immunology.sciencemag.org/cgi/content/full/5/52/eaba0759/DC1

Supplementary Materials for

Regulatory T cell control of systemic immunity and immunotherapy response in

liver metastasis

James C. Lee*, Sadaf Mehdizadeh, Jennifer Smith, Arabella Young, Ilgiz A. Mufazalov, Cody T. Mowery, Adil Daud, Jeffrey A. Bluestone*

*Corresponding author. Email: jeff.bluestone@ucsf.edu (J.A.B.); james.lee4@ucsf.edu (J.C.L.)

Published 2 October 2020, Sci. Immunol. 5, eaba0759 (2020)

DOI: 10.1126/sciimmunol.aba0759

The PDF file includes:

Fig. S1. Mice with experimental liver metastasis have a normal liver metabolic function. Fig. S2. Liver-mediated suppression of distant antitumor immunity requires adaptive immunity but not high tumor burden. Fig. S3. Activation markers are decreased on effector CD4+ and CD8+ T cells but increased on Tregs in mice with experimental liver metastasis. Fig. S4. The percentage and number of Tregs and tetramer+ CD8+ T cells are similar in distant tumors. Fig. S5. Experimental liver metastasis mediated suppression in the B16F10 tumor model. Fig. S6. KSP tetramer specifically stains MC38 tumor–targeted CD8+ T cells. Fig. S7. Suppression of distant CD8+ T cells is liver mediated and tumor antigen specific. Fig. S8. Foxp3-DTR mouse model schema. Fig. S9. DT induces profound Treg depletion in the blood and the SQ tumor of treated Foxp3-DTR mice. Fig. S10. Combination therapy with Treg-depleting anti–CTLA-4 and anti–PD-1 antibodies overcomes experimental liver metastasis immune suppression. Fig. S11. Clustering of tumor-infiltrating immune cell subsets using scRNA-seq. Fig. S12. Differential gene expression in distant MDSCs driven by the presence of liver tumor. Fig. S13. Liver tumor–mediated suppression is associated with distant increase in CD11b+ monocyte populations. Fig. S14. Increase in distant tolerogenic MDSCs is anatomically unique to liver tumor. Fig. S15. Treg or MDSC depletion can enhance tumor rejection in mice with experimental liver metastasis. Fig. S16. Treg-depleting versus nondepleting anti–CTLA-4 antibody in combination with anti–PD-1 treatment in experimental liver metastasis.

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/52/eaba0759/DC1)

Table S1. Raw data table (Excel file).

Fig. S1. Mice with experimental liver metastasis have a normal liver metabolic function. C57BL/6 mice were implanted with MC38 tumor cells and common clinical liver function tests, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, and albumin were performed on serum samples from day 14 post tumor injection. Data are shown as mean +/- s.e.m. pooled from two or more experiments.

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SQ SQ + Liver

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Fig. S2. Liver-mediated suppression of distant antitumor immunity requires adaptive immunity but not high tumor burden. (A) Total tumor weight of tumors dissected from mice sacrificed on day 20 post tumor implantation from indicated sites (I.P.= intra-peritoneum). (B) Subcutaneous tumor sizes on day 20 post tumor implantation from C57BL/6, Rag-1-/- , and NSG mice implanted with MC38 tumor cells (5 x 105) subcutaneously or subcutaneously and at the liver. Data are shown as mean +/- s.e.m. Asterisks indicating significance determined by unpaired t tests between groups are * p

Fig. S3. Activation markers are decreased on effector CD4+ and CD8+ T cells but increased on Tregs in mice with experimental liver metastasis. (A) Percentage of PD-1 and CTLA-4 positive and PD-1, CTLA-4, ICOS, Ki-67 positive subcutaneous CD8+ TILs. (B) PD-1, CTLA-4, and ICOS MFIs from Foxp3- CD4+ T cells in the subcutaneous tumor from mice with (black) and without (red) concurrent liver tumor and naïve CD4 T cells (blue) by flow cytometry. (C) Percentage of PD-1 and CTLA-4 positive and PD-1, CTLA-4, ICOS, Ki-67 positive on Tregs. Data are shown as mean +/- s.e.m., n=10 (B) and n=15 (A&C), pooled from two or more experiments. Asterisks indicating significance determined by unpaired t tests between groups are * p

Fig. S4. The percentage and number of Tregs and tetramer+ CD8+ T cells are similar in distant tumors. C57BL/6 mice re implanted ith MC38 tumor cells (5 x 105) subcutaneously (SQ) alone (black) or SQ plus in the liver (red) and TILs were harvested on day 14 post tumor implantation. (A) Percent of Foxp3+ Tregs of total CD4 T cells and (B) absolute Foxp3+ Treg count via flow cytometry. (C) Absolute KSP tetramer

+ CD8 T cell count from the SQ TILs of SQ alone (black) SQ plus liver (red) groups, and from the liver tumor TILs of the SQ plus liver group (purple). Data are shown as mean +/- s.e.m., n=8, pooled from two independent experiments.

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Fig. S5. Experimental liver metastasis mediated suppression in the B16F10 tumor model. C57BL/6 mice were implanted with B16F10 tumor cells (5 x 105) subcutaneously (black) or subcutaneously and at the liver (red). Tumor measurements on day 14 post tumor injection (n=5) as well as percentage of PD-1, CTLA-4, ICOS, Ki-67 positive and IFNγ+ subcutaneous CD8+ TILs analyzed by flow cytometry (n=10) are shown. Data are shown as mean +/- s.e.m., experiments performed twice with similar results. Asterisks indicating significance determined between groups are * p

Fig. S6. KSP tetramer specifically stains MC38 tumor–targeted CD8+ T cells. (A) Representative flow cytometric plot of KSPWFTTL- H-2Kb versus control tetramer staining on CD8+ T cells sampled from an implanted MC38 subcutaneous tumor. (B) KSP tetramer staining on CD4 versus CD8 T cells, tetramer staining on CD8 T cells from a B16 tumor sample. (C) KSP tetramer staining on CD8 T cells sampled from the tumor-draining lymph node of mice implanted with a B16F10 versus MC38 tumor.

