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1 Supplementary Materials for Androgen receptor antagonists compromise T cell response against prostate cancer leading to early tumor relapse Yang Pu, Meng Xu, Yong Liang, Kaiting Yang, Yajun Guo, Xuanming Yang,* Yang-Xin Fu* *Corresponding author. (X.Y.); (Y.-X.F.) The PDF file includes: Published 6 April 216, Sci. Transl. Med. 8, 333ra47 (216) DOI: /scitranslmed.aad5659 Fig. S1. AR antagonists suppress various tumor immunotherapies in prostate cancer. Fig. S2. The immunosuppression mediated by flutamide is not mediated by liver toxicity and is independent of AR. Fig. S3. AR antagonists inhibit the immune response by suppressing T cells. Fig. S4. High doses of antiandrogens fail to induce apoptosis in activated T cells. Fig. S5. Antiandrogens inhibit human T cell activation through off-targeting GABA-A receptor as well.

2 A B Tumor Volume (mm3) 25 Ad-null Ad-LIGHT *** tumor volume(mm 3 ) MC38 *** Anti-PDL1 Isotype ctrl Anti-PDL1+Vehicle Anti-PDL1+Flutamide Days after castration Days after treatment Supplementary Figure 1. AR antagonists suppress various tumor immunotherapies in prostate cancer. (A) 3x1 6 Myc-Cap tumor cells were subcutaneously injected to male FVB mice. Castration was performed two weeks after tumor inoculation. Ad-null, Ad-LIGHT (1x1 1 VP) were intratumorally administrated on day 7, 4 and before castration. Tumor volume was recorded twice a week.administrated on day 7, 4 and before castration. ***P <.1; Tumor volume was recorded twice a week. (B) C57BL/6 mice were injected s.c. on day with MC38 cells. Mice received flutamide (6mg/kg) or vehicle from day 7 to day 25, and anti-pdl1 or isotype Ig were given on day 1,14 and day18.***p <.1; Tumor volume was recorded twice a week

3 A ALT/GPT Day 1 **** Uninfected CTR HSV-1 HSV-1+Flu without morbidity HSV+Flu with mobidity High dose Flu AST/GPO Day 1 *** Uninfected CTR HSV-1 HSV-1+Flu without morbidity HSV+Flu with mobidity High dose Flu B tumor volume(mm 3 ) Flutamide Vehicle Castration prior to flutamide ** days after tumor innoculation Supplementary Figure 2. The immune suppression mediated by flutamide is not mediated by liver toxicity and is independent of AR. (A) B6 mice infected intramuscularly with PFU of HSV-1 were treated with 6mg/kg or high dose (3mg/kg) of flutamide from day to day 12 daily,.plasma ALT and AST levels were measured at day 1. Data are mean ± SD (n = 4). Significantly different from h mice; ***P <.1. (B) Mice being injected s.c. 2x1 6 B16-EGFR tumor at day are treated with flutamide (6mg/kg) or vehicle from day to day 12. Castration was performed five days before tumor innoculation. The growth of tumor was measured and compared twice a week. Representative data are conducted with 5 mice per group; **P <.1.

4 IL4 only IL4+CD4 CTR IL4+CD4 Flutamide IL4+CD4 Enzalutamide IL4+CD4 Abiraterone Supplementary Figure 3. AR antagonists inhibit the immune response by suppressing T cells. CD19 + CD11C + B cells from WT B6 mice were sorted by FCS, labeled with CFSE and stimulated by 2ug/ml anti CD-4 and 5ng/ml IL4 for 96 hours. B cell proliferation was assessed by analyzing the dilution of CFSE in the same number of viable cells.

5 A vehicle flutamide 1uM flutamide 3uM 7-AAD B IFN pg/ml Annexin-V Vehicle Flutamide Enzalutamide Abiraterone Leuprolide IL2 IFN TNF Supplementary Figure 4. High doses of antiandrogens fail to induce apoptosis in activated T cells. (A) Splenocytes from OTI transgenic mice were stimulated with OTI peptide in the presence of different doses of flutamide for 24 hours.cells were labeled with 7-AAD and Annexin V and then analyzed on BD Fortessa Flow Cytometry The percentage of Annexin V-positive and 7-ADDnegative cells that were in early-stage apoptosis (bottom right) and Annexin V-positive and 7- ADD-positive cells that were dead or in end-stage apoptosis (top right), are presented in each quadrant. The percentage of Annexin V-positive cells are presented at the bottom of each chart. Representative data of dual staining for Annexin V-PE binding and 7-AAD uptake from 3 independent experiments. (B) T cells were purified from B6 WT mice splenocytes by Mouse T cell isolation kit from Stemcell Technologies and stimulated with PMA (2ng/ml) and 1ug/ml ionomycin in the presence of 1uM flutamide, 1uM enzalutamide,1um abiraterone and 1uM leuprolide. After 48 hours, the concentration of cytokines in supernatants was measured by BD CBA kit.

6 * IFN-r pg/ml * Vehicle Flutamide Flutamide+Bicuculline Enzalutamide Enzalutamide+Bicuculline 1 Supplementary Figure 5. Antiandrogens inhibit human T cell activation through offtargeting GABA-A receptor as well. Human T cells were stimulated with plate bounded anti-human CD3(1ug/ml) and soluble antihuman CD28(1ug/ml). At the same time, flutamide(25um), enzalutamide(1um),gaba-a receptor antagonists bicuculline were added to the culture as indicated. After 48hrs, the concentration of indicated IFN in supernatants was measured by BD CBA kit..*p <.5; One of three representative experiments is shown.

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