Cancer Research in Switzerland - Krebsliga Schweiz

De Libero Gennaro | Lipid-specific T cells against
leukaemic blasts (KLS 02211-02-2008)
We identified highly polar lipid fractions derived from
THP1 AML and C1R LCL cell lines able to stimulate two
different CD1c-restricted T cell clones. These fractions
were obtained using two different procedures from total
cellular lipids, extracted according to the Folch method.
LC-MS-MS analysis of the fractions confirmed the presence
of several ion masses in the biologically active ones,
and some of these masses were shared between the stimulatory
fractions, independently of the procedure used.
However, the low yield and purity of active lipids achieved
using both methods did not allow us to identify the structures
of these molecules. Therefore, we developed a new
extraction procedure and established a new HPLC purification
strategy. Using this novel method, we divided total
lipids into 10 different fractions according to their polarity
and charges. Only one fraction strongly stimulated the selected
T cell clones with high efficacy and potency. The
antigenic capacity of this fraction was further confirmed
by CD1c plate-bound assays, in which soluble recombinant
CD1c molecules were attached to plastic wells,
loaded with the fraction and then used to stimulate T cell
clones. The high and specific T cell clone activation induced
by fraction-loaded CD1c complexes clearly suggested
that contained lipids behave as minimal antigens
and do not require processing by APC to become immunogenic.
The LC-MS-MS profile of the stimulatory fraction revealed
the presence of few molecular species. In order to
separate these molecules and to isolate the active ones,
we further sub-fractionated the T cell activating fraction
by HPLC and LC-MS-MS. Three out of 60 sub-fractions
showed potent stimulatory activity, and in each of them
only one predominant molecular species was present, as
indicated by the MS-MS analysis performed in real time
during the collection.
A further LC-MS-MS analysis, performed with two different
mass spectrometers (ESI ion trap and triple quadrupole)
in both positive and negative ionisation modes, indicated
that two of these molecules are characterized by
a fragmentation pattern in part similar to that of lysophosphatidic
acid. To confirm this finding and to assign exact
molecular mass to these compounds, we analyzed the active
sub-fractions with an Orbitrap mass spectrometer
equipped with a nanospray system. This allowed us to
confirm the structure of the previously identified molecules
and to determine length and features of the single
acyl chain present in the two active molecules. In a new
series of studies we performed nuclear magnetic resonance
(NMR) analysis of the antigenic lipid compounds
after extensive purification. NMR confirmed the proposed
structure and provided complementary information on
the nature of lipids.
All together, these findings show that a unique lipid with
a structure never described before accumulates in leukaemic
cells. This novel lipid family seems to be very abundant
in rapidly proliferating cells. It will be important to
investigate the metabolic pathways responsible for the
synthesis of these lipids and how they are regulated in leukaemic
cells.
Project coordinator
Prof. Dr. Gennaro De Libero
Experimentelle Immunologie
Departement Biomedizin
Universität Basel
Hebelstrasse 20
CH-4031 Basel
Phone +41 (0)61 265 23 65
gennaro.delibero@unibas.ch
Dirnhofer Stephan | Inducing cell death in apoptosisresistant
hematological tumors: Impact on diagnosis
and therapeutic intervention (OCS 01792-10-2005)
Study outline
Patients with DLBCL (diffuse large cell B cell lymphoma)
and AML (acute myeloid leukaemia) have been evaluated
for potential therapeutic markers regarding CD44 and its
interaction partners. DLBCL and AML belong to the most
abundant lymphomas and leukaemias with a wide-ranging
prognosis. A long-term goal is to develop tailor-made
therapies for defined groups of patients to allow for more
potent and specific treatment.
Study design
Our main objective was to find out how the resistance
against apoptosis of malignant hemopoietic cells (from
DLBCL and AML patients) can be favourably affected, i.e.
abolished.
Study results
We showed that co-expression of CD44 variant isoforms
(all isoforms analyzed) and CD168 (RHAMM, receptor
for hyaluronan mediated motility) identifies a subgroup of
patients with DLBCL who have a very adverse prognosis.
This prognosis was independent of the international prognostic
index (IPI). Expression of CD168 RHAMM in AML
patients turned out to be a central prognostic indicator.
When CD168 is expressed in association with CD44, active
caspase-3 (as indicator for spontaneous apoptosis)
and the oncogenic transcriptions factors STAT-3 and -5 a
very adverse prognosis is implied for these patients.
Benefit for the patient
A therapeutic application of antibodies against surface
molecules such as CD44 and CD168 could exert a blocking
effect on the resistance against apoptosis. The group
of patients in which CD44 and CD168 are co-expressed
could have better survival odds with accordingly adjusted
therapies. When patients with AML receive stem cell therapies
the beneficial graft vs. leukaemia (GvL) reaction indicates
the importance of leukaemia associated antigens
(LAA). In search for potent LAA in patients with AML,
CD168 is an auspicious candidate for specific vaccination.
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