Journal: Cancer cell

Article Title: Tumor-Repopulating Cells Induce PD-1 Expression in CD8 + T Cells by Transferring Kynurenine and AhR Activation.

doi: 10.1016/j.ccell.2018.02.005

Figure Lengend Snippet: Figure 3. IFN-g Upregulates the Expression of IDO1 and SLC1A5 to Boost Kyn Production (A) The mRNA expression of IDO1 in bulk cells (B16, A375, or HepG2) or TRCs treated with PBS or IFN-g (10 ng/mL) for 24 hr. (B) OVA-B16 TRCs were co-cultured without or with anti-CD3/CD28 bead-activated OT-1 cells for 24 hr. The expression of Ido1 was determined by qPCR. (C and D) Ido1 shRNA-(Sh1 or Sh2)-OVA B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 T cells. After 4 hr, TRC apoptosis (C) or after 24 hr, percentage of PD-1+CD8+ cells (D) was analyzed by flow cytometry. Iso, isotype control. (E) OVA-B16, A375, or HepG2 bulk cells or TRCs were treated with PBS or IFN-g (10 ng/mL) for 24 hr. The expression of SLC1A5, SLC7A5, or SLC7A8 was determined by qPCR. (F) Tryptophan (Trp) levels were determined by ELISA in cell lysates of Slc1a5 shRNA (Sh1 or Sh2)-B16 TRCs treated with or without IFN-g (10 ng/mL) for 24 hr. (G and H) Slc1a5 shRNA (Sh1 or Sh2)-OVA B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 cells. After 4 hr, TRC apoptosis (G) or after 24 hr, percentage of PD-1+CD8+ cells (H) was analyzed by flow cytometry. Iso, isotype control. (I) B16 bulk cells were transfected with vector or Flag-SLC1A5 plasmid. SLC1A5 expression and tryptophan consumption were determined by western blot (left) and ELISA (right). (J) B16 bulk cells expressing vector or SLC1A5 were treated with PBS or IFN-g (10 ng/mL) for 24 hr. Kyn levels in supernatants were determined by ELISA. *p < 0.05, **p < 0.01, by one-way ANOVA (A, C–H, and J) or Student’s t test (B and I). The data represent means ± SEM of three independent experiments. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cell Activation Cocktail (with Brefeldin A) BioLegend Cat#423303 Murine lymphocyte separation kit Solarbio Cat#P8620 Ficoll-PaqueTM PLUS GE Healthcare Life Sciences Cat#17-1440-02 BCA Kit Applygen Technologies Cat#P1511 Experimental Models: Cell Lines Mouse H22 China Infrastructure of Cell Line Resources Cat#3142C0001000000110 Mouse B16F1 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000365 Mouse B16-OVA Dr. Shengdian Wang Lab (Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) N/A Mouse ID8 Dr. Weiming Deng Lab(Tianjin Medical University) N/A Human A375 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000126 Human HepG2 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000035 Human Plat-E cell Cell Biolabs Cat#RV-101 Human HEK293T Clontech Cat#632180 Experimental Models: Organisms/Strains Mouse: OT-1 transgenic mice Dr. Hui Zhang Lab (Sun Yat-Sen University) N/A Mouse: Pmel-1 transgenic mice Dr. Ying Wan Lab (Third Military Medical University) N/A Mouse: Ahr–/– (C57BL/6) mice Dr. Jun Yan Lab (Third Military Medical University) N/A Mouse: MMTV-PyMT mice Dr. Bing Cui Lab (Chinese Academy of Medical Sciences) N/A Mouse: C57BL/6 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: C57B/6J CD45.1 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: BALB/c Mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Oligonucleotides Primers for qPCR, see Table S4 Tsingke Biological Technology N/A Primers for Chip, see Table S5 Tsingke Biological Technology N/A Recombinant DNA pGL3 Promega E1751 pGL3-PD-1 This paper N/A pGL3-PAT4 This paper N/A pGL3-SLC7A8 This paper N/A pRL-SV40 (Renilla vector) Promega E2231 pCMV6-Entry Origene PS100001 pCMV6-AHR Origene MR227590 pCMV6- NFATc1 Origene MR210848 pCMV6- SLC1A5 Origene RC200305 psPAX2 Addgene Cat#12260 pMD2.G Addgene Cat#12259 Slc36a4 Mouse shRNA Origene TG507107 Slc7a8 Mouse shRNA Origene TG502853 (Continued on next page) Cancer Cell 33, 480–494.e1–e7, March 12, 2018 e3

