CD73-mediated adenosine production promotes stem cell-like properties in mouse Tc17 cells

Abstract

The CD73 ectonucleotidase catalyses the hydrolysis of AMP to adenosine, an immunosuppressive molecule. Recent evidence has demonstrated that this ectonucleotidase is up-regulated in T helper type 17 cells when generated in the presence of transforming growth factor-β (TGF-β), and hence CD73 expression is related to the acquisition of immunosuppressive potential by these cells. TGF-β is also able to induce CD73 expression in CD8(+) T cells but the function of this ectonucleotidase in CD8(+) T cells is still unknown. Here, we show that Tc17 cells present high levels of the CD73 ectonucleotidase and produce adenosine; however, they do not suppress the proliferation of CD4(+) T cells. Interestingly, we report that adenosine signalling through A2A receptor favours interleukin-17 production and the expression of stem cell-associated transcription factors such as tcf-7 and lef-1 but restrains the acquisition of Tc1-related effector molecules such as interferon-γ and Granzyme B by Tc17 cells. Within the tumour microenvironment, CD73 is highly expressed in CD62L(+) CD127(+) CD8(+) T cells (memory T cells) and is down-regulated in GZMB(+) KLRG1(+) CD8(+) T cells (terminally differentiated T cells), demonstrating that CD73 is expressed in memory/naive cells and is down-regulated during differentiation. These data reveal a novel function of CD73 ectonucleotidase in arresting CD8(+) T-cell differentiation and support the idea that CD73-driven adenosine production by Tc17 cells may promote stem cell-like properties in Tc17 cells.

Keywords: CD73; Tc17; adenosine; cell differentiation; stem cell; tumour immunology.

Figure 1

In vitro‐generated Tc17 cells present stem‐cell‐like properties and anti‐tumour activity. Ovalbumin (OVA) ‐specific Tc1 and Tc17 cells were generated by the co‐culture of CD8⁺ T cells (from OT‐I mice), with antigen‐presenting cells for 4 days in the presence of soluble α‐CD3 and polarizing cytokines [interleukin‐6 (IL‐6), transforming growth factor‐β ₁ (TGF‐β ₁) and α‐interferon‐γ (α‐IFN‐γ) for Tc17 cells and IL‐2 plus IL‐12 for Tc1 cells]. (a) Expression of tcf7, lef1, gzmb and pfn1 in Tc1 and Tc17 cells assayed by real time PCR (n = 3). (b) Cytokine production by Tc1 or Tc17 cells was measured by CBA or ELISA after stimulation with PMA plus ionomycin for 4 hr (n = 4). (c) Tc1 and Tc17 cells were adoptively transferred (1 × 10⁶) into B16‐OVA‐bearing B6.SJL mice (CD45.1⁺). Tumour growth in Tc1, Tc17 or PBS‐treated mice was monitored every 2–3 days. (n = 6 mice per group). (d) Ten days after adoptive transfer, the mice were killed and the frequency of transferred (CD45.2⁺) cells in tumour‐draining lymph nodes (TdLN) and tumours was evaluated by flow cytometry. (e) The production of IFN‐γ, IL‐17 and GzmB was evaluated by flow cytometry on transferred cells (CD8⁺ CD45.2⁺) following in vitro activation with PMA plus ionomycin in the presence of Golgi Plug for 4 hr. (f) The expression of memory‐related markers (CD62L/CD127) and terminal differentiation‐related markers (CD44/KLRG1) was analysed on adoptively transferred Tc1 and Tc17 cells (n = 3). (g) 1 × 10⁶ OVA‐specific Tc17 or Tc1 cells were transferred into B6.SJL CD45.1 congenic mice. One day after the adoptive transfer, the mice were immunized intraperitoneally with OVA protein (1 mg). Two weeks later, mice were re‐immunized with OVA protein (0·5 mg) and 24 hr later challenged with 1 × 10⁶ B16‐OVA cells. Tumour growth was monitored every 2 days (n = 4 mice per group). Data in (d) and (e) are representative of two independent experiments (n = 3 per group). The percentage of cells within each gate or quadrant are presented in (d) and (e). n.d., not detected; *P < 0·05; **P < 0·01; ***P < 0·005 Mann–Whitney test.

