Extended Data Fig. 3: Liver metastasis induces systemic loss of antigen-specific T cells.

a Subcutaneous tumour CD8⁺ T cell, IFNγ⁺CD8⁺ T cell and Ki67⁺CD8⁺ T cell number per gram tumour. Analysed 7 days post anti-PD-L1 treatment initiated. One-way ANOVA with Tukey’s correction, mean ± SD, IgG n = 5, others n = 8. b Representative plot for H-2K^b MuLV p15E tetramer (KSP-tetramer) staining of MC38 tumour-specific CD8⁺ T cells in S.C. tumour samples. c, d Schematic for unactivated OT-I cell adoptive cell transfer (c) and absolute number of CD45.1⁺CD8⁺ OT-I T cells in indicated compartments of mice bearing MC38-Luc or MC38-OVA liver tumour (d). Analysed 14 days after tumour inoculation. Unpaired two-tailed Student’s t-test, mean ± SD, n = 10 per group. e, f Schematic (e) and quantification (f) of activated OT-I-cell distribution 2 days after adoptive cell transfer into mice bearing both subcutaneous and liver MC38-OVA tumour. Displayed as relative cell number to hepatic OT-I cell number. One-way ANOVA, *P = 0.0247, **P = 0.002, ***P = 0.0003, ****P < 0.0001, mean ± SD, n = 6 per group. g, h Representative flow plots (g) and quantification (h) of activated OT-I-cell distribution 4 days after adoptive cell transfer into MC38-OVA tumour bearing mice with MC38-Luc (n = 4) or MC38-OVA (n = 5) liver tumour (as shown in Fig. 3j); unpaired two-tailed Student’s t-test, mean ± SD. i, j Schema (i) and quantification (j) of activated OT-I-cell distribution 4 days after adoptive cell transfer into subcutaneous MC38-OVA tumour bearing mice with MC38-Luc lung tumour or MC38-OVA lung tumour. Displayed as relative cell number (normalized to MC38-Luc group). Unpaired two-tailed Student’s t-test, NS, not significant (P-value: liver, 0.37; lung, 0.42; S.C. tumour, 0.09; spleen, 0.91; tdLN, 0.74; cerLN, 0.26; liverLN, 0.22; lungLN, 0.45; blood, 0.36), mean ± SD, n = 5 per group. k Flow cytometry histogram depicting expression of LFA-1 (left) and CD44 (right) on in vitro activated OT-I cells. l Flow cytometry plots depicting expression of LFA-1 (left) and CD44 (right) expression on in vivo activated OT-I cells isolated from subcutaneous MC38-OVA tumour-draining LNs or non-draining LNs. Analysed 3 days after adoptive transfer. m Flow cytometry histogram depicting expression of LFA-1 (upper) and CD44 (bottom) on tumour specific KSP-tetramer⁺CD8⁺ cells (green) in the liver. n CFSE⁺CD8⁺OT-I cell number per gram liver tissue. OT-I cells were adoptively transferred 1-2 days after anti-ICAM-1 or HA-se. Analyzed 24 hours after transfer. One-way ANOVA, mean ± SD, control n = 5, HA-se n = 6, anti-ICAM-1 n = 6. o Pre-treatment immune cell subset blood counts of NSCLC patients receiving immunotherapy (Cohort 3) with (n = 62) or without (n = 189) liver metastases. Unpaired two-tailed Student’s t-test, box and whiskers, box represents mean and IQR, whisker represents 10-90%, outliers represent min to max. p Pre-treatment blood immune cell subset counts of NSCLC patients receiving immunotherapy (Cohort 3) with (n = 187) or without (n = 62) lung metastases. Unpaired two-tailed Student’s t-test, box and whiskers, box represents mean and IQR, whisker represents 10-90%, outliers represent min to max. Data are representative of at least two independent experiments (a-n).

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