Techniques: Expressing, Cell Culture, shRNA, Cytometry, Control, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Cancer cell

Article Title: Tumor-Repopulating Cells Induce PD-1 Expression in CD8 + T Cells by Transferring Kynurenine and AhR Activation.

doi: 10.1016/j.ccell.2018.02.005

Figure Lengend Snippet: Figure 5. AhR Upregulates SLC7A8 and PAT4 Expression for Kyn Transport (A) Resting or anti-CD3/CD28 bead-activated CD8+ T cells (1 3 104 cells) from spleens of C57BL/6 mice were lysed and Kyn levels were analyzed by ELISA. (B) Anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice were treated with PBS or Kyn (200 mM) for 24 hr. Pat4 and Slc7a8 were determined by qPCR (left) and immunostaining (right). Bar, 10 mm. (C and D) Scramble shRNA (Scr) or shRNAs against Slc7a8 and Pat4 was delivered to anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice for 24 hr, and then the cells were treated with Kyn (200 mM) for 48 hr. Kyn consumption was determined by ELISA (C), and the percentage of PD-1+ CTLs was analyzed by flow cytometry (D). Iso, isotype control. (E) OVA-B16 TRCs were co-cultured with anti-CD3/CD28 bead-activated OT-1 cells transfected with scramble shRNA (Scr), Pat4 shRNAs (Sh1 and Sh2), or Slc7a8 shRNAs (Sh1 and Sh2). After 4 hr, TRC apoptosis was analyzed by flow cytometry. (F) Pat4 and Slc7a8 mRNA expression was determined by qPCR in anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice treated with PBS, Kyn (200 mM), Kyn/CH223191 (10 mM), or Kyn/DMF (20 mM) for 48 hr. (G) Resting or anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice were treated with PBS or Kyn (200 mM) for 48 hr. ChIP-qPCR analysis was performed with AhR antibody and Slc7a8 or Pat4 promotor-specific primers. Data are presented as relative to the resting/PBS group. (H) HEK293T cells expressing Slc7a8 or Pat4 promotor-luciferase reporter PGL3 were co-transfected with AhR. Cells were treated with Kyn (200 mM) for 48 hr and analyzed by luciferase assay. **p < 0.01, by Student’s t test (B and G) or one-way ANOVA (C–F and H). The data represent means ± SEM of three independent experiments. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cell Activation Cocktail (with Brefeldin A) BioLegend Cat#423303 Murine lymphocyte separation kit Solarbio Cat#P8620 Ficoll-PaqueTM PLUS GE Healthcare Life Sciences Cat#17-1440-02 BCA Kit Applygen Technologies Cat#P1511 Experimental Models: Cell Lines Mouse H22 China Infrastructure of Cell Line Resources Cat#3142C0001000000110 Mouse B16F1 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000365 Mouse B16-OVA Dr. Shengdian Wang Lab (Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) N/A Mouse ID8 Dr. Weiming Deng Lab(Tianjin Medical University) N/A Human A375 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000126 Human HepG2 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000035 Human Plat-E cell Cell Biolabs Cat#RV-101 Human HEK293T Clontech Cat#632180 Experimental Models: Organisms/Strains Mouse: OT-1 transgenic mice Dr. Hui Zhang Lab (Sun Yat-Sen University) N/A Mouse: Pmel-1 transgenic mice Dr. Ying Wan Lab (Third Military Medical University) N/A Mouse: Ahr–/– (C57BL/6) mice Dr. Jun Yan Lab (Third Military Medical University) N/A Mouse: MMTV-PyMT mice Dr. Bing Cui Lab (Chinese Academy of Medical Sciences) N/A Mouse: C57BL/6 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: C57B/6J CD45.1 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: BALB/c Mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Oligonucleotides Primers for qPCR, see Table S4 Tsingke Biological Technology N/A Primers for Chip, see Table S5 Tsingke Biological Technology N/A Recombinant DNA pGL3 Promega E1751 pGL3-PD-1 This paper N/A pGL3-PAT4 This paper N/A pGL3-SLC7A8 This paper N/A pRL-SV40 (Renilla vector) Promega E2231 pCMV6-Entry Origene PS100001 pCMV6-AHR Origene MR227590 pCMV6- NFATc1 Origene MR210848 pCMV6- SLC1A5 Origene RC200305 psPAX2 Addgene Cat#12260 pMD2.G Addgene Cat#12259 Slc36a4 Mouse shRNA Origene TG507107 Slc7a8 Mouse shRNA Origene TG502853 (Continued on next page) Cancer Cell 33, 480–494.e1–e7, March 12, 2018 e3

