Abstract Book 2010 - CIMT Annual Meeting

072 van Esch | New targets & new leads
T cell epitope discovery through HLA class I peptide ligand exchange
and combinatorial coding
Wim van Esch 1 , Juk Yee Mok 1 , Annemieke Molenaar 1 , Arnold Bakker 2 , Mireille Toebes 2 ,
Sine Reker Hadrup 2 , Chengyi Shu 2 , Ernst Soethout 3 , Josine van Beek 3 , Huib Ovaa 4 and Ton
Schumacher 2
1 Dept. R&D, Sanquin Reagents, Amsterdam, The Netherlands
2 Div. Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands
3 Netherlands Vaccine Institute, Bilthoven, The Netherlands
4 Div. Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands
Human major histocompatibility complex class I
(HLA-I) consists of beta2-microglobulin, a polymorphic
heavy chain and a peptide ligand. Peptides
in context with HLA-I on the cell surface are
recognized by appropriate cytotoxic T cells. Our
aim is to develop a platform for high-throughput
identification of HLA-I ligands and T cell detection
using flow cytometry in order to detect new
disease-specific epitopes/T cells. To this end, conditional
ligands that can be cleaved in the HLAbound
state upon UV irradiation were designed
for different HLA alleles. Cleavage of HLA-I-bound
conditional ligand in the presence of another experimental
ligand results in peptide exchange.
Only those peptides that fit in the peptide binding
groove will be able to maintain the integrity of the
empty HLA complex, and the efficiency of HLA-I
stabilization by experimental ligands can be determined
by ELISA. Subsequently, HLA-I-binding
peptides identified in this manner are tested for
their immunological relevance. To this purpose, a
combinatorial coding technology has been developed
that allows the parallel detection of a multitude
of different T cell populations in a single sample.
Detection of antigen-specific T cells from peripheral
blood by combinatorial coding is as efficient
as detection with conventionally fluorescently-labeled
HLA multimers and allows comprehensive
screens to be performed. Using these technologies,
several screens for HLA ligands have been performed,
either manually or using robotics, using
peptides potentially associated with infectious or
non-infectious diseases. Large-scale screenings of
their immunological relevance by analysis of T cell
responses using combinatorial coding are ongoing.
Together these techniques form a highly efficient
platform for T cell epitope discovery in a broad
range of human diseases and for the monitoring of
disease- and therapy-induced T cell immunity.
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