Dysfunctional CD39POS regulatory T cells and aberrant control of T-helper type 17 cells in autoimmune hepatitis

Authors

  • Charlotte R. Grant,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
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  • Rodrigo Liberal,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
    2. Faculty of Medicine, University of Porto, Porto, Portugal
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  • Beth S. Holder,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
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  • John Cardone,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
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  • Yun Ma,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
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  • Simon C. Robson,

    1. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
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  • Giorgina Mieli-Vergani,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
    2. Pediatric Liver, Gastrointestinal, and Nutrition Center, King's College London School of Medicine at King's College Hospital, London, UK
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  • Diego Vergani,

    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
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  • Maria Serena Longhi

    Corresponding author
    1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK
    2. Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA
    • Address reprint requests to: Maria Serena Longhi, M.D., Ph.D., Institute of Liver Studies, King's College London School of Medicine, 3rd Floor Cheyne Wing, King's College Hospital, Denmark Hill, London, UK, SE5 9RS. E-mail: maria.longhi@kcl.ac.uk; fax: +44(0)2032993760.

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  • Potential conflict of interest: nothing to report.

  • See Editorial on Page 754

  • The authors acknowledge financial support from the Department of Health by the National Institute for Health Research (NIHR) comprehensive Biomedical Research Center award to Guy's & St Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London and King's College Hospital NHS Foundation Trust. C.R.G. is supported by an Alex P Mowat Ph.D. Studentship from King's College Hospital Charity, UK. R.L. is supported by a doctoral grant from the Science and Technology Foundation, Science and Higher Education Ministry, Portugal. M.S.L. was supported by the Roger Dobson Fund, King's College Hospital Charity, UK, when this project was started and is currently supported by a Clinician Scientist Fellowship from the Medical Research Council, UK.

Abstract

Autoimmune hepatitis (AIH) is an important cause of severe liver disease and is associated with both quantitative and qualitative regulatory T-cell (Treg) impairments. Tregs express CD39, an ectonucleotidase responsible for extracellular nucleotide hydrolysis, culminating in the production of immunosuppressive adenosine. Here, we describe multiple CD39pos Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39pos Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg subsets to suppress both proliferation of effector T cells and interleukin (IL)-17 production was evaluated. In AIH, CD39pos Tregs are decreased in frequency, exhibit limited adenosine triphosphate/adenosine diphosphate hydrolysis activity, and fail to suppress IL-17 production by effector CD4 T cells. Moreover, these CD39pos Tregs display a more proinflammatory profile in AIH, which is characterized by elevated CD127 positivity, and a greater propensity to produce interferon-gamma or IL-17 upon challenge with proinflammatory stimuli. Conclusions: In AIH, CD39pos Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL-17 production by effector CD4 T cells. CD39pos Tregs show plasticity and are unstable upon proinflammatory challenge, suggesting that defective immunoregulation in AIH might result not only from reduced Treg number and function, but also from increased conversion of Tregs into effector cells. (Hepatology 2014;59:1007–1015)

Abbreviations
ADP

adenosine diphosphate

AIH

autoimmune hepatitis

AMP

adenosine monophosphate

ANA

anti-nuclear antibody

APC

allophycocyanin

AST

aspartate aminotransferase

ATP

adenosine triphosphate

autoAb

autoantibody

CFSE

carboxy fluorescein succinimidyl ester

Cy7

cychrome-7

DC

disease control

FACS

fluorescence-activated cell sorting

FCM

flow cytometry

FCS

fetal calf serum

FITC

fluorescein isothyocyanate

HSs

healthy subjects

IFN-γ

interferon-gamma

IL

interleukin

mAbs

monoclonal antibodies

MMF

mycophenolate mofetil

NS

not significant

PBMCs

peripheral blood mononuclear cells

PBS

phosphate-buffered saline

PE

phycoerythrin

rh

recombinant human

SEM

standard error of the mean

SMA

smooth muscle antibody

TGF-β

transforming growth factor beta

Th

T-helper cell

TLC

thin-layer chromatography

Treg

regulatory T cell.

Autoimmune hepatitis (AIH) is an inflammatory disease of the liver, characterized by female preponderance, interface hepatitis on histology, hypergammaglobulinemia, and serum autoantibody positivity.[1, 2] Several lines of evidence indicate that in AIH, numerical and functional regulatory T-cell (Treg) defects are likely to play a permissive pathogenic role, allowing effector CD4 and CD8 T lymphocytes to initiate and perpetuate liver damage.[3-5]

The reasons for Treg functional impairment in AIH are unclear. Previous studies in both mice and humans have highlighted a number of mechanisms used by Tregs to mediate suppression, including release of anti-inflammatory cytokines and modulation of antigen-presenting cell function.

