Dysfunctional CD39(POS) regulatory T cells and aberrant control of T-helper type 17 cells in autoimmune hepatitis

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 CD39(pos) Treg defects that potentially contribute to the impaired immunoregulation that is characteristic of AIH. We have examined the frequency and phenotype of CD39(pos) Tregs by flow cytometry and measured their ectonucleotidase activity. The capacity of CD4(pos) CD25(high) , CD4(pos) CD25(high) CD39(pos) , and CD4(pos) CD25(high) CD39(neg) subsets to suppress both proliferation of effector T cells and interleukin (IL)-17 production was evaluated. In AIH, CD39(pos) 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 CD39(pos) 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, CD39(pos) Tregs are decreased in number, fail to adequately hydrolyze proinflammatory nucleotides and do not efficiently suppress IL-17 production by effector CD4 T cells. CD39(pos) 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.

Figure 1.. Characterisation of CD39^pos Tregs.

(A) Frequency of CD4^posCD25^highCD39^pos Tregs in one representative autoimmune hepatitis (AIH) patient, one disease control (DC) patient and one healthy subject (HS). Plots show gated CD4^pos lymphocyte populations. (B) Frequency of CD4^posCD25^highCD39^pos Tregs in 31 AIH patients, 8 DC patients and 25 HS. (C) Frequency of FOXP3^pos cells within CD4^posCD39^posCD25^high, CD4^posCD39^pos CD25^med and CD4^posCD39^posCD25^low populations in 31 AIH patients, 8 DC patients and 25 HS.

Figure 2.. CD39 enzymatic activity of Tregs in AIH and HS.

(A) Ability of immunomagnetically isolated CD4^posCD25^pos and CD4^posCD25^neg cells from 3 autoimmune hepatitis (AIH) patients and 4 healthy subjects (HS) to produce free phosphate – the bi-product of ATP hydrolysis – after the addition of exogenous ATP. (B) CD39 ADPase enzymatic activity of immunomagnetically isolated CD4^posCD25^pos and CD4^posCD25^neg cells was assessed by thin layer chromatography at 5, 10, 20, 40 and 60 minute time-points following incubation with 14C-radiolabelled ADP substrate. Image representative of 3 independent experiments.

Figure 3.. Suppressive ability of CD39^pos Tregs.

The ability of FACS-sorted CD4^posCD25^high, CD4^posCD25^highCD39^pos and CD4^posCD25^highCD39^neg Treg populations to suppress the proliferation (A) or IL17 production (B) of CD4^posCD25^neg responder cells. For suppression of proliferation, data refer to 4 healthy subjects (HS) and 4 autoimmune hepatitis (AIH) patients. For suppression of IL17 production, data refer to 6 HS and 10 AIH patients.

Figure 4.. Impairment of CD39^pos Tregs in AIH.

In health, CD39^pos Tregs produce adenosine and adequately control autoreactive T cell effector functions. In AIH, multiple CD39^pos Treg defects – a reduction in frequency, an inability to suppress IL17 production by effector cells, and a propensity to convert to IFNγ or IL17 producing effectors – contribute to impaired immunosuppression and autoimmune liver damage.