Abstract Book 2010 - CIMT Annual Meeting

040 Kotsiou | Cellular therapy
Application of MHC class I single chain trimers for leukaemia
immunotherapy
Eleni Kotsiou 1,2 , Julian Dyson 2 , Keith G Gould 1
1
Department of Immunology, Wright-Fleming Institute, St Mary’s campus, Imperial College,
London W2 1PG, UK
2
Immunobiology Section, Commonwealth Building, Hammersmith Hospital, Imperial College,
London W12 ONN, UK
84
Major Histocompatibility Complex (MHC) class I
single chain trimers (SCTs) consist of antigenic
peptide, β2-microglobulin and MHC heavy chain
all joined together in a single polypeptide molecule
via flexible linkers. SCTs are extremely valuable
tools for the stimulation of CD8+ T cell responses
because of their enhanced stability compared to
conventional MHC molecules and can therefore be
used for the expansion of peptide specific CD8+ T
cells either in vitro or in vivo.
We have expressed the human minor histocompatibility
(H) antigens, HA-1 and HA-2 (expressed only
on haematopoietic cells and restricted by human
leukocyte antigen (HLA) A2) and the mouse HY
minor transplantation antigens originating from
the Smcy and Uty genes (restricted by H2-Db) in the
SCT format. For the HLA-A2 SCTs, different forms
were constructed by modifying the CD8 binding
region (CD8 enhanced and non-binding variants)
whereas in both mouse and human SCTs we introduced
cysteine residues in the heavy chain and
first linker that have been shown to form a disulfide
bond, anchoring the peptide more efficiently
(disulfide-trap (dt) variants).
All human and mouse SCTs had good cell surface
expression as determined by specific antibody
staining of TAP2- deficient CHO cell transfectants.
Soluble forms of HA-1 and HA-2 SCTs were obtained
by retroviral transduction of human embryonic
kidney (HEK) 293 T cells, and subsequent purification
of the soluble protein from culture superna-
tant. As the HA-1 and HA-2 minor H antigens are
only expressed on normal and malignant haematopoietic
cells, they are good candidates for therapeutic
targeting. For this reason soluble SCTs were
used for the in vitro expansion of allo-restricted
CD8+ T cells specific for HA-1 and HA-2 minor H
antigens from HLA-A2 negative individuals. Furthermore
the minor male specific transplantation
antigens DbUty and DbSmcy were expressed as
SCTs and dtSCTs and will be introduced into bone
marrow dendritic cells by retroviral transduction.
Their potential to expand antigen specific CD8+
T cells in vitro and in vivo as a vaccination model
will be assessed. Further results will be presented
illustrating the applications and functions of MHC
class I SCTs.