More recently, metabolic disruption of effector cell function by Tregs has also been explored.[6] Central to this mode of suppression is expression by Tregs of the ectoenzyme, CD39, which catalyzes the degradation of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP). AMP is subsequently converted to the immunomodulatory nucleoside, adenosine, by CD73, an ectoenzyme that works in tandem with CD39.[7-9]

At variance with the murine setting, CD39 in humans is expressed not only by conventional Tregs, but also by cytokine (i.e., interleukin (IL)-4, IL-5, IL-17, and interferon-gamma [IFN-γ]), producing effector memory cells.[8, 10] Again, unlike mice, CD73 is poorly expressed by human Tregs, suggesting that, in humans, AMP conversion to adenosine is mediated by paracrine mechanisms or by the presence of CD73 on target or neighboring cells.[10]

Compared to their CD39neg counterpart, CD39pos Tregs have been shown to be phenotypically stable upon proinflammatory challenge[10] and to display preferential suppression over T-helper (Th)17 immunity.[11]

Defective numbers of CD39pos Tregs have been reported in patients with multiple sclerosis,[8, 11] where these cells are also impaired in their ability to suppress IL-17 production.[11] Defective CD39pos Treg function has been also described in systemic lupus erythematosus.[12] Additionally, the presence of a CD39 single-nucleotide polymorphism has been reported in Crohn's disease and found to be associated with low CD39 messenger RNA expression levels and disease susceptibility.[13]

Given the key role of CD39 in governing Treg-suppressive function, we aimed to explore whether impairment of Tregs, previously observed in AIH, resided in alterations of CD39 expression. To this end, we investigated the frequency, phenotypic, and functional signature of CD39pos Tregs in AIH as well as their stability upon proinflammatory challenge, a feature particularly relevant to the development of immunotherapeutic strategies aimed at reconstituting immunotolerance through Treg adoptive transfer.

Patients and Methods

Patients and Controls

Forty-one patients with anti-nuclear (ANA)- and/or anti-smooth muscle antibody (SMA)-positive AIH (25 females) were studied. At the time of or close to diagnosis, a liver biopsy showed interface hepatitis in all patients. Patients with bile duct changes characteristic of sclerosing cholangitis on retrograde cholangiography were excluded from analysis. The median age of patients included in the study was 14 years (range, 6-27). Of 14 patients with active disease, defined by the presence of abnormal aspartate aminotransferase (AST) levels, 3 were studied before immunosuppressive treatment was started. Twenty-seven patients were studied during drug-induced remission (defined by normalization of AST levels). Treatment consisted of prednisolone (5-15 mg/day) with or without azathioprine (25-150 mg/day) or mycophenolate mofetil (MMF; 500-2,000 mg/day, n = 10). Demographic and biochemical data are shown in Table 1. Eight subjects with liver disorders of nonautoimmune and nonviral etiology served as disease control (DC) patients (7 females; median age: 15 years; range, 6-25). Of this group, 2 patients had nonalcoholic fatty liver disease, 1 had α-1 antitrypsin deficiency, 1 had Gilbert's syndrome, 1 had Wilson's disease, 1 had congenital portosystemic shunt, 1 had Alagille's syndrome, and 1 had hepatic adenoma. Twenty-five healthy subjects (HSs) served as normal controls (15 females; median age: 35 years; range, 22-50). The age difference between AIH/DC patients and HSs was derived from ethical constraints in obtaining blood from healthy children. The study was approved by the ethical committee of King's College Hospital (London, UK), and written consent was obtained from each AIH patient and HS enrolled in the study.

Table 1. Autoimmune Hepatitis Patient Demographics and Laboratory Data
  %AgeASTBilirubinIgG% ANA Positive% SMA Positive
Laboratory DataNo.Female(Years)(nv: <50 IU/L)(nv: <20 µmol/L)(nv: 6.5-17 g/L)(Median)(Median)
  1. Data are presented as median (range), unless otherwise stated.

  2. Abbreviation: IgG, immunoglobulin G; nv, normal value.

  3. a

    Four patients were studied before starting treatment and 5 were studied after treatment initiation, but before the normalization of AST levels.

  4. b

    P < 0.005; ‡P < 0.05 comparing AST, bilirubin, and IgG levels in patients with active disease and those at remission.

Active disease147112 (9-18)112 (54-1,074)b25 (8-82)b15.6 (2.4-25.9)‡44 (1/40)33 (1/640)
Near presentationa97811 (9-15)117 (61-1,074)39 (8-82)19.0 (2.4-25.9)60 (1/40)40 (1/400)
Relapse56012 (9-18)87 (54-224)14 (10-24)14.0 (10.0-19.5)025 (1/640)
Remission275515 (6-27)27 (16-46)9 (4-42)10.7 (0.9-19.0)9 (1/60)9 (1/120)

Cell Separation

Peripheral blood mononuclear cells (PBMCs) were isolated as described previously.[3] Viability of mononuclear cells, determined by trypan blue exclusion, exceeded 98%.

