’s multiple comparisons test, P-value: S.C. tumour 0.003, tdLN 0.0045, liverLN 0.0048, cerLN 0.0002, blood 0.0005, mean ± SD, n = 4 per group. e viSNE analysis of indicated marker as detected by CyTOF. Displayed on aggregated samples. Related to Fig. 4c. f Subcutaneous MC38 tumour growth in mice with subcutaneous and liver tumours, treated with anti-PD-L1, anti-CD4, or the combination. Two-way ANOVA with Tukey’s correction, mean ± SD, S.C. +IgG n = 5, S.C. + anti-PD-L1 n = 5, S.C. +liver n = 8, S.C. + anti-PD-L1+ anti-CD4 n = 9. g MC38 subcutaneous tumour growth in mice with subcutaneous and liver tumours, treated with anti-PD-L1, or in combination with hepatic CD4⁺ adoptive cell transfer (ACT). Two-way ANOVA with Tukey’s correction, mean ± SD, S.C. + IgG n = 9, S.C. + anti-PD-L1 n = 10, S.C. + liver+IgG n = 10, S.C. + liver+anti-PD-L1 n = 8, S.C + liver+CD4 ACT n = 8. Data are representative of at least two independent experiments (a-d).

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