Synergy between the ectoenzymes CD39 and CD73 contributes to adenosinergic immunosuppression in human malignant gliomas

Abstract

Background: The importance of ectoenzymes CD39 and CD73 in mediating adenosinergic immunosuppression has been recognized, but their roles in human malignant glioma-associated immunosuppression remain largely unknown.

Methods: In this study, the ectoenzyme characteristics of malignant glioma cells and infiltrating CD4(+) T lymphocytes isolated from newly diagnosed malignant glioma patients were investigated. The ectoenzyme activities of both cell populations were determined by nucleotide hydrolysis assay. The immunosuppressive property of the CD39-CD73 synergic effect was evaluated via responder T-cell proliferation assay.

Results: We observed that CD39(-)CD73(+) glioma cells and infiltrating CD4(+)CD39(high)CD73(low) T lymphocytes exhibited 2 distinct but complementary ectoenzyme phenotypes, which were further verified by enzyme activity assay. The nucleotide hydrolysis cascade was incomplete unless CD39 derived from T lymphocytes and CD73 collaborated synergistically. We demonstrated that increased suppression of responder CD4(+) T-cell proliferation suppression was induced by CD4(+)CD39(+) T cells in the presence of CD73(+) glioma cells, which could be alleviated by the CD39 inhibitor ARL67156, the CD73 inhibitor APCP, or the adenosine receptor A2aR antagonist SCH58261. In addition, survival analysis suggested that CD73 downregulation was a positive prognostic factor related to the extended disease-free survival of glioblastoma patients.

Conclusions: Our data indicate that glioma-derived CD73 contributes to local adenosine-mediated immunosuppression in synergy with CD39 from infiltrating CD4(+)CD39(+) T lymphocytes, which could become a potential therapeutic target for treatment of malignant glioma and other immunosuppressive diseases.

Keywords: CD39-CD73-adenosinergic immunosuppression; glioma microenvironment; infiltrating T lymphocytes; malignant glioma; synergic effect.

Fig. 1.

Phenotypic characteristics of the glioma cells. (A) Detection of ENTPD/CD39 family members and NT5E/CD73 by RT-PCR (left) and quantitative (q)RT-PCR (right) in glioma cell lines U-87 MG and T98G. Human peripheral blood mononuclear cells (PBMCs) were used as a positive control and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. The values of each group are expressed as mean percents ± SD for 3 qRT-PCT assays. (B) Surface expressions of ectoenzymes CD39 and CD73 on U-87 MG, T98G, and U-251 glioma cells were assessed by flow cytometry. Representative results are shown on the left. Numbers in graphs represent the percentages of positively stained cells (black histograms) relative to those labeled with isotype antibodies (gray histograms), which are further summarized as mean percents ± SD on the right. (C) Surgical specimens of malignant gliomas were stained with anti-CD39 (left) and anti-CD73 (right) antibodies for immunohistochemistry. Representative images indicate the preferential expression of CD73 over CD39 in human malignant glioma specimens. Immune detection was revealed by diaminobenzidine and then counterstained with hematoxylin. The images were taken at 400× magnification. (D) Glioblastoma mRNA microarray data were obtained from the TCGA database and analyzed via the cBio Cancer Genomics Portal. The comparison of median disease-free survival between patients with CD73 mRNA z-score < −1 (red curve) and those with CD73 mRNA z-score≥−1 (blue curve) indicated that downregulation of CD73 was a positive prognostic factor. P < .05.

Fig. 2.

Ectoenzyme characterization of glioma-infiltrating CD4⁺ T lymphocytes. (A) Surface expressions of CD39 and CD73 on the peripheral CD4⁺ T lymphocytes from healthy donors (n = 10) and patients with newly diagnosed malignant glioma (n = 9); the matched tumor-infiltrating CD4⁺ T lymphocytes were determined by flow cytometry. Scatter plot summarizing flow cytometry data shows percentage of CD4+ T cells expressing CD39 (left) and CD73 (right). Mean percents ± SD indicated by horizontal lines and bars are given. ***P < .001. (B) Dot plots show expression of CD39 and CD73 in a representative individual from each group.

Fig. 3.