Flow Cytometry

PBMCs were stained with allophycocyanin (APC) cychrome-7 (Cy7)-conjugated anti-CD4, APC-conjugated anti-CD73, phycoerythrin (PE)-Cy7-conjugated anti-CD39 (all eBioscience, Hatfield, UK), PE-conjugated anti-CD25, fluorescein isothyocyanate (FITC)-conjugated anti-CD127, PE-conjugated anti-CD45RO, and FITC-conjugated anti-CD62L (all BD Biosciences, Discovery Labware, Oxford, UK) monoclonal antibodies (mAbs). Cells were incubated at 4°C in the dark for 30 minutes and washed with phosphate-buffered saline (PBS), supplemented with 1% fetal calf serum (FCS), before analysis by flow cytometry (FCM) on a Becton Dickinson fluorescence-activated cell sorter (FACSCanto II; Beckton Dickinson Immunocytochemistry Systems, San Jose, CA). FACSDiva software was used for analysis. The percentage of cells positive for FOXP3 or intracellular CD152 was determined after fixation and permeabilization with Cytofix/Cytoperm (BD Biosciences) and the addition of APC-conjugated anti-FOXP3 (eBioscience) or APC-conjugated anti-CD152 (BD Biosciences) mAbs.

The percentage of IFN-γ, IL-17, IL-10, and transforming growth factor beta (TGF-β)-positive cells was determined after exposure to phorbol 12-mystrate 13-acetate (10 ng/mL) and ionomycin (500 ng/mL; both from Sigma-Aldrich, Gillingham, UK) and after the addition of brefeldin A (10 µg/mL; Sigma-Aldrich) for 5 hours. Cells were then stained using PE-conjugated anti-IFN-γ, PE-conjugated anti-IL-10 (both BD Biosciences), FITC-conjugated anti-IL-17 (eBioscience), or peridinin chlorophyll protein complex–conjugated anti-TGF-β (R&D Systems, Abingdon, UK).

Cell Stimulation

PBMCs were seeded at 1 × 106 cells/ml in 96-well round-bottomed plates in RPMI-1640 presupplemented with 2 mM of L-glutamine and 1% antibiotic-antimycotic solution (both from Gibco, Invitrogen, Paisley, UK) and 10% FCS. Cells were exposed to anti-CD3/anti-CD28 T-cell expander (ratio bead/cell: 1/2; Dynal Invitrogen, Oslo, Norway) and recombinant human (rh)IL-2 (30 U/mL; EuroCetus, Amsterdam, the Netherlands), a protocol chosen on the basis of previous experiments.[14] To test whether the phenotype of CD39pos Tregs remained stable upon proinflammatory challenge, cells were treated with rhIL-6 (0.04 µg/mL) and rhIL-1β (0.01 µg/mL; both R&D Systems) and cultured at 37°C in 5% CO2 for 5 days. Cells were washed in PBS/1% FCS, and FCM was performed as described above.

Cell Purification

For coculture assays, CD4pos cells were isolated from the total PBMC population using immunomagnetic beads (Dynal Invitrogen), as previously described.[3, 14] CD4pos T cells were then stained with FITC- or APC-Cy7-conjugated anti-CD4 (eBioscience), APC- or PE-conjugated anti-CD25 (BD Bioscences), and PE- or PE-Cy7-conjugated CD39 (eBioscience). CD4pos cells were then sorted into CD25high, CD25highCD39pos (CD39pos Tregs), CD25highCD39neg (CD39neg Tregs), and CD25neg subsets by fluorescence-activated cell sorting (FACS) using a Becton Dickinson cell sorter (FACSAria II; Beckton Dickinson Immunocytochemistry Systems). Purity of CD25high, CD25highCD39pos, and CD25highCD39neg populations exceeded 95%, and purity of CD25neg cells exceeded 98%.

For experiments assessing CD39 enzymatic activity, CD4posCD25pos and CD4posCD25neg populations were isolated immunomagnetically as described previously.[3, 14] The purity of immunomagnetically isolated populations exceeded 85%.

Measurement of Enzymatic Activity

Enzymatic activity of immunomagnetically isolated CD4posCD25neg and CD4posCD25pos cells was measured indirectly by quantifying the concentration of free phosphate using the colorimetric Sensolyte malachite green phosphate assay kit (AnaSpec, Seraing, Belgium). Populations were washed in saline solution containing 0.9% (w/v) NaCl—to remove residual phosphate-containing media—and plated at 2 × 105 cells/mL, before exposure to 10 µM of ATP (Sigma-Aldrich) for 15 minutes. Phosphate concentration was quantified at 600 nm using an absorbance plate reader after comparison with a standard curve.

