Introduction In autoimmune hepatitis (AIH) CD4+CD25+ regulatory T-cells (T-regs) are defective in their ability to control CD4 T-cell effector function. T-regs express Galectin9 (Gal9), a β-galactosidase-binding-protein that inhibits Th1-mediated immune-responses by binding the T-cell-immunoglobulin-and-mucin-domain3 (Tim-3) on CD4 effector cells. In AIH T-regs express reduced levels of Gal9.
Aim To characterise lineage-specific transcription factor and cytokine profiles of peripheral-blood-derived Gal9+ T-regs in AIH.
Method 34 ANA/SMA+ patients (24 females; median age: 14.6 years) and 17 healthy subjects (HS, 12 females, median age: 29 years) were studied. The phenotype of circulating T-regs was determined by cytofluorimetry using CD4, CD25 and CD127 monoclonal antibodies. The frequency of cells positive for Gal9, FOXP3, T-bet, GATA3 and RORC and that of cells producing IFNγ, IL-10, TGF-β and IL-17 were determined by intracellular staining. T-reg suppressor function was evaluated in a proliferation assay following co-culture with CD25-Tim-3+ and CD25-Tim-3− autologous target cells.
Results Within Gal9+ cells the frequency of: (1) FOXP3+ cells was lower in AIH than in HS (14.4±2 vs 42.8±3.1, p<0.001); (2) T-bet+, GATA3+ and RORC+ cells was similar in AIH and HS; (3) IL-10-producing cells was lower in AIH than in HS (5.1±0.6 vs 9.1±0.5, p<0.001) but higher than in the Gal9− T-reg fraction for both (AIH: 5.1±0.6 vs 2.3±0.2, p=0.001; HS: 9.1±0.5 vs 3.3±0.4, p<0.001); (4) TGF-β-producing cells was lower in AIH than in HS (6.4±0.7 vs 8.1±0.4, p=0.04); (5) IFNγ- and IL-17-producing cells was higher in AIH than in HS (IFNγ: 4.4±0.6 vs 2.1±0.3, p=0.002; IL-17: 4.1±0.6 vs 1.8±0.2, p=0.002). Treatment with anti-IL-10 neutralising antibodies reduced T-reg ability to suppress CD25-Tim-3+ cell proliferation (AIH: 42% inhibition in the absence of antibodies vs 36% in their presence, p=0.06; HS: 56% vs 48%, p=0.04), while did not affect CD25−Tim-3− cell proliferation.
Conclusion A skewing towards a pro-inflammatory phenotype and a reduced proportion of FOXP3+ and IL-10-producing cells within Gal9+ T-regs may contribute to defective immunoregulation in AIH. The reduction of Gal9+T-reg suppression following anti-IL-10 blockade both in health and AIH, suggests a role for IL-10 in Gal9+T-cell immune-regulatory function.
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