CD4+ T cells were identified as CD3+CD8− by surface staining. Intracellular IL-17 (FITC labelled IL-17A,
eBioscience, San Dieago, CA, USA) and IFN-γ (PE-labelled, BD Pharmingen) cytokines were measured using a fixation and permeabilisation kit 15 in a standard ICS assay. Absolute IL-17 numbers were determined as the percentage of cells staining positive for IL-17 secretion multiplied by the absolute CD4+ T-cell count of the patient/control at the time of sampling. FoxP3 expression was determined using anti-human FoxP3 staining set (Clone PCH101, eBioscience). Briefly, MG-132 cells were surface stained with FITC-labelled CD4+ (clone SK3 BD Pharmingen) and PE-labelled CD25 (clone MEM-181,
AbD Serotec, Oxford, UK). Cells were then washed and fix/permeabilised and stained using Fix/Permeabilisation Foxp3 staining kit for FoxP3 or the appropriate isotype control antibody 15. Absolute numbers of Treg cells were determined as the percentage of cells staining for defined Treg cell markers multiplied by the absolute CD4+ T-cell count of the patient/control at the time of sampling. Statistical analysis was performed using Graphpad PRISM software (Graphpad Prism, version 4, CA, USA). Unpaired multiple comparison tests were performed using non-parametric Kruskal–Wallis test. Paired analysis was performed using Student’s t test. p-Values of 0.05 and below were considered statistically significant. G.T. was supported by an educational grant from Gilead Pharmaceuticals. MAPK inhibitor The authors Dichloromethane dehalogenase acknowledge financial support from the Department of Health via the National Institute for Health Research
(NIHR) comprehensive Biomedical Research Centre award to Guy’s & St Thomas’ NHS Foundation Trust in partnership with King’s College London and King’s College Hospital NHS Foundation Trust. We thank our patients for their active participation in the study. The author contributions were as follows: Experiments were conceived and designed by G.T., B.P. and A.V. and performed by G.T. Data were analysed by G.T. and A.V. The manuscript was prepared by G.T., A.V. and B.P. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“T regulatory (Treg) cells are critical for maintaining immune homeostasis and establishing tolerance to foreign, non-pathogenic antigens including those found in commensal bacteria and food. Because of their multiple suppressive mechanisms, Tregs represent a promising strategy for engineering tolerance to self and non-self antigens in chronic inflammatory diseases.