Abstract Book 2010 - CIMT Annual Meeting

050 Klobuch | Cellular therapy
T cell receptor RNA transfer into non-reactive human
T lymphocytes turns them into potent CMV-specific effector
cells
Sebastian Klobuch 1 , Katrin Besold 2 , Bodo Plachter 2 , Mirjam H. M. Heemskerk 3 , Niels
Schaft 4 , Ralf-Holger Voss 1 , Matthias Theobald 1 , Wolfgang Herr 1 , Simone Thomas 1
1 Department of Hematology & Oncology, University Medical Center, Mainz, Germany
2
Institute of Virology, University Medical Center, Mainz, Germany
3
Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands
4 Department of Dermatology, University Hospital of Erlangen, Erlangen, Germany
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Cytomeglovirus (CMV)-associated disease is a
life-threatening complication in patients after allogeneic
hematopoietic stem cell transplantation
(HSCT). Although antiviral drug therapy is successfully
used to reduce the risk of CMV disease,
long-term virus control requires the reestablishment
of protective antiviral T cell immunity in
the host. The latter is challenging, particularly if
the donor is CMV seronegative or possesses only
a weak CMV-specific memory T cell response and
thus, no CMV-reactive T cells are being transferred
from donor to recipient during HSCT. Grafting
nonreactive T cells of CMV seronegative donors by
virusantigen specific T cell receptors (TCR) may be
an efficient means to transfer CMV specific T cell
function into allogeneic HSCT recipients. In this
study, we have reprogrammed T cells of CMV-seronegative
donors with human TCR recognizing the
immunodominant HLA-A*0201- binding epitope
495-503 derived from the CMV pp65 protein. To
overcome the limitations of retroviral TCR gene
transduction that hamper clinical translation, we
used in vitro transcribed RNA encoding CMV-specific
TCR for electroporation of non-reactive human
T cells. After RNA transfection of anti-CD3 stimulated
peripheral blood mononuclear cells (PBMCs),
TCR expression levels were sufficient to trigger
IFN-g secretion and cytolytic activity against pp65
peptide-pulsed target cells and moreover against
human fibroblast upon CMV infection. Due to the
instability of the introduced RNA molecules, TCR
expression and effector function was measureable
for a time period of at least 3 days. We also observed
that TCR RNA transfection of CD4+ T cells
turned them into potent CMVpp65/HLA-A*0201specific
T helper cells. This was demonstrated by
co-incubating them with immature dendritic cells
(DC), which resulted in maturation of DC only in
the presence of the CMV pp65 epitope.
Next, we transfected pure naïve and memory CD8+
T cell subsets isolated from peripheral blood of
CMV-seronegative donors. Although 90% of naïve
CD8+ T cells were CMVpp65/HLA-A*0201 tetramer
positive after electroporation, they mediated only
marginal lysis toward CMV-infected fibroblasts.
In contrast, memory CD8+ T cells showed strong
TCR expression and cytotoxicity upon antigen recognition
up to one week. In summary, our data demonstrate
that non-reactive human T cells can be
easily redirected with CMVpp65 TCR RNA, thereby
gaining CMV-specific T cell effector function for a
considerable time period.
We therefore believe that CMVpp65 TCR RNA has
the potential to be further developed as a therapeutic
‘off-the-shelf’ reagent for CMV-seropositive
patients who undergo allogeneic HSCT from CMVseronegative
donors.