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Immunostaining, shRNA, Cytometry, Control, Cell Culture, Transfection, ChIP-qPCR, Luciferase

Journal: Cancer cell

Article Title: Tumor-Repopulating Cells Induce PD-1 Expression in CD8 + T Cells by Transferring Kynurenine and AhR Activation.

doi: 10.1016/j.ccell.2018.02.005

Figure Lengend Snippet: Figure 6. TCR Signaling Facilitates Kyn-induced PD-1 Upregulation (A) Splenic CD8+ T cells from C57BL/6 mice were treated with Kyn (200 mM) for the indicated time. The percentage of PD-1+ cells was determined by flow cytometry. Iso, isotype control. (B) Flow cytometry analysis for percentage of PD-1+ resting or anti-CD3/CD28 bead-activated Ahr/ CD8+ T cells. (C) Percentage of PD-1+ cells in resting and anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice at indicated time points was determined by flow cytometry. (D) Splenic CD8+ T cells from C57BL/6 mice were treated with PBS or Kyn for 24 hr. Some cells were pretreated with anti-CD3/CD28 beads for 4 hr, and then treated with Kyn (Pre/Kyn) for 24 hr. Percentage of PD-1+ cells was determined by flow cytometry. Iso, isotype control. (E) Slc7a8 and Pat4 mRNA levels measured by qPCR in resting or anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice. (F) Anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice were treated with 1 mM CsA for 48 hr. Pat4 and Slc7a8 mRNA was determined. (G) Anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice were treated with Kyn (200 mM) and CsA (1 mM) for 48 hr. Kyn levels in cell lysates were determined by ELISA. (H) Resting or anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice were treated with PBS, Kyn (200 mM), CsA (1 mM), or Kyn/CsA for 48 hr. Percentage of PD-1+ cells was determined by flow cytometry. Iso, isotype control. (I) ChIP-qPCR analysis of resting or anti-CD3/CD28 bead-activated CD8+ T cells from spleens of C57BL/6 mice with NFATc1 antibody and Pat4 or Slc7a8 promoter primers. (J) Splenic antigen-presenting cells from C57BL/6 mice pulsed with OVA257-264 (2 mg/mL) were co-cultured with Kyn (200 mM)-treated (right) or untreated (left) OT-1 cells in the presence of immunoglobulin G (IgG) or PD-1 (10 mg/mL) neutralizing antibody for 72 hr. IFN-g expression was determined by flow cytometry. **p < 0.01, by Student’s t test (A and E–G) or one-way ANOVA (D and H–J). The data represent means ± SEM of three independent experiments. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cell Activation Cocktail (with Brefeldin A) BioLegend Cat#423303 Murine lymphocyte separation kit Solarbio Cat#P8620 Ficoll-PaqueTM PLUS GE Healthcare Life Sciences Cat#17-1440-02 BCA Kit Applygen Technologies Cat#P1511 Experimental Models: Cell Lines Mouse H22 China Infrastructure of Cell Line Resources Cat#3142C0001000000110 Mouse B16F1 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000365 Mouse B16-OVA Dr. Shengdian Wang Lab (Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) N/A Mouse ID8 Dr. Weiming Deng Lab(Tianjin Medical University) N/A Human A375 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000126 Human HepG2 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000035 Human Plat-E cell Cell Biolabs Cat#RV-101 Human HEK293T Clontech Cat#632180 Experimental Models: Organisms/Strains Mouse: OT-1 transgenic mice Dr. Hui Zhang Lab (Sun Yat-Sen University) N/A Mouse: Pmel-1 transgenic mice Dr. Ying Wan Lab (Third Military Medical University) N/A Mouse: Ahr–/– (C57BL/6) mice Dr. Jun Yan Lab (Third Military Medical University) N/A Mouse: MMTV-PyMT mice Dr. Bing Cui Lab (Chinese Academy of Medical Sciences) N/A Mouse: C57BL/6 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: C57B/6J CD45.1 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: BALB/c Mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Oligonucleotides Primers for qPCR, see Table S4 Tsingke Biological Technology N/A Primers for Chip, see Table S5 Tsingke Biological Technology N/A Recombinant DNA pGL3 Promega E1751 pGL3-PD-1 This paper N/A pGL3-PAT4 This paper N/A pGL3-SLC7A8 This paper N/A pRL-SV40 (Renilla vector) Promega E2231 pCMV6-Entry Origene PS100001 pCMV6-AHR Origene MR227590 pCMV6- NFATc1 Origene MR210848 pCMV6- SLC1A5 Origene RC200305 psPAX2 Addgene Cat#12260 pMD2.G Addgene Cat#12259 Slc36a4 Mouse shRNA Origene TG507107 Slc7a8 Mouse shRNA Origene TG502853 (Continued on next page) Cancer Cell 33, 480–494.e1–e7, March 12, 2018 e3

