Extended Data Fig. 5: Hepatic myeloid cells induce activated T-cell apoptosis via the Fas/FasL pathway.

a Gating strategy for hepatic CD11b⁺F4/80⁺ cells. b Relative cell number of intrahepatic CD11b⁺F4/80⁺ following indicated treatment. Samples were analysed after two doses of anti-CSF-1 and clodronate liposome treatment. Data were normalized to control mice receiving PBS liposomes and IgG. One-way ANOVA, mean ± SD, PBS-lipo+IgG n = 9, Clo-lipo+IgG n = 10, PBS-lipo+anti-CSF-1 n = 9, Clo-lipo+anti-CSF-1 n = 11, S.C. n = 8. c Frequency of CD11b⁺F4/80⁺ cells (left), absolute number of CD11b⁺F4/80⁺ cells (middle) and ratio of CD11b⁺F4/80⁺ cells to CD8⁺ T cells (right) in the liver from mice bearing both MC38 subcutaneous tumour and liver tumour. Samples were collected after two doses of anti-CSF-1 and clodronate liposome treatment. Unpaired two-tailed Student’s t-test, mean ± SD, PBS-lipo+IgG n = 9, Clo-lipo+anti-CSF-1 n = 8. d Absolute number of intrahepatic dendritic cells following two doses of anti-CSF-1 and clodronate liposome treatment. Dendritic cells were gated as CD45⁺F4/80⁺CD11c⁺MHCII⁺ cells. Unpaired two-tailed Student’s t-test, mean ± SD, PBS-lipo+IgG n = 9, Clo-lipo+anti-CSF-1 n = 8. e Frequency of CD11b⁺F4/80⁺ cells (left), absolute number of CD11b⁺F4/80⁺ cells (middle) and ratio of CD11b⁺F4/80⁺ cells to CD8⁺ T cells (right) in the subcutaneous tumour from mice bearing both MC38 subcutaneous tumour and liver tumour. Samples were collected after two doses of anti-CSF-1 and clodronate liposome treatment. Unpaired two-tailed Student’s t-test, mean ± SD, n = 7 per group. f MC38 subcutaneous tumour growth in mice with only S.C. tumours treated with anti-PD-L1, clodronate liposome and anti-CSF-1, or the combination. Two-way ANOVA with Tukey’s correction, mean ± SD, n = 8 per group. g Schematic for clodronate liposome, anti-CSF-1, and OT-I adoptive transfer. h MC38 subcutaneous tumour growth in mice with S.C. and liver tumours treated with anti-PD-L1, clodronate liposome, anti-CSF-1, anti-CD8, or the combination. Two-way ANOVA with Tukey’s correction, mean ± SD, n = 6 per group. i Frequency of annexin V⁺7-AAD⁺ OT-I cells co-cultured in the presence of OVA peptide with hepatic F4/80⁺ cells isolated from liver tumour bearing mice at indicated ratios for 48 hours; Activated OT-I cells were labeled with CFSE before co-culture. One-way ANOVA with Dunnett’s multiple comparisons test, mean ± SD, n = 7 biologically independent samples. j Frequency of annexin V⁺7-AAD⁺ OT-I cells (CFSE labeled) after co-cultured in the presence of OVA peptide with hepatic F4/80⁺ cells in indicated conditions for 48 hours. One-way ANOVA with Tukey’s correction, mean ± SD, n = 3 biologically independent samples. k Flow cytometry histogram of Fas expression on hepatic OT-I (left) and KSP-tetramer⁺CD8⁺ T cells (right). Unactivated OT-I cells were transferred into mice bearing MC38-OVA subcutaneous tumour and liver tumour. Phenotype of transferred OT-I cells and endogenous KSP-tetramer⁺CD8⁺ T cells were analysed 12 days after adoptive transfer. l Frequency of annexin V⁺7-AAD⁺ OT-I cells co-cultured with MC38-OVA tumour cells and hepatic F4/80⁺ cells isolated from liver tumour bearing mice with and without TNFα blockade. Activated OT-I cells were labeled with CFSE before co-culture. Unpaired two-tailed Student’s t-test, mean ± SD, n = 4 biologically independent samples. m Quantification of H-2K^b-OVA mean fluorescent intensity (MFI) on hepatic CD11b⁺F4/80⁺ cells recovered from mice bearing subcutaneous MC38-OVA tumour with or without liver MC38-OVA tumour. Unpaired two-tailed Student’s t-test, mean ± SD, n = 5 per group. n, o Quantification of FasL (n) and H-2K^b (o) MFI on lung CD11b⁺F4/80⁺ cells recovered from mice bearing subcutaneous MC38 tumour with (n = 9) or without (n = 6) lung MC38 tumour, in comparison with hepatic CD11b⁺F4/80⁺ cells recovered from mice bearing subcutaneous MC38 tumour with (n = 9) or without (n = 6) liver MC38 tumour. Tissues were collected 10 days after tumour inoculation. One-way ANOVA, mean ± SD. Data are representative of at least two independent experiments (b-o).

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