Phenotypical analysis of ectoenzymes CD39/CD73 and adenosine receptor 2A in CD4+ CD25high Foxp3+ regulatory T-cells in psoriasis

Abstract

Background: CD39 and CD73 are two novel cell surface markers of CD25^high Foxp3⁺ regulatory T-cells (Tregs). Concordant expression of these two ectoenzymes not only discriminate Tregs from other cell populations, but also generates pericellular adenosine, which has been reported to suppress proliferation of activated T effector (Teff) cells. Because it is currently unclear whether human ectoenzymes (CD39/CD73) are involved in the impaired suppressive activity of Tregs in psoriasis, we examined the frequencies and phenotypes of CD39/CD73-expressing Tregs and related receptor adenosine receptor 2A (A_2A R) in peripheral blood of patients with different types of psoriasis.

Methods: Peripheral blood mononuclear cells (PMBC) were prepared from patients with three different types of psoriasis (psoriasis vulgaris, pustular psoriasis and erythrodermic psoriasis). CD4⁺ cells were separated from PBMC by negative selection on midiMACS columns, and the frequencies and phenotypes of CD39 and CD73 expressing Tregs, and A_2A R expressing Teff were all determined by flow cytometry analysis. Blood from healthy volunteers served as controls.

Results: The expression of single CD73⁺ Tregs was markedly reduced (approximately 50%) in psoriasis vulgaris, compared to normal controls. In pustular psoriasis, the mean numbers of CD39⁺ Tregs and A_2A R⁺ Teff was significantly lower than in normal controls. Among three different types of psoriasis, CD39 expression was strikingly reduced in the blood Treg population of pustular psoriasis patients. Decreased CD73⁺ Tregs levels were observed in psoriasis vulgaris compared to pustular psoriasis and erythrodermic psoriasis.

Conclusions: The differences in the expression of CD39^- and CD73^- Tregs may be a factor in the pathogenesis of psoriasis.

Keywords: A2AR; CD39; CD73; Tregs; ectoenzymes; psoriasis.

Figure 1

The role of CD39 and CD73 in Tregs. Tregs express CD39 and CD73. CD39 belongs to the ectonucleoside triphosphate diphosphohydrolase family, an ectoenzyme that hydrolyzes extracellular ATP/UTP, ADP/UDP to adenosine monophosphate (AMP). AMP is then degraded to nucleosides (adenosine) by CD73 (ecto‐5′‐nucleotidase), which in turn binds the adenosine receptor 2A (A_2A R) expressed on T effector cells to elevate the intracellular cAMP which suppresses the proliferation of T effector cells.

Figure 2

The purification of CD4⁺ T‐cell isolation. Flow cytometric analyses of purity of CD4⁺ T‐cells after negative selection. Control samples were stained with isotype‐matched control antibody. (a) The negative fraction contained less than 5% CD4 T‐cells. (b) The histogram shows that the purity of the CD4⁺ T‐cells was almost 100%.

Figure 3

The proportion of CD39 and CD73 expressing CD25^highFoxp3⁺Tregs in peripheral blood of healthy controls. Expression levels of CD39 and CD73 on normal CD25^highFoxp3⁺ regulatory T‐cells (Tregs; n = 10). (a) Histogram depicts CD4⁺ (negative bead selection) cells defined by CD25 and Foxp3. CD25^highFoxp3⁺ Tregs, CD25^medFox3⁻ and CD25⁻Foxp3⁻T‐cells were gated and further analyzed for expression of CD39 and CD73. (b) CD4⁺ CD25^highFoxp3⁺ Tregs showed significantly higher expression levels of CD39 (P < 0.01) and lower proportion CD73 (P < 0.05) compared to CD25^midFoxP3⁻ and CD25⁻ pool. (*P < 0.05, **P < 0.01, Student's t‐test).

Figure 4

The proportion of CD39 and CD73 expressing CD25^highFoxp3⁺ Tregs in peripheral blood of different types of psoriasis patients. (a) Representative diagrams showed CD39 and CD73 expressing regulatory T‐cells in peripheral blood of one healthy and one patient with psoriasis. The psoriasis vulgaris patient showed markedly reduced CD73 expression level in Tregs. (b) Cumulative data (n = 10) demonstrate a statistically significant lower single CD73 expression level of Tregs in psoriasis vulgaris patients compared to healthy controls (7 ± 2% vs 13 ± 4%, P < 0.01). Also, Tregs showed significant reduced double CD39/CD73 expression level (P < 0.05). While in pustular psoriasis patients, cumulative data (n = 10) demonstrate a 50% decrease in single CD39 expression in Tregs compared to healthy control subjects (30 ± 9% vs 60 ± 14%, P < 0.01). No significant expression differences were observed on single CD73 and double CD39/CD73 expression level (*P < 0.05, **P < 0.01, Student's t‐test). (c) Correlation between CD73⁺ Treg cells number, CD39⁺ Treg cells number and the psoriasis area and severity index score of psoriatic vulgaris patients (n = 10, r ² = 0.247 and r ² = 0.0045, respectively).

Figure 5

Proportion of adenosine receptor 2A (A_2A R>) in Teff cells among different types of psoriasis patients. (a) CD4⁺ cells were identified based on their characteristic properties shown in the forward scatter (FSC) and sideward scatter (SSC). CD4⁺ CD25⁻ T‐cells were gated and further analysed for expression of A_2A R. (b) Data shown are A_2A R ⁺/CD4⁺ CD25⁻ ratios in peripheral blood of normal controls, psoriasis vulgaris (PV), pustular psoriasis, and psoriatic erythroderma patients, representing a summary of 10 (normal), 10 (PV), 10 (PE) and 10 pustular psoriasis with PV (PP) independent experiments. Data are mean ± SD. (***P < 0.0001, Bonferroni correction).