Techniques: Cytometry, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, ChIP-qPCR, Cell Culture, Expressing

Journal: Cancer cell

Article Title: Tumor-Repopulating Cells Induce PD-1 Expression in CD8 + T Cells by Transferring Kynurenine and AhR Activation.

doi: 10.1016/j.ccell.2018.02.005

Figure Lengend Snippet: Figure 7. Kyn Upregulates PD-1 Expression In Vivo via AhR (A–C) 50 mg Kyn was injected intraperitoneally (i.p.) into mice once per day for 5 days (n = 5). The percentage of PD-1+ cells in splenic and mesenteric CD8+ T cells was determined by flow cytometry (A). Splenic CD8+ T cells were immunostained for AhR and DAPI; bar, 10 mm (B). Pat4 and Slc7a8 mRNAs were determined by qPCR (C). (D) 50 mg Kyn was injected i.p. into Ahr/ mice once per day for 5 days (n = 5). The percentage of PD-1+ cells in splenic and mesenteric CD8+ T cells was determined by flow cytometry. (E) The percentage of PD-1+ cells was analyzed by flow cytometry in resting or anti-CD3/CD28 bead-activated CD8+ T cells from the Ahr/ mice with Kyn treatment from (D) (n = 5). (F) Ahr/ mice were injected with anti-CD3 antibody (5 mg, i.p.) and percentage of PD-1+ CD8+ splenic T cells was determined by flow cytometry (n = 5). (G) CD3 antibody-pretreated mice were treated with Kyn (50 mg/mouse) (n = 5). 48 hr later, the percentage of PD-1+ CD8+ splenic T cells was determined by flow cytometry. (H) C57BL/6 mice bearing 5 3 5 mm OVA-B16 melanoma were adoptively transferred with CFSE-labeled OT-1 cells (4 3 106/mouse, every 3 days) on three occasions (n = 5). At the same time, mice were treated with either PBS or Kyn (50 mg/mouse, once per day) for 9 days. CFSE+ CD8+ T cells were isolated from tumor for flow-cytometric analysis of the percentage of PD-1+ cells.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cell Activation Cocktail (with Brefeldin A) BioLegend Cat#423303 Murine lymphocyte separation kit Solarbio Cat#P8620 Ficoll-PaqueTM PLUS GE Healthcare Life Sciences Cat#17-1440-02 BCA Kit Applygen Technologies Cat#P1511 Experimental Models: Cell Lines Mouse H22 China Infrastructure of Cell Line Resources Cat#3142C0001000000110 Mouse B16F1 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000365 Mouse B16-OVA Dr. Shengdian Wang Lab (Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) N/A Mouse ID8 Dr. Weiming Deng Lab(Tianjin Medical University) N/A Human A375 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000126 Human HepG2 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000035 Human Plat-E cell Cell Biolabs Cat#RV-101 Human HEK293T Clontech Cat#632180 Experimental Models: Organisms/Strains Mouse: OT-1 transgenic mice Dr. Hui Zhang Lab (Sun Yat-Sen University) N/A Mouse: Pmel-1 transgenic mice Dr. Ying Wan Lab (Third Military Medical University) N/A Mouse: Ahr–/– (C57BL/6) mice Dr. Jun Yan Lab (Third Military Medical University) N/A Mouse: MMTV-PyMT mice Dr. Bing Cui Lab (Chinese Academy of Medical Sciences) N/A Mouse: C57BL/6 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: C57B/6J CD45.1 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: BALB/c Mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Oligonucleotides Primers for qPCR, see Table S4 Tsingke Biological Technology N/A Primers for Chip, see Table S5 Tsingke Biological Technology N/A Recombinant DNA pGL3 Promega E1751 pGL3-PD-1 This paper N/A pGL3-PAT4 This paper N/A pGL3-SLC7A8 This paper N/A pRL-SV40 (Renilla vector) Promega E2231 pCMV6-Entry Origene PS100001 pCMV6-AHR Origene MR227590 pCMV6- NFATc1 Origene MR210848 pCMV6- SLC1A5 Origene RC200305 psPAX2 Addgene Cat#12260 pMD2.G Addgene Cat#12259 Slc36a4 Mouse shRNA Origene TG507107 Slc7a8 Mouse shRNA Origene TG502853 (Continued on next page) Cancer Cell 33, 480–494.e1–e7, March 12, 2018 e3

Techniques: Expressing, In Vivo, Injection, Cytometry, Labeling, Isolation

Journal: Cancer cell

Article Title: Tumor-Repopulating Cells Induce PD-1 Expression in CD8 + T Cells by Transferring Kynurenine and AhR Activation.

