Article Text

PDF

P94 Rapamycin helps maintain the regulatory phenotype of cytochrome P450IID6-specific Treg expanded from patients with autoimmune hepatitis type 2 by reducing the number of IFNγ+ cells
  1. B Holder,
  2. C R Grant,
  3. Y Ma,
  4. G Mieli-Vergani,
  5. D Vergani,
  6. M S Longhi
  1. Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, UK

Abstract

Introduction Control of T cell reactivity to cytochrome P450IID6 (CYP2D6) is key to immune-tolerance restoration in autoimmune hepatitis type 2 (AIH-2). CD4+CD25+ regulatory T cells (Treg), central to autoreactive T cell regulation, are impaired in AIH-2. Cell therapy based on CYP2D6-specific Treg (CYP-Treg) could provide specific control over effectors of liver damage in AIH-2. We have generated CYP-Treg from AIH-2 patients and demonstrated that these cells exert greater suppression than polyclonal Treg. Whether CYP-Treg can undergo expansion maintaining their functional phenotype is untested.

Aim To assess CYP-Treg functional phenotype over 2-week expansion in AIH-2 patients.

Methods 48 CYP-Treg cell lines were obtained from 12 AIH-2 patients positive for the predisposing HLA-DR7 and DR3 alleles; 36 Treg cell lines specific for a DR7 or DR3-restricted influenza-haemagglutinin (HA) peptide were generated from 9 DR7+ or DR3+ healthy subjects (HS) and used as controls. CYP- and HA-Treg were obtained after co-culture with peptide-pulsed semi-mature DCs. T-reg were expanded for 2 weeks in the presence of: (1) IL2 (300 U/ml); (2) IL2+rapamycin (RP) to enhance Treg function; (3) IL2+IL6/IL1b, cytokines mimicking the proinflammatory milieu of AIH-2. Treg phenotype was determined by flow cytometry; frequency of cytokine-producing cells by intracellular staining.

Results Before expansion, the frequency of CD127- and FOXP3+ cells exceeded 80% in both CYP- and HA-Treg. Compared to HA-Treg, CYP-Treg contained higher numbers of IFNg (6.4±1 vs 3.6±1.2, p=0.09), IL2 (9.5±2.6 vs 2.6±0.5, p=0.02), IL17 (7.1±1 vs 3±1.2, p=0.026), IL10 (9.1±2 vs 3.3±2.6, p=0.03) and TGFb (10.4±2 vs 3.6±0.7, p=0.001) producing cells. After expansion with IL2, CYP- and HA-Treg maintained a similarly high frequency of FOXP3+ and CD127 cells, while frequency of IFNg+ cells increased markedly (CYP-Treg: from 6.5±1 to 27±3, p<0.0001; HA-Treg: from 3.8±1.5 to 11±1.3, p<0.01). Exposure to RP decreased the frequency of IFNg+ cells by 36% (p=0.04) in HS and by 30% (p=0.15) in AIH-2. Exposure of Treg to IL6/IL1b had no effect on their phenotype and cytokine production.

Conclusion Compared to HA-Treg, CYP-Treg contain higher numbers of cytokine-producing cells, possibly reflecting a higher activation state of their precursors. After expansion, antigen-specific Treg retain a classical T-reg phenotype (CD127− and FOXP3+) even upon exposure to pro-inflammatory stimuli, but contain a high proportion of IFNg+ cells. Reduction of IFNg+ cells in the presence of RP suggests a role for this drug in the expansion of antigen-specific T-reg for immunotherapy in AIH-2.

Statistics from Altmetric.com

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.