Genetic Analysis

Methods

Genetic analysis

For sequencing 100 ng genomic DNA was amplified and amplimers were sequenced using the Big Dye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Life Technologies,Darmstadt, Germany). The sequencing products were separated on an Applied Biosystems3130xl Genetic Analyzer. Primer sequences are available on request().

RNA isolationandcDNAsynthesis

A Ficollgradientof an EDTA blood sample from the patient was performedand MNCs wereisolated. RNA was isolatedusing the RNeasy ® Mini Kit [QIAGEN GmbH, Hilden]. cDNA was synthesizedwithSuperScript ® II Reverse Transcriptase [Invitrogen, Carlsbad, California, USA].

PCR andSequencing

ForcDNAanalysisincluding exon 8 of the FOXP3 geneprimerswerelocated in exon 7 (f-primer) and exon 9 (r-primer) of the FOXP3 geneand PCR amplification was performed. PCR productswerepurifiedwith the useofQIAamp DSP DNA Blood Mini Kit [QIAGEN GmbH, Hilden]. The purified PCR productsweresequencedby Big Dye Terminator v1.1 Cycle Sequencing Kit [Applied Biosystems, Darmstadt, Germany] on an Applied Biosystems Prism 3100 Genetic Analyzer.

Gelelectrophoresis

PCR productswereloaded on 1% agarosegels [Seakem® LE Agarose (LONZA, Basel, Swiss)]. Forsizing a 100bp DNA ladder [Invitrogen, Carlsbad, USA] was used.

Immunological analysis

PBMC were isolated by Percoll density gradient centrifugation (PanBiotech). T reg cells were quantified by flow cytometry using the T regphenotyping kit from Beckton Dickinson according to the instructions of the manufacturer. Data acquisition was performedwith a Gallios flow cytometer (Beckman Coulter). Data were analyzed using FlowJo version 7.2.5 analysis software (Tree Star). For the Treg suppression assay, PBMC of the patient and a healthy control were separated into CD4+CD25- responder cells and CD4+CD25+ Treg cells by MACS (Regulatory CD4+ CD25+ T cell isolation kit; MiltenyiBiotec). 2x104 T responder cells were cocultured for 4 days in IMDM + 10% human AB serum (Lonza) supplemented with 1 mg/ml anti-CD3 (clone Hit3a) either alone, in the presence of 2x104 irradiated allogeneic PBMC (allo) or in the presence of allo cells and 2x104Treg cells. After 2 days, cells were pulsed with BrdU(1:1000). BrdU incorporation was detected by Europium-labeled anti-BrdU antibodyafter overnight incubation according to the manufacturer’s protocol (Delfia® cellproliferation kit, Perkin Elmer). Eu-fluorescence was measured with an EnVisionreader (Perkin Elmer). Mean fluorescence of triplicate values were normalized to thefluorescence of a standard Europium solution (Perkin Elmer).