CD73 is a phenotypic marker of effector memory Th17 cells in inflammatory bowel disease

Abstract

Purinergic signaling and associated ectonucleotidases, such as CD39 and CD73, have been implicated in the pathogenesis of inflammatory bowel disease (IBD). CD39 is known to be a Treg memory cell marker, and here we determine the phenotype and function of CD73(+) CD4(+) T lymphocytes in patients with IBD. We describe elevated levels of CD73(+) CD4(+) T cells in the peripheral blood and intestinal lamina propria of patients with active IBD. The functional phenotype of these CD73(+) CD4(+) T cells was further determined by gene expression, ecto-enzymatic activity, and suppressive assays. Increased numbers of CD73(+) CD4(+) T cells in the periphery and lamina propria were noted during active inflammation, which returned to baseline levels following anti-TNF treatment. Peripheral CD73(+) CD4(+) T cells predominantly expressed CD45RO, and were enriched with IL-17A(+) cells. The CD73(+) CD4(+) cell population expressed higher levels of RORC, IL-17A, and TNF, and lower levels of FOXP3 and/or CD25, than CD73(-) CD4(+) T cells. Expression of CD73 by peripheral CD4(+) T cells was increased by TNF, and decreased by an anti-TNF monoclonal antibody (infliximab). In vitro, these peripheral CD73(+) CD4(+) T cells did not suppress proliferation of CD25(-) effector cells, and expressed higher levels of pro-inflammatory markers. We conclude that the CD73(+) CD4(+) T-cell population in patients with active IBD are enriched with cells with a T-helper type 17 phenotype, and could be used to monitor disease activity during treatment.

Figure 1

CD73 expression by CD4⁺ T lymphocytes in patients with IBD. (A) Box and whisker plots showing proportion (by flow cytometry) of peripheral blood CD4⁺ T cells expressing CD73 in healthy donors (white) and patients with clinically quiescent IBD (light gray) and clinically active IBD (dark gray). Data are shown as median, interquartile range, and the range of ten patients/controls per group and are pooled from 30 experiments performed. *p < 0.05 by Student’s t-test. (B) Box and whisker plots showing proportion (by flow cytometry) of LP CD4⁺ T cells expressing CD73 in healthy donors (white) and patients with inactive IBD (light gray) and active IBD (dark gray). Data are shown as median, interquartile range, and the range of ten patients/controls per group and are pooled from 30 experiments performed. *p < 0.05 by Student’s t-test. (C–E) Colonic biopsies were subjected to immunofluorescent staining for CD4 (bright green), CD73 (red), and co-localized CD4/CD73 (yellow), with Hoechst nuclear counterstain (blue). Representative sections from biopsied tissue from (C) an apparently healthy individual, (D) a patient with inactive IBD, and (E) a patient with active IBD. Magnification 20×. Data shown are representative of three samples examined from six experiments performed.

Figure 2

Characterization of CD73⁺ human peripheral blood CD4⁺ cells. (A) Flow cytometry study of CD45RO status (memory) in peripheral blood CD4⁺ T cells from a patient with active Crohn’s disease. The expression of CD73 in relation to the memory marker CD45RO was evaluated (left). The percentage of CD73⁺ cells amongst CD45RO^high cells (top) and CD45RO^low cells (bottom) was also evaluated. Data shown are representative of three independent experiments. (B) Histogram of CD25 expression in CD39⁻CD73⁺ (left) and CD39⁺CD73⁻ (center) and CD39⁺CD73⁺ (right) CD4⁺ T cells. Data shown are representative of three independent experiments.

Figure 3

Phenotype of CD73⁺ cells. (A) Representative example of a flow cytometry density plot of CD25 and FOXP3 expression in the population of LP CD73⁺CD39⁺CD4⁺ T cells (left) and CD73⁻CD39⁺CD4⁺ T cells (right). The proportion of double-positive cells is indicated as a percentage in the top right quadrant. Data shown are representative of three independent experiments. (B) Representative histogram of CD25^loCD4⁺ T-cell numbers in the absence of other cells (control), or in the presence of CD73⁺CD4⁺ T cells (center), or CD73⁻CD4⁺ T cells (right). CD25^loCD4⁺ T cells (2 × 10⁴/well) were cultured with the same amount of CD73⁺CD4⁺ T cells, or CD73⁻CD4⁺ T cells and stimulated with anti-CD3/28 beads for 3 days. Data shown are representative of three independent experiments. (C) Mean percentage proliferation of CD25^loCD4⁺ T cells (2 × 10⁴/well) in control medium (white column), or co-cultured with equal numbers of CD73⁺CD4⁺ T cells (gray column) or CD73⁻CD4⁺ T cells (black column) after stimulation with anti-CD3/28 beads for 3 days. Data are shown as mean + SD of nine samples pooled from three independent experiments performed. *p < 0.05 by t-test.

