(A) Increased survival of CD73 KO mice challenged with ID8 ovarian tumors is T cell dependent. Female C57BL/6 WT, CD73 KO, or CD73 KO mice depleted of CD8⁺ T cells by anti-CD8 mAb 53.6.7 were inoculated i.p. with 1 × 10⁷ ID8 cells (n = 6). (B and C) Suppression of tumor growth in CD73 KO mice. Mice (n = 3 or 5 per group) were inoculated with 10⁶ EG7 (B) or B16-SIY (C) cells. White symbols, CD73 KO; black symbols, WT; each symbol represents 1 tumor-bearing mouse. (D and E) In CD73 KO mice (n = 5 per group), depletion of CD4⁺ T cells, CD8⁺ T cells, or NK cells was achieved by twice-weekly i.p. injection of control Ig or anti-CD4, anti-CD8, or anti–asialo GM1 depleting Abs, respectively, beginning 1 day prior to EG7 (D) or B16-SIY (E) challenge. (F) Survival of WT or CD73 KO mice (n = 5–8 per group) after i.p. inoculation with 10⁷ ID8-OVA-SiCD73 (CD73-silenced) or ID8-OVA-SiNS (CD73-nonsilenced control) tumor cells. (G and H) Tumor growth was enhanced by both BM-derived and non–BM-derived host cells expressing CD73. Chimeric mice (n = 5 per group), generated by BM reconstitution, were injected s.c. with 10⁶ EG7 (G) or B16-SIY (H) cells. Data (mean ± SD) are representative of 3 independent experiments (A and F), at least 10 independent experiments (B and C), or 2 (G and H) independent experiments. *P < 0.05 vs. all other groups or as denoted by brackets.

(A) Representative flow cytometric analyses of antitumor T cells in spleen from EG7-bearing WT or CD73 KO mice 14 days after tumor inoculation. (B) The H-2K^b-OVA/SIINFEKL pentamer was used to track OVA-specific CD8⁺ T cells. Pentamer⁺CD8⁺ and CD8⁺IFN-γ⁺ T cell frequency in response to SIINFEKL peptide is summarized (n = 5). (C) The H-2K^b-SIYRYYGL-IgG dimer was used to track SIY-specific CD8⁺ T cells in spleen from B16SIY-tumor bearing WT or CD73 KO mice 14 days after tumor inoculation. Dimer⁺CD8⁺ and CD8⁺IFN-γ⁺ cell frequency in response to SIYRYYGL peptide is summarized (n = 5). (D) Representative flow cytometric analyses of in vivo antigen-specific killing capacity of antitumor T cells from EL4- or EG7-bearing WT and CD73 KO mice. Equal numbers (10⁷ cells each) of CFSE^hi SIINFEKL peptide-pulsed and CFSE^lo non–peptide-pulsed WT splenocytes were adoptively transferred into tumor-bearing mice. Numbers denote percentage of SIINFEKL peptide-pulsed target cell killing in spleen, calculated as described in Methods. (E) Percent killing for EG7-bearing mice (n = 5). (F) Percent killing of adoptively transferred SIYRYYGL peptide-pulsed splenocytes from parallel experiments involving B16-SIY–bearing mice (n = 5). Data (mean ± SD) are representative of 3 independent experiments. *P < 0.05.

(A) Percent CD31⁺CD45^– TECs in B16-SIY tumors (n = 5). (B) TECs in B16-SIY tumors expressed CD73. (C) Expression of the adhesion molecules ICAM-1, VCAM-1, and P-selectin on TECs from B16-SIY–bearing mice (n = 5). Data are representative of 2 independent experiments. *P < 0.05.

(A and B) Purified OT-I CD8⁺CD90.1⁺ T cells (2 × 10⁶) were injected i.v. into WT mice (n = 3 per group) with or without 10⁶ splenic CD4⁺CD25⁺ Tregs derived from EG7-bearing WT or CD73 KO mice. The next day, 10⁶ EG7 cells were injected s.c. into the T cell recipients. 3 days later, percent transferred T cells in spleen and DLN (A) and percent IFN-γ⁺ cells among the transferred cells in spleen (B) were determined by flow cytometry (n = 3). (C) For CD25⁺ T cell depletion, splenic T cells were further purified by negative selection using magnetic bead–conjugated anti-CD25 Ab. Rag1^–/– mice (n = 5 per group) were challenged with 10⁶ B16-SIY cells 24 hours after receiving 10⁷ total T cells or CD25-depleted T cells from naive WT or CD73 KO mice. (D) Treg depletion using DT inhibited tumor growth. WT or CD73 KO mice (n = 5 per group) were challenged with 10⁶ B16-SIY cells. 3 days later, mice were treated with DT (2 μg/mouse twice weekly). (E) Adoptive transfer of WT Tregs, but not CD73 KO Tregs, reversed the tumor-inhibiting advantage of host CD73 deficiency. CD4⁺CD25⁺ Tregs derived from WT or CD73 KO mice were injected i.v. into CD73 KO mice. The next day, 10⁶ B16-SIY cells were challenged. Data (mean ± SD) are representative of 3 (A and B) or 2 (C–E) independent experiments. *P < 0.05; **P < 0.01.

