Contamination of synthetic HuD protein spanning peptide pools with a CMV-encoded peptide.
To detect HuD-specific T cells in patients with Hu-antibody associated paraneoplastic neurological syndromes (Hu-PNS), we used short-term stimulation assays with HuD protein spanning peptide pools (PSPP) with purities of at least 70% and found reproducible false-positive CD8+ T-cell responses in three of 127 individuals (two healthy controls and one Hu-PNS patient), which all shared HLA-A*2402 and HLA-B*1801. After testing the 15-mer peptides of the HuD antigen separately, we discovered that the same three 15-mers yielded the CD8+ T cell response in those three individuals. This highly unusual result could not be reproduced when using new batches of peptides with a higher level of purity (>82% and >95%). Therefore, we assumed this response was not directed against the HuD peptides and analyzed the HuD 15-mers by Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS), which showed the presence of a cytomegalovirus (CMV)-encoded peptide (AIAEESDEEEAIVAY) as a contaminant. The three responding individuals all were CMV-seropositive and the contaminating peptide appeared to fit in the binding groove of HLA-B*18. Our data reveal that synthetic PSPP may contain immunogenic contaminations which may cause false positive results in T-cell stimulation assays.
de Graaf
Marieke T
MT
Department of Neurology, Erasmus University Medical Center, Rotterdam, The Netherlands.
de Beukelaar
Janet W
JW
Burgers
Peter C
PC
Luider
Theo M
TM
Kraan
Jaco
J
Smitt
Peter A Sillevis
PA
Gratama
Jan W
JW
D000595:Amino Acid Sequence; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003587:Cytomegalovirus / Q000276:immunology*; D051959:ELAV Proteins / Q000737:chemistry / Q000276:immunology*; D006650:Histocompatibility Testing; D006801:Humans; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D051959:ELAV Proteins; D010455:Peptides