Thin-layer chromatography (TLC) was performed, as described previously,[7] to visualize the hydrolysis of radiolabeled ADP to AMP and its subsequent conversion to adenosine. Immunomagnetically isolated CD4posCD25pos or CD4posCD25neg cells (2.5 × 105) were exposed to 2 mCi/mL of [C14]ADP (PerkinElmer, Cambridge, UK) in the presence of 10 mM of Ca2+ and 5 mM of Mg2+. Aliquots were collected at reaction times of 5, 10, 20, 40, and 60 minutes before analysis of [C14]ADP hydrolysis products by TLC. Samples were loaded onto silica gel matrix plates (Sigma-Aldrich), and [C14]ADP derivatives were separated using an appropriate solvent mixture.[15]

Coculture Assays

Once purified, CD25neg responder cell populations were seeded overnight in 96-well round-bottomed plates in the presence of anti-CD3/anti-CD28 T-cell expander (ratio bead/cell: 1/2; Dynal Invitrogen) and rhIL-2 (30 U/mL; EuroCetus). CD25high, CD39pos, or CD39neg Tregs were then added to autologous CD25neg responder cells at a ratio of 1:8.[14] Parallel cultures of CD25neg responder cells in the absence of Tregs were performed. To analyze the proliferation of effector cells, for the final 18 hours of culture, cells were pulsed with 0.25 µCi/well 3H-thymidine (PerkinElmer) and harvested using a multichannel harvester. The amount of incorporated 3H-thymidine was measured using a β-counter. In preliminary experiments, in which cells from 4 AIH patients and 4 HSs were tested, proliferation was also analyzed using the CellTrace carboxy fluorescein succinimidyl ester (CFSE) cell proliferation kit (Molecular Probes, Paisley, UK). For analysis of cytokine production, cells were stained with FITC- or APC-Cy7-conjugated anti-CD4, FITC- or PE- conjugated anti-IL-17 (all eBioscience) and APC- or PE- conjugated anti-IFN-γ (IQ Products [Groningen, the Netherlands] and BD Biosciences) and analyzed as described above.

Statistical Analysis

The normality of variable distribution was assessed by Kolmogorov-Smirnov's goodness-of-fit test; once the hypothesis of normality was accepted (P < 0.05), comparisons were performed using paired or unpaired Student t tests for linked or unlinked data, respectively. A one-way analysis of variance, followed by Tukey's multiple comparisons test, was used to compare the means of multiple samples. Results are expressed as mean ± standard error of the mean (SEM), unless otherwise stated, and P values <0.05 were considered significant. Data were analyzed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA) and SPSS software (IBM; Hampshire, UK).

Results

Enumeration and Characterization of CD4posCD25highCD39pos Regulatory T Cells

The frequency of circulating CD4posCD39pos cells was similar in AIH patients, DC patients, and HSs (9.7 ± 2.7, 10.8 ± 2.4, and 11.1 ± 2.0, respectively, P = not significant [NS]). However, the frequency of CD4posCD39posCD25high (hereafter denoted as CD39pos Tregs) was markedly reduced in AIH patients, compared to DC patients or HSs (Fig. 1A,B). The frequency of CD39pos Tregs was similar in AIH patients with active and inactive disease (4.2 ± 1.3 versus 3.5 ± 0.7; P = NS). Male AIH patients had fewer CD39pos Tregs, compared to their female counterparts (2.4 ± 0.6 versus 5.1 ± 0.9; P = 0.03). The difference between males and females was not observed in the HS population (9.4 ± 2.8 versus 6.8 ± 1.8; P = NS). To test the influence of age on the frequency of CD39pos Tregs, AIH patients were subdivided into those <13 or ≥13 years of age. Frequency of CD39pos Tregs did not differ when this comparison was made (4.7 ± 1.4 versus 3.3 ± 0.3; P = NS). CD25high cells contained higher proportions of cells positive for CD39, compared to the CD25medium(med) and CD25neg populations in HSs (CD25high: 43.0 ± 4.8 versus CD25med: 26.0 ± 5.3 [P < 0.05] and versus CD25neg: 14.6 ± 3.2 [P < 0.001]), DC patients (CD25high: 46.7 ± 8.4 versus CD25med: 23.4 ± 3.6 [P < 0.05] and versus CD25neg: 15.2 ± 3.5 [P < 0.01]), and AIH patients (CD25high: 40.7 ± 4.6 versus CD25med: 22.8 ± 3.8 [P < 0.05] and versus CD25neg: 12.6 ± 2.6 [P < 0.001]). In AIH patients, DC patients, and HSs, the frequency of CD4posCD39pos cells positive for FOXP3 was greater in the CD25high subset, compared to the CD25med or CD25neg populations (Fig. 1C).