Phenotypic characterization of CD4⁺CD39⁺ T lymphocytes. (A and B) Surface expressions of CD26 (A) and CD73 (B) in peripheral CD4⁺CD39⁺ and CD4⁺CD39⁻ T-cell subsets were determined by flow cytometry. Left panel, ectoenzyme expression histograms gated on each subset from a representative sample. Right panel, bar graphs summarizing data obtained. (C) Surface expressions of CD39 and CD73 in natural CD4⁺Foxp3⁺ Tregs (nTregs) from GBM patient peripheral blood mononuclear cells (PBMCs) were verified. (D) Bar graphs summarizing the nTreg CD39/CD73 surface data (n = 7). (E) After in vitro induction of adaptive CD4⁺Foxp3⁺ Tregs (iTregs), CD39/CD73 surface expressions were determined by flow cytometry. (F) Bar graphs summarizing the iTreg CD39/CD73 expression from 3 independent experiments. Numbers in histogram graphs represent the percentages of positively stained cells (black histograms) compared with isotype controls (gray histograms). Bars in bar graphs represent mean percents + SD. *P < .05, ***P < .001.

Fig. 4.

Ectoenzyme activity measurement by determining the Pi generated during nucleotide hydrolysis. (A and B) 10⁵ U-87 MG and T98G glioma cells were assessed for 5′-nucleotidase (A) or ENTPDase (B) activity by adding exogenous AMP or ATP in the presence or absence of 100 µM APCP or 250 µM ARL67156, respectively. After 30 min incubation, cell supernatants were collected, and corresponding concentrations of Pi were determined. Baseline phosphate release was determined by the supernatant harvested from cells incubated without nucleotide substrate. (C–D) 10⁵ CD4⁺CD39⁺/CD4⁺CD39⁻ T cells sorted by flow cytometry were assessed for ENTPDase (C) or 5′-nucleotidase (D) activity. (E) 5′-nucleotidase activity of soluble 5′-nucleotidase (s5′-NT) purified from Crotalus atrox venom was verified. (F) Exogenous AMP was added to freshly sorted CD4⁺CD39⁺/CD4⁺CD39⁻ T cells in the presence of 2 kU/mL s5′-NT. After 30 min incubation, Pi concentration was determined. (G) Exogenous ATP was added to freshly sorted CD4⁺CD39⁺/CD4⁺CD39⁻ T cells in the presence of 2 kU/mL s5′-NT. After 30 min incubation, Pi concentration was determined. All the data are presented as mean percents ± SD acquired from 3 independent experiments. *P < .05, **P < .01, ***P < .001.

Fig. 5.

CD4⁺CD39⁺ T cells induce more significant proliferation suppression in the presence of CD73⁺ glioma cells. (A) CFSE-labeled U-87 MG glioma cells were treated with 20 µg/mL mitomycin C (Mito C) for 2 h. After 3 days, the proliferation percent of U-87 MG cells was evaluated by flow cytometry. (B) CFSE-labeled CD4⁺CD39⁻ responder T cells (RC) were cocultured with autologous CD4⁺CD39⁺ suppressor T cells (S) at the ratio of 1 : 1 in the presence or absence of preseeded U-87 MG glioma cells (U87). Microbeads coated with anti-human CD2/3/28 antibodies were used to stimulate T-cell proliferation. The effects of ARL67156, APCP, and the adenosine receptor A_2aR antagonist SCH58261 were tested as described in Materials and Methods. After 4 days, cells were harvested and analyzed via flow cytometry. Representative plots are shown. The numbers represent the percentages of proliferating CFSE-labeled responder T cells. (C) The suppression percents obtained in 3 independent experiments were summarized and presented as mean percents ± SD. *P < .05.

Fig. 6.

Schematic diagram showing the synergy between ectoenzymes CD39 and CD73 demonstrated in the human malignant glioma microenvironment. Extracellular ATP is hydrolyzed by CD39 on infiltrating T lymphocytes (including Foxp3⁺CD25⁺ Tregs and Foxp3⁻CD25⁻ Tinds) to AMP and then further converted rapidly to anti-inflammatory adenosine by glioma-derived CD73. Thus, ectoenzymes CD39 and CD73 derived from different cell populations contribute to the generation and accumulation of extracellular adenosine, which activates adenosine receptor A_2aR expressed by immune cells, elevates the intracellular cAMP level, and initiates downstream signaling of protein kinase A/cAMP response element binding protein and Epac/phospholipase C. CD39/CD73-mediated adenosinergic immunosuppression not only inhibits the activation and function of pro-inflammatory immune cells, such as infiltrating CD4⁺CD39⁻/CD8⁺ effector T cells, M1 macrophages/microglia, and dendritic cells, but also promotes the induction of CD39⁺ Treg cells and tumor-supportive M2 macrophages/microglia.