Expansion and de novo generation of potentially therapeutic regulatory T cells in patients with autoimmune hepatitis.
CD4+CD25+ regulatory T cells (T-regs) are central to the maintenance of immune tolerance and represent an immune intervention candidate in autoimmune hepatitis (AIH), a condition characterized by impaired T-reg number and function. We investigated whether T-regs can be expanded from the existing CD4+CD25+ T cell pool and generated de novo from CD4+CD25- T cells in AIH patients and healthy controls. Purified CD4+CD25+ and CD4+CD25- T cells from 24 patients with type 1 AIH and 22 healthy controls were cultured for up to 5 weeks with anti-CD3/anti-CD28 T cell expander and high-dose interleukin-2 (IL-2). Cell phenotypes, suppressor ability, forkhead winged/helix transcription factor box P3 (FOXP3) gene, and protein expression were assessed weekly by cytofluorimetry, proliferation assay, real-time polymerase chain reaction (PCR), and immunoblot. During culture, the number of CD4+CD25+ T cells derived from the existing T-reg pool (expanded T-regs) and generated de novo from CD4+CD25- T cells (newly generated T-regs) increased constantly up to week 4 in both healthy controls and, to a lesser extent, in AIH patients. Expanded T-regs retained conventional T-reg phenotype and, compared with baseline, demonstrated more vigorous suppressive function and increased FOXP3 gene and protein expression. Newly generated T-regs not only acquired T-reg phenotype but underwent greater growth and were more resistant to apoptosis than expanded T-regs. Their suppressive function augmented throughout culture, reaching a peak at week 4, preceded by a peak FOXP3 gene and protein expression at week 2. Suppressor function and FOXP3 expression of both expanded and newly generated T-regs were higher in normal controls than in AIH patients.


CONCLUSIONS
Functionally enhanced T-regs can be expanded and generated de novo in patients with AIH. This finding may assist in reconstituting impaired immune regulation and restoring peripheral tolerance through T-reg infusion in this condition.
Longhi
Maria Serena
MS
Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, Denmark Hill, London, UK.
Meda
Francesca
F
Wang
Pengyun
P
Samyn
Marianne
M
Mieli-Vergani
Giorgina
G
Vergani
Diego
D
Ma
Yun
Y
D000293:Adolescent; D000328:Adult; D015703:Antigens, CD / Q000097:blood; D017209:Apoptosis; D002648:Child; D051858:Forkhead Transcription Factors / Q000235:genetics; D005786:Gene Expression Regulation / Q000276:immunology; D019693:Hepatitis, Autoimmune / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D053645:Interleukin-2 Receptor alpha Subunit / Q000097:blood; D008213:Lymphocyte Activation; D012016:Reference Values; D050378:T-Lymphocytes, Regulatory / Q000166:cytology / Q000276:immunology* / Q000473:pathology
D015703:Antigens, CD; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D053645:Interleukin-2 Receptor alpha Subunit