Figure 1.

Characterization of CD39pos Tregs. (A) Frequency of CD4posCD25highCD39pos Tregs in 1 representative AIH patient, 1 DC patient, and 1 HS. Plots show gated CD4pos lymphocyte populations. (B) Frequency of CD4posCD25highCD39pos Tregs in 31 AIH patients, 8 DC patients, and 25 HSs. (C) Frequency of FOXP3pos cells within CD4posCD39posCD25high, CD4posCD39posCD25med, and CD4posCD39posCD25low populations in 31 AIH patients, 8 DC patients, and 25 HSs.

AIH patients treated with prednisolone and MMF had a lower frequency of CD39pos Tregs, compared to those treated with prednisolone alone (5.6 ± 1.9 versus 1.7 ± 0.5; P = 0.01) or with prednisolone and azathioprine (4.7 ± 1.1 versus 1.7 ± 0.5; P = 0.03).

Compared to HSs and DC patients (Table 2), CD39pos Tregs from AIH patients contained a lower frequency of cells negative for CD127 (the lack of which distinguishes bona fide Tregs from effector T cells[16, 17]), lower proportions of cells positive for the memory cell marker, CD45RO, and a similar frequency of cells positive for the Treg function-associated markers, FOXP3, CD152, or CD62L. Approximately 10% of CD39pos Tregs expressed CD73 in AIH patients, DC patients, and HSs.

Table 2. Phenotypic Signature and Cytokine Profile of CD39posTregs
 HSDCAIHP ValueaP ValuebAIH ActiveAIH RemissionP Value
  1. Data are presented as mean ± SEM. FOXP3, CD127, CD45RO, CD73, IL-17, IFN-γ, IL-10, and TGF-β data refer to 31 AIH patients (22 inactive and 9 active), 8 DC patients, and 25 HSs. CD152 and CD62L data refer to 10 AIH patients (4 inactive and 6 active), 8 DC patients, and 4 HSs.

  2. a

    P < 0.05, comparing HSs and AIH patients.

  3. b

    P < 0.05, comparing DC patients and AIH patients.

FOXP3pos47.4 ± 3.343.9 ± 7.343.3 ± 2.9NSNS37.7 ± 4.545.6 ± 3.6NS
CD127neg92.3 ± 1.495.3 ± 0.983.0 ± 3.00.010.05076.3 ± 4.785.8 ± 3.6NS
CD45ROpos91.3 ± 2.493.3 ± 2.080.8 ± 3.40.020.00375.0 ± 8.583.3 ± 3.3NS
CD73pos13.8 ± 2.910.3 ± 1.711.2 ± 2.5NSNS9.1 ± 2.512.0 ± 3.4NS
CD152pos18.3 ± 5.227.9 ± 5.520.2 ± 2.3NSNS21.4 ± 3.618.4 ± 2.6NS
CD62Lpos50.0 ± 6.155.5 ± 7.149.9 ± 5.0NSNS47.2 ± 3.654.1 ± 12.0NS
IL-17pos11.4 ± 2.712.9 ± 3.315.7 ± 2.5NSNS19.9 ± 4.814.1 ± 3.0NS
IFN-γpos12.3 ± 2.717.6 ± 2.99.2 ± 2.0NSNS6.1 ± 1.710.6 ± 2.6NS
IL-10pos11.2 ± 2.021.1 ± 5.013.1 ± 2.8NSNS11.3 ± 6.113.5 ± 3.2NS
TGF-βpos9.7 ± 2.511.6 ± 2.811.2 ± 2.4NSNS13.4 ± 2.810.3 ± 3.1NS

The frequency of CD39pos Tregs producing the proinflammatory cytokines, IFN-γ and IL-17, or the anti-inflammatory cytokines, TGF-β or IL-10, was similar in AIH patients, DC patients, and HSs (Table 2).

Given that the frequency and the phenotype profile of CD39pos Tregs in HSs and DC patients were comparable, only cells from HSs were used for the following experimental sections.

Phenotypic Stability of CD39pos Regulatory T Cells

After stimulation of PBMCs with anti-CD3/anti-CD28 T-cell expander (Table 3), the frequency of CD39pos Tregs positive for FOXP3 or CD127 increased to a similar extent in AIH patients and HSs. Frequency of CD45ROpos CD39pos Tregs increased in HSs, whereas frequency remained unchanged upon stimulation in AIH patients. Frequencies of IFN-γ- and IL-17-producing cells within CD39pos Tregs remained stable in HSs, whereas they increased in AIH patients upon stimulation. The increase in the frequency of CD39pos Tregs expressing IL-17 or IFN-γ was greater in AIH patients, compared to HSs (Table 3).

