Human T-cells recognise N-terminally Fmoc-modified peptide
Introduction
Since the discovery that short peptides can substitute for protein antigens [1], synthetic peptides have been used to probe and manipulate the immune system. Peptide libraries have been used to identify T-cell epitopes in infectious [2] and autoimmune diseases [3], [4], Peptide vaccines have also been developed for the treatment of cancers [5], the prevention of parasitic [6] and viral infections [7]. The widespread use of synthetic has been made possible by improvements in automated methods for peptide synthesis that use Fmoc1 amino acids as building blocks. Once peptide synthesis is completed the remaining, N-terminal Fmoc group is removed by treatment with piperidine [8].
We used a panel of synthetic peptides covering the entire sequence of human pre-proinsulin to clone human pre-proinsulin-specific T-cells from individuals at risk of T1D2. T1D is an autoimmune disease caused by progressive T-cell mediated destruction of insulin-producing β cells in the pancreas [9]. Human T-cell responses to β-cell autoantigens have been reported [3], [10], [11], [12] but are imprecise and poorly characterised.
Surprisingly, from the peripheral blood of an individual at risk for T1D, we isolated a CD4+ T-cell clone that specifically recognised a proinsulin peptide with an N-terminal Fmoc adduct. Here we demonstrate that human T-cells, specific for Fmoc, can be expanded following exposure to synthetic peptides. This finding has important implications for the application of synthetic peptides.
Section snippets
Blood donors and cell purification
Blood was obtained by venupuncture from an asymptomatic, GAD3 antibody-positive male with informed consent and ethics committee approval. His HLA type was A2, −; B13, 27; DR1, 4; DQ5, 8. Peripheral blood mononuclear cells (PBMC) were isolated over Ficoll/Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden), washed twice in PBS and resuspended in IMDM medium supplemented with 5% pooled male human serum, 2 mM glutamine (Glutamax, Gibco, Rockville, MA, USA), 5×10−5 M
Detection and cloning of PPI peptide-specific T-cells
Our original aim was to isolate T-cell clones from pre-diabetic donors that were specific for human pre-proinsulin. PBMC were prepared from a suitable donor and cultured with one of 20 pools of pre-proinsulin varimer peptides (Table 1). ELISpot assays were used to detect rare peptide-specific T-cells after stimulation with peptide pools and expansion in IL-2 and IL-15. A sample of each line was tested in an ELISpot assay with the appropriate peptide pool. Seven lines gave more spots than the
Discussion
T-cell responses to contaminants in synthetic peptides are a widely acknowledged, yet under-reported problem. Responses to peptide contaminants have been reported following priming of murine CD8+ T-cells to the La autoantigen [14] and human CD4+ T-cells to acetylcholine receptor peptides [21], [22]. We isolated a human CD4+, TCRα + T-cell clone restricted by HLA DQB1*0301-2 (DQ8) that recognises an Fmoc adduct of a human proinsulin peptide.
During peptide synthesis, Fmoc is used to block the
Note added in proof
Analysis by mass spectrometry of a pepset containing 72, 15mer peptides comprising the sequence of human proinsulin showed that 11 or 15% of the peptides contained species that were 222 Da heavier than the target peptide, consistent with Fmoc adducts.
Acknowledgements
This work was funded by a Centre Programme Grant from the Juvenile Diabetes Research Foundation and the Australian National Health and Medical Research Council and the Wellcome Trust. AWP is a CR Roper Fellow of the Faculty of Medicine, Dentistry and Health Sciences at the University of Melbourne. We are grateful to the blood donors for their generosity.
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