Figure S6

SQ TILs

CD8

24.8 1.6

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KSPWFTTL KSPWFTTL CD8CD4

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Fig. S7. Suppression of distant CD8+ T cells is liver mediated and tumor antigen specific. (A) TILs were harvested on day 14 post tumor implantation. Percent of ICOS+ KSP tetramer+ CD8 T cells in the subcutaneous (SQ) tumor of SQ only, SQ plus lung, and SQ plus liver tumor groups as indicated. (B) Percent of cells for MC38 SQ only, MC38 SQ plus B16 liver, and MC38 SQ plus MC38 liver tumor groups. Representative data n=5 from one of two or more experiments are shown as mean +/- s.e.m. Asterisks indicating significance determined between groups are * p

Fig. S8. Foxp3-DTR mouse model schema.

Figure S8

Fig. S9. DT induces profound Treg depletion in the blood and the SQ tumor of treated Foxp3-DTR mice. TILs were harvested on day 14 post tumor implantation. Percent of Foxp3+ Tregs of total CD4 T cells in the blood (A) and subcutaneous tumor (B) of mice treated with DT or –DT (PBS) control as indicated. Data are shown as mean +/- s.e.m., n=10, representative data from one of three experiments. Asterisks indicating significance determined between groups are **** p

Fig. S10. Combination therapy with Treg-depleting anti–CTLA-4 and anti–PD-1 antibodies overcomes experimental liver metastasis immune suppression. (A) Representative whole mouse bioluminescent image of implanted MC38 tumors from 9H10 and 9H10 + anti-PD-1 treatment groups as described in Figure 4 are shown. (B) Representative liver images.

Figure S10

Day 20 post tumor injection

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Fig. S11. Clustering of tumor-infiltrating immune cell subsets using scRNA-seq. (A) Unbiased clustering (UMAP) of CD45+ subcutaneous (SQ) tumor-infiltrating cells identified by scRNAseq. Four panels on the right show select sample gene markers used to identify cell subsets (B) Reclustering (UMAP) of monocyte/myeloid cells in the SQ tumor showing distinct subclusters.

Figure S11

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Fig. S12. Differential gene expression in distant MDSCs driven by the presence of liver tumor. Heatmap displaying the top 20 differentially upregulated genes by subcutaneous tumor infiltrating MDSCs mice with or without concurrent liver tumor.

Figure S12SQ + Liver SQ

Cluster 6- MDSCs

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Fig. S13. Liver tumor–mediated suppression is associated with distant increase in CD11b+ monocyte populations. (A) Unbiased clustering (t-SNE) of subcutaneous (SQ) tumor infiltrating CD45+ immune cells from mice with or without concurrent liver tumor by flow cytometry. (B) C57BL/6 mice were implanted with B16F10-OVA tumor cells (5 x 105) at SQ (black) or SQ plus liver (red) sites. Day 14 SQ tumor sizes are shown. (C) Activated CD8 T cell to Treg ratio within the SQ tumor sample of SQ (black) or SQ plus liver (red) groups. (D) Percentage of OVA tumor-antigen (H-2Kb/SIINFEKL) specific CD11b monocytes within the SQ tumor sample of SQ (black) or SQ plus liver (red) groups. (B, C, and D) Data analyzed by unpaired t tests and shown as mean +/- s.e.m. n=5. Representative data is shown from 2 independent experiments. Asterisks indicating significance determined between groups are * p

Fig. S14. Increase in distant tolerogenic MDSCs is anatomically unique to liver tumor. (A) Percentage of CD11b+ cells in the subcutaneous (SQ) tumor of mice with concurrent kidney, intraperitoneal (IP), or liver tumor by flow cytometry. (B) ) MFI of CD80/86 from CD11b+ cells in the SQ tumor from mice with concurrent kidney, intraperitoneal (IP), or liver tumor. Representative data from 2 independent experiments. . Data analyzed by unpaired t tests, and shown as mean +/- s.e.m. Asterisks indicating significance determined between groups are **p

Fig. S15. Treg or MDSC depletion can enhance tumor rejection in mice with experimental liver metastasis. (A) Day 14 subcutaneous (SQ) tumor size from liver-tumor bearing mice that were treated with liposomal clodronate (CLL) (blue) or vehicle control (red). (B) Day 20 SQ tumor size from liver-tumor bearing mice treated with isotype control (red), 9D9 IgG2a (blue), or 9D9 IgG2b (black). CR= complete rejection with no measurable SQ tumor at endpoint. Representative data from 2 independent experiments. Data analyzed by unpaired t tests, and shown as mean +/- s.e.m. Asterisks indicating significance determined between groups are * p

Fig. S16. Treg-depleting versus nondepleting anti–CTLA-4 antibody in combination with anti–PD-1 treatment in experimental liver metastasis. (A) Representative whole mouse bioluminescent image of implanted MC38 tumors from isotype control, anti-CTLA-4 9D9 IgG2a + anti-PD-1, and anti-CTLA-4 9D9 IgG2b + anti-PD-1 treatments. (B) Day 20 subcutaneous (SQ) tumor size from liver-tumor bearing mice that were treated by the indicated combinations. N=5 for all groups. CR= complete rejection with no measurable SQ tumor at endpoint. Tumor growth curves were analyzed by two-way ANOVA with Sidak’s multiple comparisons, all others were analyzed by unpaired t tests. Asterisks indicating significance determined between groups are **p