Table 3. Percentage of FOXP3pos, CD127neg, CD45ROpos, or CD73pos Within CD39pos Cells and Frequency of IFN-γ- or IL-17-Producing CD39pos Cells After Stimulation of PBMCs With Anti-CD3/CD28 T-Cell Expander
 HSAIH
 Frequency at BLFrequency After StimulationP ValueSize of ChangeFrequency at BLFrequency After StimulationP ValueSize of Change
  1. Data are presented as mean ± SEM and refer to 6 AIH patients with inactive disease and 9 HSs.

  2. a

    P < 0.05, when comparing magnitude of change in frequencies between HSs and AIH patients.

  3. Abbreviation: BL, baseline.

FOXP3pos33.2 ± 3.881.3 ± 9.2<0.00148.1 ± 9.135.3 ± 6.973.4 ± 10.00.0325.4 ± 10.2
CD127pos1.8 ± 0.85.7 ± 1.40.0103.3 ± 1.62.6 ± 1.811.6 ± 2.90.016.0 ± 2.2
CD45ROpos87.3 ± 5.198.7 ± 0.80.03011.4 ± 4.490.5 ± 5.298.1 ± 1.1NS5.1 ± 3.6
CD73pos24.0 ± 5.310.4 ± 3.6NS−13.6 ± 6.817.5 ± 7.727.2 ± 13.9NS6.5 ± 12.4
IFN-γpos7.3 ± 2.66.6 ± 2.4NS−0.7 ± 2.98.3 ± 5.328.3 ± 15.1NS20.0 ± 11.5a
IL-17pos8.0 ± 1.28.8 ± 2.0NS0.9 ± 1.93.8 ± 1.812.3 ± 3.70.035.7 ± 2.3a
Table 4. Percentage of FOXP3pos, CD127neg, CD45ROpos, or CD73pos Within CD39pos Cells and Frequency of IFN-γ or IL-17-Producing CD39pos Cells After Exposure of PBMCs to IL-1β and IL-6
 HSAIH
 Frequency at BLFrequency After StimulationP ValueSize of ChangeFrequency at BLFrequency After StimulationP ValueSize of Change
  1. Data are presented as mean ± SEM and refer to 6 AIH patients with inactive disease and 9 HSs.

  2. a

    P < 0.05, when comparing magnitude of change in frequencies between HSs and AIH patients.

  3. Abbreviation: BL, baseline.

FOXP3pos33.2 ± 3.873.9 ± 5.1<0.00140.7 ± 7.735.3 ± 6.962.7 ± 13.1NS27.4 ± 11.1
CD127pos1.8 ± 0.81.3 ± 0.8NS−0.5 ± 1.22.6 ± 1.814.3 ± 12.0NS11.7 ± 10.3
CD45ROpos87.3 ± 5.190.9 ± 2.7NS3.6 ± 4.790.5 ± 5.281.5 ± 6.3NS−9.0 ± 7.2
CD73pos24.0 ± 5.36.4 ± 2.00.010−17.6 ± 6.017.5 ± 7.725.9 ± 7.7NS8.3 ± 11.9a
IFN-γpos7.3 ± 2.668.9 ± 15.00.00454.0 ± 13.48.3 ± 5.369.0 ± 10.10.00260.7 ± 10.4
IL-17pos8.0 ± 1.214.9 ± 4.4NS5.3 ± 5.33.8 ± 1.817.9 ± 2.70.00314.1 ± 2.6

Interestingly, in AIH patients, the size of the increase in frequency of IFN-γpos CD39pos Tregs correlated with serum AST concentration (r2 = 0.82; P < 0.05).

Exposure of PBMCs to IL-1β and IL-6 (Table 4) increased the frequency of FOXP3pos CD39pos Tregs in HSs, but not AIH patients, but had no effect on the frequency of CD39pos cells expressing CD127 or CD45RO. The frequency of CD73pos CD39pos Tregs decreased significantly in HSs, whereas it increased in AIH patients, though not significantly. Though the frequency of CD39pos Tregs producing IFN-γ increased to a similar extent in both AIH patients and HSs, the frequency of those producing IL-17 increased in AIH patients, but remained stable in HSs.