(A) Representative flow cytometric analysis of tumor-infiltrating immune cells from EG7-bearing WT or CD73 KO mice collected 14 days after tumor inoculation. (B and C) Percent CD4⁺TCRVβ⁺, CD8⁺TCRVβ⁺, Gr1⁺CD11b⁺, and CD49b⁺NK1.1⁺ cells in tumor infiltrates of WT or CD73 KO mice collected 14 days after inoculation with EG7 (B) or B16-SIY (C) tumor cells (n = 5). (D) Frequency of pentamer⁺CD8⁺ cells specific for the OVA epitope SIINFEKL in infiltrates from mice in B, as determined by flow cytometry (n = 5). (E) Frequency of dimer⁺CD8⁺ cells specific for the epitope SIYRYYGL in infiltrates from mice in C, as determined by flow cytometry (n = 5). (F) Representative immunohistochemical staining using anti-CD8 mAb on EG7 tumor tissue sections obtained from A. Original magnification, ×10 (top and middle); ×40 (bottom). (G) Representative dot plots of Foxp3 expression in B16-SIY tumor-infiltrating CD4⁺ cells. Percent Foxp3⁺ cells is indicated within plots and summarized (n = 3). Data (mean ± SD) are representative of 3 independent experiments. *P < 0.05; **P < 0.01.

The H-2K^b-OVA/SIINFEKL pentamer was used to stain OVA-specific CD8⁺ T cells in spleens of EG7 tumor–bearing WT and CD73 KO chimeric mice 14 days after tumor inoculation. (A) Frequency of pentamer⁺CD8⁺ cells (n = 5). (B) Percent in vivo OVA-specific killing (n = 5). (C and D) Frequency of pentamer⁺CD8⁺ (C) and CD8⁺IFN-γ⁺ (D) cells in tumor infiltrates, as determined by flow cytometry (n = 5). Data are representative of 2 independent experiments.*P < 0.05.

Activated OT-I CD90.1 cells were labeled with CFSE and adoptively transferred into EG7-tumor bearing WT or CD73 KO mice. (A) Representative flow cytometric analyses of OT-I cells in the spleen, DLN, and EG7 tumor 24 hours after transfer. (B) Frequency and absolute number of CFSE⁺CD8⁺ OT-I cells among different tissues from EG7-bearing mice 24 hours after T cell transfer (n = 5). (C) Frequency and absolute number of CFSE⁺CD8⁺ 2C cells among different tissues from B16-SIY–bearing mice 24 hours after T cell transfer (n = 5). (D) Frequency and absolute number of CFSE⁺CD8⁺ OT-I cells among tumor tissues from EG7-bearing WT mice treated as indicated and assessed 24 hours after T cell transfer (n = 5). (E) Frequency and absolute number of CFSE⁺CD8⁺ 2C cells among tumor tissues from B16-SIY–bearing WT mice treated as indicated and assessed 24 hours after T cell transfer (n = 5). Data (mean ± SD) are representative of 3 independent experiments. *P < 0.05; **P < 0.01.

(A and B) WT (A) or CD73 KO (B) mice (n = 5 per group) were challenged s.c. with 10⁶ B16-SIY cells. 1 week later, mice were treated with APCP (20 mg/kg i.v.) once daily for 1 week, then twice weekly, and tumor volume was measured. (C and D) WT mice (n = 5 per group) were s.c. challenged with 10⁶ B16-SIY cells. 10 days later, mice were treated with either APCP as described above (C) or 100 μg TY/23 (D) 2 times weekly and adoptively transferred with 5 × 10⁶ 2C T cells 13 days after tumor challenge as indicated. Tumor volumes were measured at the indicated times. (E) Female WT mice were inoculated i.p. with 10⁷ ID8 cells. 1 week later, tumor-bearing mice were left untreated or transferred with 10⁷ ID8-reactive T cells i.p. and/or treated with TY/23 as described above. Survival of the mice (n = 5–8 per group) was measured. Data are representative of 2 independent experiments. *P < 0.05; **P < 0.01.