doi: 10.1016/j.ccell.2018.02.005

Figure Lengend Snippet: Figure 8. Kyn-AhR Regulates PD-1 in Patients (A) The percentage of PD-1+CD8+ T cells from peripheral blood of healthy volunteers (n = 50) or patients with breast (n = 84), colon (n = 28), or lung (n = 15) cancer, or from breast (n = 9) or colon (n = 17) tumor tissues. Iso, isotype control. (B) Kyn levels measured by ELISA in plasma from healthy volunteers (n = 31) and patients with breast (n = 73), lung (n = 10), or colon (n = 26) cancer, or in lysates of fresh human breast (n = 11) or colon (n = 8) tumor tissues and the adjacent normal tissues. (C) Correlation between PD-1 expression and Kyn levels in healthy people (left, n = 37) and breast cancer patients (right, n = 62). (D) Peripheral CD8+ T cells from patients with breast (n = 8) or lung cancer (n = 8) were treated with anti-CD3/CD28 beads plus Kyn (200 mM) or Kyn/DMF (20 mM) for 48 hr. The percentage of PD-1+ CD8+ T cells was determined by flow cytometry. Iso, isotype control. (E) CD8+ T cells from breast (n = 6) or lung cancer (n = 6) patients were treated with DMF (20 mM) for 48 hr. PD-1 expression was determined. Iso, isotype control. (F and G) PD-1high CD8+ or PD-1dim CD8+ T cells were sorted from peripheral blood mononuclear cells of healthy donors (n = 8) or patients with breast (n = 8) or lung (n = 8) cancer. Real-time PCR-analyzed AHR mRNA is shown (F). Cells were also immunostained with anti-AhR and DAPI (G). Bar, 10 mm. (H) CD8+ T cells from healthy donors (n = 6) or breast cancer (n = 6) patients were subjected to ChIP-qPCR analysis of AhR binding to the PD-1 promotor. Data are presented as relative to the healthy donor group. Neg, negative control. (I) CD8+ T cells from breast cancer patients (n = 6) were treated with PBS, Kyn (200 mM), Kyn/DMF (20 mM), or Kyn/CH223191 (50 mM) for 48 hr. PAT4 and SLC7A8 mRNAs were determined by qPCR. Data are presented as relative to PBS-treated cells. (J) PAT4 or SLC7A8 expression in PD-1high and PD-1dim CD8+ T cells from breast (n = 8) or lung (n = 8) cancer patients determined by qPCR. (K) ChIP-qPCR analysis of PD-1high or PD-1dim CD8+ T cells isolated from breast cancer patients (n = 6) with anti-AhR and SLC7A8 or PAT4 promotor primers. Data are presented as relative to the PD-1dim group. **p < 0.01, ***p < 0.001, by one-way ANOVA (A, B, D, H, I, and K), Student’s t