ATP/ADP Hydrolysis

Immunomagnetically isolated CD4posCD25pos Tregs from AIH patients were less able to hydrolyze exogenous ATP, compared to HSs (Fig. 2A). In HSs, but not AIH patients, CD25pos cells generated greater concentrations of phosphate, compared to CD25neg cells (Fig. 2A), as reflected by higher CD39 expression (3.65 ± 0.40 versus 24.53 ± 4.61; P = 0.002).

Figure 2.

CD39 enzymatic activity of Tregs in AIH patients and HSs. (A) Ability of immunomagnetically isolated CD4posCD25pos and CD4posCD25neg cells from 3 AIH patients and 4 HSs to produce free phosphate—the by-product of ATP hydrolysis—after the addition of exogenous ATP. (B) CD39 ADPase enzymatic activity of immunomagnetically isolated CD4posCD25pos and CD4posCD25neg cells was assessed by TLC at 5-, 10-, 20-, 40-, and 60-minute time points after incubation with 14C-radiolabeled ADP substrate. Image is representative of three independent experiments.

Analysis of [C14]-radiolabeled ADP hydrolysis by TLC (Fig. 2B) revealed that CD4posCD25pos cells from HSs were able to hydrolyze ADP into AMP and, at the longer reaction time of 60 minutes, these cells could generate extracellular nucleosides.

In contrast, AMP generation was less pronounced in CD4posCD25pos cells from AIH patients. CD4posCD25neg cells in HSs and AIH patients degraded ADP less efficiently than the CD4posCD25pos populations, failing to produce extracellular adenosine.

Suppressive Ability of CD39pos Tregs

Preliminary experiments, in which both 3H-thymidine and CFSE were used to analyze the suppressive ability of immunomagnetically isolated CD4posCD25pos cells, confirmed reports[3, 4, 14] that Tregs from AIH patients are less able to suppress the proliferation of autologous CD4posCD25neg responder cells, compared to HSs (Supporting Fig. 1). Because CFSE- and 3H-thymidine-based assays gave comparable results, given the requirement for fewer cells, 3H-thymidine was used to measure the proliferation of FACS populations, which had comparatively low yield compared to those magnetically isolated.

CD4posCD25high Tregs from HSs and AIH patients and CD39neg Tregs from HSs were able to suppress the proliferation of responder T cells (one-sample t tests, when comparing suppression in the presence and absence of Tregs; P = 0.04, P = 0.04, and P = 0.05, respectively). Percent suppression of proliferation by CD25high cells was lower in AIH patients than in HSs. In HSs, CD39pos Tregs were poor suppressors of proliferation, compared to conventional CD4posCD25high Tregs and CD39neg Tregs (Fig. 3A).

Figure 3.

Suppressive ability of CD39pos Tregs. Ability of FACS-sorted CD4posCD25high, CD4posCD25highCD39pos, and CD4posCD25highCD39neg Treg populations to suppress proliferation (A) or IL-17 production (B) of CD4posCD25neg responder cells. For suppression of proliferation, data refer to 4 HSs and 4 AIH patients. For suppression of IL-17 production, data refer to 6 HSs and 10 AIH patients.

In HSs, CD4posCD25high, CD39pos, and CD39neg Tregs were able to suppress the production of IL-17 (one-sample t tests; P = 0.001, P = 0.01, and P = 0.007, respectively), whereas in AIH patients, only the CD25high population was able to suppress IL-17 production (one-sample t test; P = 0.006). Both the CD25high and CD39pos Tregs were less able to suppress IL-17 production in AIH patients, compared to HSs (Fig. 3B).

Discussion

In the current study, we show that Tregs expressing the ectonucleotidase, CD39, are present at low levels and are also dysfunctional in AIH.

Phenotypic analysis has indicated that the expression of CD39 is associated with classical Treg features (i.e., high CD25 and FOXP3 and low CD127 expression). CD39pos Tregs effectively suppress CD4 T-cell IL-17 production while exerting poor control over target cell proliferation, suggesting that this Treg subgroup may have a specific role in dampening Th17 immunity. Low frequencies of CD39pos Tregs and inability to adequately control IL-17-mediated immunoreactivity have been described also in patients with multiple sclerosis.[8, 11] Moreover, low CD39 expression has been reported in inflammatory bowel disease, where it is associated with a CD39 polymorphism,[13] suggesting a genetically encoded defect of immune regulation in this condition. Future studies should explore whether CD39 polymorphisms account for the observed Treg/effector cell imbalance in AIH and therefore contribute to disease initiation and/or perpetuation.

A comparison between health and disease has revealed that CD39pos Tregs from AIH patients are impaired in number, in their ability to hydrolyze ATP and ADP and in their suppressive function, indicating that, in AIH, CD39pos Treg impairment occurs at multiple levels.

