Human CD4+ CD39+ regulatory T cells produce adenosine upon co-expression of surface CD73 or contact with CD73+ exosomes or CD73+ cells

Abstract

While murine CD4(+) CD39(+) regulatory T cells (T(reg)) co-express CD73 and hydrolyze exogenous (e) adenosine triphosphate (ATP) to immunosuppressive adenosine (ADO), surface co-expression of CD73 on human circulating CD4(+) CD39(+) T(reg) is rare. Therefore, the ability of human T(reg) to produce and utilize ADO for suppression remains unclear. Using mass spectrometry, we measured nucleoside production by subsets of human CD4(+) CD39(+) and CD4(+) CD39(-)CD73(+) T cells or CD19(+) B cells isolated from blood of 30 volunteers and 14 cancer patients. CD39 and CD73 expression was evaluated by flow cytometry, Western blots, confocal microscopy or reverse transcription-polymerase chain reaction (RT-PCR). Circulating CD4(+) CD39(+) T(reg) which hydrolyzed eATP to 5'-AMP contained few intracytoplasmic granules and had low CD73 mRNA levels. Only ∼1% of these T(reg) were CD39(+) CD73(+) . In contrast, CD4(+) CD39(neg) CD73(+) T cells contained numerous CD73(+) granules in the cytoplasm and strongly expressed surface CD73. In vitro-generated T(reg) (Tr1) and most B cells were CD39(+) CD73(+) . All these CD73(+) T cell subsets and B cells hydrolyzed 5'-AMP to ADO. Exosomes isolated from plasma of normal control (NC) or cancer patients carried enzymatically active CD39 and CD73(+) and, when supplied with eATP, hydrolyzed it to ADO. Only CD4(+) CD39(+) T(reg) co-incubated with CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes produced ADO. Thus, contact with membrane-tethered CD73 was sufficient for ADO production by CD4(+) CD39(+) T(reg). In microenvironments containing CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes, CD73 is readily available to CD4(+) CD39(+) CD73(neg) T(reg) for the production of immunosuppressive ADO.

Keywords: adenosine; ectonucleotidases; exosomes; human tTreg; immune suppression; pTreg.

Fig. 1

Lymphocyte subpopulations expressing ectonucleotidases. (a) Co-expression of CD39 and CD73 on CD4⁺ T cells and CD19⁺ B cells in peripheral blood lymphocytes obtained from a representative normal donor (NC). The gates were set based on side- and forward-scatter. (b) Box-plots showing the frequency of CD4⁺CD73⁺CD39(–) T cells and CD4⁺CD39⁺CD73(–) regulatory T cells (T_reg) in the peripheral circulation of 29 NC. Box-plots show medians and quartiles for 25 and 75% as boxes and values for 0% and 100% as whiskers. (c) The percentages of CD4⁺CD39⁺ T_reg and CD4⁺CD73⁺ T cells in the peripheral blood of NC do not correlate with each other.

Fig. 2

Microscopic examinations of isolated CD4⁺CD25⁺ T cells. The cells were separated from normal peripheral blood mononuclear cells (PBMC) by magnetic antibody-coated beads, as described in Methods. (a) Separated cells were stained for surface expression of CD39 and CD73. Only rare CD39⁺CD73⁺ cells were seen (see arrow). Note distinct surface staining for CD39 (evenly distributed) and CD73 (a cap formation). (b) CD4⁺CD25⁺ T cells were permeabilized prior to staining and examined by confocal microscopy. In row 1, a CD4⁺CD39⁺CD73^neg regulatory T cells (T_reg) cell shows uniform staining (green) localized to the surface compartment. Next to it is a CD4⁺CD39⁺CD73^± cell which contains few CD73⁺ (red) cytoplasmic granules. In row 2, a CD4⁺CD73⁺CD39^neg T cell filled with CD73⁺ (red) cytoplasmic granules. Next to it is a CD4⁺CD73⁺CD39^± T cell which contains many red granules plus some CD39⁺ (green) elements. In row 3, a CD39⁺CD73⁺ B cell and a rare double-positive CD4⁺CD39⁺ T_reg cell. These cells contain CD73 in cytoplasmic granules (red) and strong surface-associated CD39 (green). This series of representative images was assembled after microscopic evaluations of at least five different slides. Control cells were stained with isotype control primary antibodies. (c) A representative Western blot of CD4⁺CD39⁺, CD4⁺CD73⁺ and CD19⁺ B cells isolated from PBMC of a normal control (NC) and evaluated for expression of CD39 and CD73. Note that isolated CD4⁺CD39⁺ cells are negative for CD73. Data are from one of three independent experiments performed with isolated cell subsets. (d) The relative ratio of CD39 and CD73 mRNA expression levels in the isolated CD4⁺ T cell subsets and B cells of NCs. The subsets were tested for CD39 and CD73 mRNA expression by reverse transcription–polymerase chain reaction (RT–PCR). The data are presented as relative ratios of Ct values for CD39/CD73 mRNA levels and CD73/CD39 mRNA levels normalized to CD39⁺CD73⁺ T_reg as 1·0. Representative data from one of three independent mRNA measurements performed with cell subsets obtained from different NCs are shown.

