CD73 on tumor cells impairs antitumor T-cell responses: a novel mechanism of tumor-induced immune suppression

Abstract

CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a cosignaling molecule on T lymphocytes and an adhesion molecule that is required for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and is upregulated in cancerous tissues. Because the ecto-5'-nucleotidase activity of CD73 catalyzes AMP breakdown to immunosuppressive adenosine, we hypothesized that CD73-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that combining tumor CD73 knockdown and tumor-specific T-cell transfer cured all tumor-bearing mice. In striking contrast, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. Moreover, blockade of the A2A adenosine receptor with a selective antagonist also augmented the efficacy of adoptive T-cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting the enzymatic activity of tumor CD73.

Figure 1. Adenosine generated by tumor CD73 suppresses T cell proliferation

(a) CD73 expression on various ID8 ovarian cancer cells was analyzed by flow cytometry (dashed line, unstained/ solid line, stained). The efficiency of CD73 knockdown was confirmed by the measurement of CD73 enzymatic activity. (b) Adenosine directly suppressed T cell proliferation. Data are mean ± SD of triplicate wells. (c) Adenosine generated by tumor CD73 suppressed T cell proliferation. ID8 cells were incubated with conditioned medium as indicated. CFSE-labeled splenocytes were then incubated with the above conditioned medium or regular complete medium (No CM) in the presence of 1 μg/ml anti-CD3 mAb for 72 h. The proliferation (CFSE dilution) was evaluated by flow cytometry by gating on CD8⁺ or CD4⁺ cells. (d) Knockdown of CD73 expression promoted T cell proliferation. ID8SiNS and ID8SiCD73 cells were incubated with conditioned medium as indicated. Splenocytes were then incubated with the above conditioned medium in the presence of 1 μg/ml anti-CD3 mAb for 72 h. Data are mean ± SD of triplicate wells. Data are representative of three independent experiments (c,d). *, p<0.05; **, p<0.01.

Figure 2. Tumor CD73 impairs antigen specific T cell responses

ID8-OVA-SiNS or ID8-OVA-SiCD73 cells were incubated with conditioned medium as indicated. After 3 days in culture, the floating cells were harvested and examined for the CFSE dilution (a) and intracellular IFN-γ staining (b) by flow cytometry by gating on CD8⁺ cells. Cells incubated with conditioned medium alone were used as control (untreated). (c) ID8-OVA cells were incubated with conditioned medium as indicated. OT-I splenocytes were added into the above tumor cell cultures. After 3 days in culture, the floating cells were harvested and examined for the CFSE dilution by gating on CD8⁺ cells. (d) Knockdown of CD73 expression promotes tumor cell killing by OT-I CTL in vitro. The killing of ID8-GFP, ID8-OVA-SiNS, ID8-OVA-SiCD73 and APCP-treated ID8-OVA-SiNS by OT-I CTL was measured in a standard ⁵¹Cr release assay. Data are representative of three independent experiments (a,b,c) or two independent experiments (d). *, p<0.05; **, p<0.01.

Figure 3. Tumor CD73 promotes apoptosis of antigen-specific T cells

(a) ID8-OVA-SiNS or ID8-OVA-SiCD73 were incubated with conditioned medium as indicated. Pre-activated OT-I splenocytes were added into the above tumor cell cultures, or only incubated with or without NECA (b). After 3 days in culture, the floating cells were harvested and examined for apoptosis by double staining with annexin V and propidium iodide (PI) and subjected to flow cytometry by gating on CD8⁺ cells. (c) Female C57BL/6 mice were inoculated i.p. with 5×10⁶ ID8-OVA or ID8-OVA-SiCD73 cells. Three days later, pre-activated OT-I CD90.1 splenocytes were injected i.p. into each tumor-bearing mouse. Peritoneal exudate cells (PEC) were collected and counted at 24 h. The percentage and number of transferred cells (CD90.1⁺ CD8⁺) was shown. (d) Apoptotic cells were calculated as the percentage of annexin V⁺ cells gated in the CD90.1⁺ CD8⁺ fraction. Data represented as mean ± SD of cells from 3 mice of each group. Data are representative of three independent experiments (a,b) or two independent experiments (c,d). *, p<0.05.

Figure 4. Knockdown of CD73 expression improves anti-tumor T cell responses

(a) The proliferation of transferred CFSE-labeled OT-I CD90.1 cells in spleens, mesenteric lymph nodes (MLN) and PEC from the indicated tumor-bearing mice was measured by gating on CD8⁺ CD90.1⁺ cells at d2, d4 and d6. The activation marker CD44, CD69 or intracellular IFN-γ staining on these transferred cells (CD8⁺ CD90.1⁺) was also measured at d2, d4 and d6 (data not shown). (b) Percentage of divided cells among the CD8⁺ CD90.1⁺ cells, as assessed by CFSE dilution, was measured and plotted. Data represented as mean ± SD of cells from 3 mice of each group. *, p<0.05, compared to ID8-OVA-SiCD73-PEC. (c) Tumor-bearing mice were injected i.p. with 1×10⁷ OT-1 splenocytes. 5 days later, OVA-I-pulsed (CFSE-Hi) or no peptide-pulsed (CFSE-Lo) target cells from C57BL/6 mice were i.p. transferred into these tumor-bearing mice. Spleens, MLN and PEC were harvested 24 h later and analyzed for CFSE fluorescence. The number in each histogram indicated the percentage of CFSE-Hi population in all CFSE⁺ cells. Panel (d) showed the compiled data of percentage of killing. *, p<0.05, n=6. Data are representative of two (a,b,c,d) independent experiments.

Figure 5. Knockdown of CD73 expression increases overall survival of tumor-bearing mice

Female C57BL/6 wild type mice (a) or Rag 1^−/− mice (d) were inoculated i.p. with 1×10⁷ ID8SiNS or ID8SiCD73 cells. (b) Female C57BL/6 mice were treated with APCP one week after ID8 tumor challenge. (c) Female C57BL/6 mice were inoculated i.p. with 1×10⁷ ID8-OVA-SiNS or ID8-OVA-SiCD73 cells, and survival of the mice (5-8 each group) was measured. *, p<0.05; **, p<0.01. Data are representative of two (b,d) independent experiments or three independent experiments (a,c).

Figure 6. Blockade of adenosinergic pathway augments adoptive T cell immunotherapy

(a) Female C57BL/6 mice were inoculated i.p. with 1×10⁷ ID8-GFP or ID8-OVA cells. One week later, ID8-OVA tumor-bearing mice were left untreated or i.p. transferred with 1×10⁷ OT-I T cells. (b) Female C57BL/6 mice were inoculated i.p. with 1×10⁷ ID8-OVA-SiNS or ID8-OVA-SiCD73 cells. One week later, 1×10⁷ OT-I T cells were i.p. infused into those tumor-bearing mice. **, p<0.01. (c, d) Female C57BL/6 mice were inoculated i.p. with 1×10⁷ ID8-OVA cells. One week later, tumor-bearing mice were left untreated or i.p. transferred with 1×10⁷ OT-I T cells. Meanwhile, tumor-bearing mice were treated with SCH58261 (c) or caffeine (d) given as drinking water (0.1% wt/vol) at d7 and i.p infused with 1×10⁷ OT-I T cells at d8. Mice treated with SCH58261 (c) or caffeine (d) alone were used as control. Mice were treated with SCH58261 three times weekly. Survival of the mice (5-8 each group) was measured. *, p<0.05; **, p<0.01. Data are representative of 3 (a,b,c) independent experiments.