download report - Istituto Pasteur

P a r t i c i p a n t s :
Ricciarda Galandrini, Angela Gismondi, Stefania
Morrone, Marco Cippitelli, Rossella Paolini, professors;
Giovanni Bernardini, Alessandra Zingoni, Cristina
Cerboni, Alessandra Soriani, researchers; Francesca Di
Rosa, CNR researcher; Cinzia Fionda, Rosa Molfetta,
Helena Stabile, post-doc fellows; Michele Ardolino,
Giuseppe Sciumè, Elisabetta Parretta, PhD students.
Report of activity
Principal investigator: Angela Santoni
Professor of Immunology
Dipartimento di Medicina Sperimentale
Tel/Fax: (+39) 06 44340632
angela.santoni@uniroma1.it
Our proposal is aimed at analyzing the molecular
mechanisms underlying the activation of NK cell
functional program. In particular in the last year
(2007-2008), we focused our attention on:
The analysis of the role of phosphatidylinositol
4,5-bisphosphate (PIP2)-dependent pathways
in the development of natural cytotoxicity
Receptor-triggered activation of PI3K and PLC
gamma and signalling components controlling
cytoskeleton rearrangement are emerging as critical
events for the development of NK cell cytotoxicity.
We have recently demonstrated that the prototype of
NK activating receptors CD16, is coupled to phosphatidylinositol
4phosphate-5kinase type I (PI5KI)
alpha (Galandrini et al., Blood 2005). More recently
we have analyzed the requirement of PIP2 in the
generation of NK cell cytotoxicity. By live confocal
imaging of primary human NK cells expressing the
chimeric protein GFP-PH we observed the consumption
of a pre-existing PIP2 pool which is critically
required for the activation of cytolytic machinery,
during effector-target cell interaction.
We identified type I phosphatidylinositol4phosphate-
5kinase (PI5KI) α and γ isoforms as the enzymes
responsible for PIP2 synthesis in NK cells. In addition,
by means of shRNA-driven gene silencing we
demonstrated that both enzymes are required for the
proper activation of NK cytotoxicity and for inosi-
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Cellular and molecular immunology - AREA 5
Molecular mechanism underlying the activation of NK cell
regulatory and effector functions
tol-3,4,5trisphosphosphate generation upon receptor
stimulation. In an attempt to elucidate the specific
step controlled by PI5KIs we found that lytic granule
secretion but not polarization resulted impaired
in PI5KI α- and γ-silenced cells.
Collectively, our findings delineate a novel mechanism
implicating PI5KIα and γ isoforms in the synthesis
of PIP2 pools critically required for IP3dependent
Ca 2+ response and lytic granule release.
A paper containing these results has been published
in Blood 2008, 111:4165-72, and was highlighted in
the Blood preview by Dr. M. Colonna.
The analysis of Cdc42/WASP pathway in the
development of natural cytotoxicity
In collaboration with Prof. Luigi Notarangelo
(University of Brescia), using cultured NK cells from
Wiskott-Aldrich patients as effector cells, we have
previously reported a marked impairment of natural
and CD16-mediated cytotoxic functions (Gismondi et
al., Blood 2004). The inhibition of NK cell cytolytic
activity was associated with reduced ability of WAS
or XLT NK cells to form conjugates with susceptible
target cells and to accumulate F-actin upon binding.
More recently we have focused our attention on the
molecular mechanisms that control WASp function
in NK cells triggered through beta1 or beta2 integrins
or chemokine receptors as it has been reported
that chemokines can augment NK cell cytotoxicity
and play a major role in the control of immunological
synapse.
We observed that receptor engagement rapidly
results in activation of Cdc42 and induction of
WASp tyrosine phosphorylation. Moreover, we
have observed that upon beta1 or beta2 integrin or
chemokine receptor ligation, WASp can associate
with the Src kinase Fyn and the focal adhesion
kinase Pyk-2. Finally, we found that the inhibition
of NK cell functions observed in NK cells from
WASp patients was associated with a reduced ability
of WAS or XLT NK cells to up-regulate the