Figure 4

Gene expression profile of activated peripheral blood CD4⁺ cells clustered by CD73 expression. (A) Mean mRNA levels by PCR (AUs, y axis) of RORC, T-bet, GATA-3 and FOXP3 in activated CD73⁺CD4⁺ peripheral blood T cells (dark columns, n = 4 samples) or CD73−CD4⁺ T cells (clear columns, n = 4 samples) isolated by flow cytometry and sorted according to CD73 expression. Activation of CD4⁺ T cells was performed by 24 h of ex vivo activation with antibodies to CD3/CD28. Data are shown as mean + SD of 32 samples pooled from two independent experiments performed. *p < 0.05 by t-test. (B) Mean mRNA levels by PCR (AUs, y axis) of IL-17A, TNF, IFN-γ, and IL-10 in activated CD73⁺CD4⁺ T cells (dark columns, n = 4 samples) or CD73⁻CD4⁺ T cells (clear columns, n = 4 samples) isolated by flow cytometry and sorted according to CD73 expression. Activation of CD4⁺ T cells was performed by 24 h of ex vivo activation with antibodies to CD3/CD28. Data are shown as mean + SD of 32 samples pooled from two independent experiments performed. *p < 0.05 by Student’s t-test. (C) Mean percentage IL-17⁺ cells (by flow cytometry) amongst CD73⁺CD4⁺ T cells (dark column) or CD73⁻CD4⁺ T cells (clear column) in peripheral blood from patients with Crohn’s disease. Data are shown as mean + SD of six samples pooled from three independent experiments performed. *p < 0.05 by Student’s t-test.

Figure 5

CD73 expression in CD4+ cells. (A) Bar chart of the percentage CD73⁺ expression in CD4⁺ T cells from healthy peripheral blood (n = 3 samples) treated with TNF (0, 20, 200 ng/mL) for 12 h without (black columns) or with (white columns) infliximab 1000 µg/mL). Data are shown as mean + SD of 18 samples pooled from three independent experiments performed. *p < 0.05 by Student’s t-test. (B) Bar chart of the percentage of annexin-positive CD73⁺CD4⁺ T cells after in vitro treatment with infliximab at concentrations of 0, 50, 1000 µg/mL. CD4⁺ T cells from healthy peripheral blood (n = 3 samples) were treated with infliximab for 12 h, then CD73 and annexin were detected by flow cytometry. Data are shown as mean + SD of nine samples pooled from three independent experiments performed. *p < 0.05 by Student’s t-test. (C) Representative example of a flow cytometry density plot of CD39 (y-axis) and CD73 (x-axis) expression in peripheral blood CD4⁺ T cells cultured alone (Th0) or in the presence of IFN-α or IFN-γ. Data shown are representative of three independent experiments.

Figure 6

Influence of infliximab (IFX) on clinical outcomes and CD73⁺ cells (A) X–Y plot of percentage CD73⁺CD4⁺ T cells to all CD4⁺ T cells (x-axis), and clinical score (HBI) (y-axis) in patients with Crohn’s disease (n = 22) before treatment. Diagonal line represents linear regression line. (B) Line graph of clinical scores (HBI) for enrolled patients before (0), and 14 days (14), after an infusion of infliximab 5mg/kg. *p-value < 0.05 by Student’s t-test comparison of means, n = 13 patients. (C) Ratio of CD73⁺CD4⁺ T cells to all CD4⁺ T cells in peripheral blood of enrolled patients before (0), and 14 days (14) and 45 days (45), after an infusion of infliximab 5 mg/kg. Horizontal line indicates mean. * indicates p < 0.05 for ANOVA and comparison of means with Bonferroni correction, n = 13 patients.