Fig. 3

Co-expression of CD39 and CD73 and adenosine (ADO) production by in-vitro-generated regulatory T cells (T_reg) (Tr1) cells. (a) Representative density plots show surface co-expression of the two ectoenzymes in Tr1 cells. Autologous CD4⁺CD25^neg cells cultured in parallel with Tr1 served as controls and were not co-incubated with dendritic cells (DC), tumour cells or a mix of cytokines used for Tr1 generation. (b) Fluorescence microscopy of freshly harvested Tr1 stained for surface CD39 and CD73. The merged image shows co-expression of the two enzymes on a large proportion of Tr1. Representative data from one of five Tr1 cultures established with CD4⁺ T cells obtained from different normal controls (NC). Magnification ×200. (c) The frequency and mean fluorescence intensity (MFI) for CD39 and CD73 in Tr1. Data are means ± standard deviation (s.d.) from three independent experiments. (d) Aliquots of inucible T_reg (iT_reg) expressing CD39 and CD73 were incubated in the presence of exogenous adenosine triphosphate (eATP) and concentrations of 5′-AMP or immunosuppressive adenosine (ADO) in supernatants were measured by mass spectrometry. Data are from one of two independent iT_reg cultures tested for ADO and 5′-AMP production.

Fig. 4

Characteristics of exosomes isolated from human plasma. (a) A representative Western blot of exosomes isolated from plasma of a normal control (NC) and floated on a continuous sucrose gradient following ultracentrifugation. Individual 1-ml fractions were collected and loaded on gels for electrophoresis. Exosomes expressing CD81 are located in fractions 6 and 7. (b) Transmission electron microscopy of exosomes collected from the sucrose gradient shown in (a) as fraction 6. Negative stain with uranyl acetate. (c) Exosomes in fraction 6 were examined in a NanoSight instrument to determine their size and particle concentration.

Fig. 5

Characteristics of exosomes fractionated from plasma of normal control (NC) or head and neck squamous cell carcinoma (HNSCC) patients. (a) Western blots of isolated exosomes demonstrating the presence of CD39 and CD73. CD81 is a tetraspanin used as an exosome marker. Note that exosomes obtained from HNSCC patients' plasma carry higher levels of both ectoenzymes than those obtained from NCs' plasma. (b) 5′-AMP and adenosine (ADO) production by isolated CD39⁺CD73⁺ exosomes (10ug protein) incubated with exogenous adenosine triphosphate (eATP) (20 μm). Exosomes produced more 5′-AMP and ADO than B cells or CD4⁺CD39⁺ T_reg (25 000 cells/well). Representative data from three experiments performed with exosomes and cells of different NC. (c) Box-plots show levels of 5′-AMP and ADO produced by exosomes obtained from NC (n = 6) or patients with HNSCC (n = 6). B cells (25 000/well) were used as a positive control. Cells were incubated in the presence of eATP (20uM) for 60 min. The baseline values are for exosomes incubated in the absence of ATP.

Fig. 6

Production of 5′-AMP and adenosine (ADO) by CD4⁺ T cell subsets. Production of exogenous 5′-AMP (a), and ADO (b) was measured by mass spectrometry after incubation of CD4⁺CD39⁺, CD4⁺CD73⁺ or CD4⁺CD39⁺ T_reg and CD4⁺CD73⁺ T cells mixed at a 1:1 ratio in the presence of eATP. (a) Only CD4⁺CD39⁺ T cells alone or CD4⁺CD39⁺ T cells supplemented with CD4⁺CD73⁺ T cells hydrolyzed eATP to 5′-AMP. (b). Only CD4⁺CD39⁺ T_reg and CD4⁺CD73⁺ T cells combined together hydrolyzed eATP to ADO. The data are means ± standard deviation (s.d.) from independent experiments performed with lymphocyte subsets obtained from three different normal controls (NC).