Figure 2

In vitro‐generated Tc17 cells express high levels of CD73 and hydrolyse AMP to adenosine. (a) CD39 and CD73 expression by Tc1 and Tc17 cells generated in vitro (obtained from C57BL/6 mice), as evaluated by flow cytometry (representative of seven independent experiments). (b) CD39 and CD73 expression by endogenous Tc1 and Tc17 cells obtained from peripheral lymph nodes of C57BL/6 mice (n = 3). The geometric mean of CD39 and CD73 is presented in each histogram. (c) 5 × 10⁴ Tc1 or Tc17 cells were incubated with exogenous AMP (10 μm) for 1 hr and the supernatant was analysed by HPLC for the presence of AMP and adenosine using anion exchange chromatography (n = 4). Standards were prepared from the stock solution the same day of the analysis using HPLC. Results are expressed as % of hydrolysed AMP and calculated as (100 × Adenosine peak area)/(Adenosine peak area + AMP peak area). ns, non‐significant; *P < 0·05; Mann–Whitney test.

Figure 3

Adenosine induces stem cell markers in Tc17 cells. CD8⁺ T cells from C57BL/6 mice were co‐cultured for 4 days with antigen‐presenting cells and were activated with soluble α‐CD3 and polarizing cytokines [interleukin‐6 (IL‐6), transforming growth factor‐β ₁ (TGF‐β ₁) and α‐interferon‐γ (α‐IFN‐γ)] in the presence of the A2AR antagonist, SCH 58261(5 μm) or DMSO as the vehicle (n = 3). (a) The expression of tcf7, lef1, gzmb and pfn1 was measured using real time PCR. (b) The expression of surface markers was analysed by flow cytometry. (c) IFN‐γ and IL‐17 intracellular staining of Tc17 cells differentiated in the presence of SCH 58261 (5 μm) or DMSO and stimulated with PMA and ionomycin in the presence of Golgi Plug for 4 hr. SCH, SCH 58261; *P < 0·05; **P < 0·01 two‐tailed t‐test.

Figure 4

CD73 is down‐regulated in Tc17 cells during differentiation to Tc1‐like cells. (a) In vitro‐generated Tc17 cells (obtained from C57BL/6 mice) were stained with Violet cell‐trace dye and then re‐stimulated with plate‐bound α‐CD3 and α‐CD28 for 2 days in the absence of polarizing cytokines. CD73 expression and GzmB production was analysed by flow cytometry (n = 1). Numbers inside the contour plots represent the percentage of GzmB⁺ cells obtained for each region. (b) Tc17 cells generated in vitro from OT‐I mice were adoptively transferred to CD45.1⁺ B16‐OVA tumour‐bearing mice. Ten days after the adoptive transfer, CD45.2⁺ transferred cells were analysed for interferon‐γ (IFN‐γ) production and CD73 expression (n = 3). CD73 geometric mean was analysed in IFN‐γ ⁺ and IFN‐γ ^– transferred populations and compared with endogenous total CD8⁺ T cells (histograms).

Figure 5

CD73 expression in CD8⁺ T cells is down‐regulated during effector differentiation. (a) CD39 and CD73 expression was analysed on naive (TN), central memory (TCM) and effector memory (TEM) splenic CD8⁺ T cells from C57BL/6 mice (n = 4). (b) Total CD8⁺ tumour‐infiltrating lymphocytes were analysed based on CD39 and CD73 expression. The expression of memory‐related markers (CD62L/CD127) and terminal differentiation‐related markers (CD44/KLRG1/IFN‐γ/GzmB) was analysed on CD39‐CD73⁺ (grey histograms) and CD39⁺ CD73^– subsets (light grey histograms) (n = 4). TN, naive T cell; TCM, central memory T cell; TEM, effector memory T cell. The percentages of cells within each gate or quadrant are presented in (a) and (b). *P < 0·05 Mann–Whitney test.

Figure 6

CD73‐driven adenosine production by Tc17 acts in an autocrine loop to sustain stem‐cell programme and restrain effector differentiation. (a) CD73 expression by Tc17 cells enables the production of extracellular adenosine that signals through adenosine receptor A2A (A2AR) in an autocrine fashion. Adenosine signalling sustains Tc17 phenotype [interleukin‐17 (IL‐17) production and TCF/LEF expression] and inhibit the acquisition of Tc1‐like effector molecules [interferon‐γ (IFN‐γ) and GzmB]. (b) CD73‐mediated adenosine production sustains the stem‐cell‐like programme on Tc17 cells, allowing these cells to survive and persist as long‐lived memory T cells. Upon antigen encounter, some Tc17 cells lose CD73 expression and adenosine production. In the absence of autocrine adenosine signalling, Tc17 cells can differentiate into short‐lived effector Tc1‐like cells (IFN‐γ ⁺ GzmB⁺).