test (E–G and J), or Pearson’s correlation test (C). The data represent means ± SEM. See also Figure S8 and Tables S1, S2, and S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Cell Activation Cocktail (with Brefeldin A) BioLegend Cat#423303 Murine lymphocyte separation kit Solarbio Cat#P8620 Ficoll-PaqueTM PLUS GE Healthcare Life Sciences Cat#17-1440-02 BCA Kit Applygen Technologies Cat#P1511 Experimental Models: Cell Lines Mouse H22 China Infrastructure of Cell Line Resources Cat#3142C0001000000110 Mouse B16F1 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000365 Mouse B16-OVA Dr. Shengdian Wang Lab (Key Laboratory of Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) N/A Mouse ID8 Dr. Weiming Deng Lab(Tianjin Medical University) N/A Human A375 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000126 Human HepG2 China Infrastructure of Cell Line Resources Cat#3111C0001CCC000035 Human Plat-E cell Cell Biolabs Cat#RV-101 Human HEK293T Clontech Cat#632180 Experimental Models: Organisms/Strains Mouse: OT-1 transgenic mice Dr. Hui Zhang Lab (Sun Yat-Sen University) N/A Mouse: Pmel-1 transgenic mice Dr. Ying Wan Lab (Third Military Medical University) N/A Mouse: Ahr–/– (C57BL/6) mice Dr. Jun Yan Lab (Third Military Medical University) N/A Mouse: MMTV-PyMT mice Dr. Bing Cui Lab (Chinese Academy of Medical Sciences) N/A Mouse: C57BL/6 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: C57B/6J CD45.1 mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Mouse: BALB/c Mice Center of Medical Experimental Animals of Chinese Academy of Medical Science N/A Oligonucleotides Primers for qPCR, see Table S4 Tsingke Biological Technology N/A Primers for Chip, see Table S5 Tsingke Biological Technology N/A Recombinant DNA pGL3 Promega E1751 pGL3-PD-1 This paper N/A pGL3-PAT4 This paper N/A pGL3-SLC7A8 This paper N/A pRL-SV40 (Renilla vector) Promega E2231 pCMV6-Entry Origene PS100001 pCMV6-AHR Origene MR227590 pCMV6- NFATc1 Origene MR210848 pCMV6- SLC1A5 Origene RC200305 psPAX2 Addgene Cat#12260 pMD2.G Addgene Cat#12259 Slc36a4 Mouse shRNA Origene TG507107 Slc7a8 Mouse shRNA Origene TG502853 (Continued on next page) Cancer Cell 33, 480–494.e1–e7, March 12, 2018 e3

Techniques: Control, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing, Cytometry, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Negative Control, Isolation