A potential limitation of this study is the use of a heterogenous AIH population, including patients under different treatment regimens. This has been overcome, to some extent, by the size of the patient group, which has enabled us to observe interesting, novel associations. We have, for example, noted that the frequency of CD39pos Tregs was markedly decreased in AIH patients receiving prednisolone and MMF, compared to those treated with prednisolone alone or in combination with azathioprine, raising the possibility that these treatment regimens differentially affect the frequency of this regulatory T-cell subset. Alternatively, the lower CD39pos Treg frequencies observed in the MMF-treated group may reflect the fact that these patients have a particularly severe form of disease characterized by a more marked impairment in immune regulation and the mechanisms governing it. Also of note, CD39pos Treg defects were particularly pronounced in male AIH patients. Because this gender difference was not observed in healthy subjects, it is possible that hormonal differences, particularly the presence of estrogen, can partially overcome the CD39pos Treg defect in AIH.

In AIH patients, particularly in those with active disease, CD39pos Tregs contain low proportions of CD127neg lymphocytes, suggesting that CD39pos Tregs from AIH patients, in addition to being numerically defective, are also skewed toward a proinflammatory profile. Lower CD127neg cell frequencies within CD39pos Tregs are paralleled by lower proportions of CD45ROpos cells. Though the reasons for the CD45RO decrease are unclear, it should be recalled that, in humans, CD39 is mainly expressed on memory cells10; therefore, low frequencies of CD45ROpos lymphocytes may reflect the numerical CD39pos Treg impairment.

In AIH, CD39pos Tregs are also less able to hydrolyze ATP and ADP, this defect ultimately resulting in reduced production of AMP and immunosuppressive adenosine. Persistently high levels of proinflammatory ATP and ADP may contribute to the perpetuation of inflammation. Inefficient CD39 hydrolytic activity is likely to account for the decreased ability of Tregs to control CD4 effector cell function, in particular, the production of IL-17, which is involved in AIH liver damage.[18, 19]

Expression of CD39 has previously been linked to Treg lineage stability.[10, 11] Characterization of CD39pos Treg phenotype before and after stimulation with anti-CD3/anti-CD28, a classical T-cell stimulus, and with IL-6 and IL-1β, cytokines mimicking the proinflammatory environment in AIH, has shown that CD39pos Tregs from AIH patients are less stable than in HSs, because they undergo a marked increase in production of IL-17 and IFN-γ. These data suggest that Treg impairment in AIH might derive from an increased rate of Treg conversion into effector cells (Fig. 4).

Figure 4.

Impairment of CD39pos Tregs in AIH. In health, CD39pos Tregs produce adenosine and adequately control autoreactive T-cell effector functions. In AIH, multiple CD39pos Treg defects—a reduction in frequency, an inability to suppress IL-17 production by effector cells, and a propensity to convert to IFN-γ- or IL-17-producing effectors—contribute to impaired immunosuppression and autoimmune liver damage.

Of note, in contrast to HSs, CD73 expression by CD39pos Tregs in AIH patients remained elevated after proinflammatory challenge. That CD73 is strictly linked to activation was previously shown in a study by Doherty et al., who reported high CD73 expression levels on CD4 T cells from Crohn's disease patients with more active disease.[20]

The findings of a reduced stability of CD39pos Tregs in AIH patients should be taken into consideration when developing immunotherapeutic strategies aimed at reestablishing immune homeostasis through adoptive Treg transfer. Thus, protocols for Treg expansion should include treatment with agents or molecules (e.g., retinoic acid and/or rapamycin) aimed at boosting Treg properties while inhibiting their conversion to pathogenic Th1 and Th17 effector cells. Because CD39pos Tregs exhibit potent IL-17-suppressive properties in HSs, possible mechanisms for boosting CD39 expression in AIH should be explored. A potential candidate is retinoic acid, which is able to boost CD39 expression by naïve T cells (Robson, unpublished observation).

Previous investigations have shown accumulation of CD39pos Tregs in liver of patients with chronic hepatitis B infection.[21] In the experimental cancer setting, hepatic growth of melanoma metastatases is inhibited in CD39null mice, whereas CD39pos Tregs inhibit antitumor immunity.[22] Future studies should examine the frequency and tissue localization of liver-infiltrating CD39pos Tregs in AIH and explore whether defective CD39 expression by circulating Tregs is also reflected in the inflamed liver. These findings would have important implications for the development of adoptive Treg therapy for AIH.

In conclusion, this study has shown that, in AIH, there is a numerical decrease in CD39pos Tregs. These CD39pos Tregs are impaired in their enzymatic and suppressive abilities and, upon proinflammatory challenge, are less stable than in HSs. Defective immune regulation in AIH may derive not only from impaired Treg number and function, but also from an increased rate of Treg conversion into effector cells.

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